2021 Book EmergingTrendsInPlantPathology

Krishna
P. Singh
Shamarao Jahagirdar
Birinchi Kumar Sarma Editors
Emerging
Trends
in Plant
Pathology
Emerging Trends in Plant Pathology
Krishna P. Singh • Shamarao Jahagirdar •
Birinchi Kumar Sarma
Editors
Emerging Trends in Plant

Pathology
Editors
Krishna P. Singh Shamarao Jahagirdar
Plant Pathology Department of Plant Pathology
G.B. Pant University University of Agricultural Sciences Dharwad
of Agriculture & Technology Dharwad, Karnataka, India
Pantnagar, India
Birinchi Kumar Sarma

Department of Mycology
and Plant Pathology
Banaras Hindu University
Varanasi, Uttar Pradesh, India
ISBN 978-981-15-6274-7 ISBN 978-981-15-6275-4 (eBook)

https://doi.org/10.1007/978-981-15-6275-4
# Springer Nature Singapore Pte Ltd. 2021

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To the Legend. . . .
(Dr. Y. L. Nene)
Foreword
Plant pathology as a discipline of agricultural science has played pivotal role over the
years in understanding plant diseases and in mitigating losses through cultural and
technological innovations. This is despite the disease scenarios that kept on evolving
and changing due to biotic, abiotic and edaphic factors. The plant pathologists have
eventually contributed significantly towards food security and in ameliorating the
livelihood of farmers across the globe. The science of plant pathology has been an
innovative and ever-emerging discipline in its scope, importance and technologies.
There have been many such innovations and advancements in each and every aspect
of plant pathology starting from the identification of the pathogen, underlining the
molecular mechanism of pathogenicity and resistance and also the management
strategies. With the commencement of the concept of sustainable agriculture, plant
disease management has become more important and has shifted from the traditional
chemical-based to more eco-friendly integrated disease management strategies with
more focus on the biocontrol and other green technologies. The latest innovations in
the field of detection and diagnosis, host resistance, disease forecasting and plant
biotechnology have helped us in better management of the diseases, but challenges
are still many more.
This first edition of Emerging Trends in Plant Pathology edited by K. P. Singh,
B. K. Sarma and Shamarao Jahagirdhar provides a comprehensive description and
highlights of the latest innovation and trends in the field of plant pathology and allied
fields. The focus is on understanding both the basic and applied aspects of plant
pathology and plant disease management. I hope the book would be of special
vii
viii Foreword
interest to both academics and professionals, working in the fields of plant pathol-
ogy, microbiology, biotechnology and plant breeding, as well as the plant protection
sciences. This book is a comprehensive reference for all those curious to understand
the latest advancements in their field of specialization.
I congratulate the editors and contributors for their dedicated effort to bring out
such a classic reference book for the scientific fraternity.
Executive Secretary, APAARI Ravi Khetarpal

Asia-Pacific Association of Agricultural
Research Institutions, 4th Floor, FAO Annex Building,
202/1 LarnLuang Road, Klong Mahanak Sub-District,
PomprabSattrupai District
Bangkok, Thailand
Preface
The science of plant pathology is essential for reliable food production through
management of plant diseases. Dynamics in the evolution of new races of plant
pathogens and changes in the global climatic scenario have made plant pathology an
ever-emerging discipline in its scope, importance and technologies. During the past
decade tremendous advancements and innovations took place in every aspect of
plant pathology starting from identification of pathogens to their management.
Additionally, with commencement of the concept of sustainable agriculture, plant
disease management has become more important and disease management strategies
have shifted from the traditional chemical based to more eco-friendly strategies.
Further, advancements in molecular biology studies have armed the researchers to
develop newer strategies for plant disease management. Therefore, recently more
focus has been on the applications of biocontrol agents, development of transgenic
cultivars, plant genome editing to other green technologies. Recent innovations in
the field of detection and diagnosis of plant diseases, host resistance, disease
forecasting and plant biotechnology have helped in developing strategies to manage
the diseases better and address the challenges still on the way.
In this first edition of Emerging Trends in Plant Pathology we have compiled
chapters to reflect on the recent trends and innovations in the field of plant pathology.
Emphasis was given to understanding both basic and applied aspects of modern tools
and techniques developed for detection and diagnosis of plant diseases that has
helped identifying pathogens associated with a disease in a very short time. Quicker
detection and diagnosis leads to identification of many new and emerging plant
pathogens, and it is of great help in designing effective management strategies
against them. The book has therefore also focused on the host-pathogen systems at
molecular level without considering the hosts and their pathogens as separate
entities. Chapters were also compiled to elaborate our understanding on host resis-
tance to plant pathogens and the mechanisms of actions of R and Avr genes of the
host and pathogen, respectively. Additionally, plant diseases and their epidemics are
highly influenced by environmental conditions and crop microclimate. The book
also includes chapters covering broad overviews of the recent advancements in
disease forecasting, remote sensing, GIS and GPS applications that help accurate
prediction of plant diseases and thereby saving crop losses from pathogens. The
book also highlights the developments in the area of biological control of plant
ix
x Preface
pathogens and use of microbial consortium which have received much attention in
the past decade due to promotion in organic farming and sustainable agriculture
globally. Further, the new-generation fungicides are considered far more
eco-friendly and very effective at low concentration and are highly target specific.
In this book, we have also focused on advancements in the use of secondary
metabolites from microbes and novel plant extracts as eco-friendly pesticides. We
also compiled chapters on the use of transgenics, cisgenics and genome editing that
are being increasingly used for plant disease management.
This book is very timely in providing essential and comprehensive source
materials, as it includes most relevant areas on emerging trends in plant pathology
and their role in crop protection.
Pantnagar, Uttarakhand, India Krishna P. Singh

Varanasi, Uttar Pradesh, India Shamarao Jahagirdar
Dharwad, Karnataka, India Birinchi Kumar Sarma
Contents
1 Emerging Plant Diseases: Research Status and Challenges . . . . . . . 1

Dipannita Mitra
2 Emerging Plant Diseases Under Changing Climate Scenario . . . . . . 19
Muhammad Priyadi and Pooja Upadhyay
3 Emerging Important Nematode Problems in Field Crops
and Their Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Mujeebur Rahman Khan, Irfan Ahamad, and Mohammad Haniph Shah
4 Modern Tools for Detection and Diagnosis of Plant Pathogens . . . . 63
Madhurababu Kunta, Jong-Won Park, W. Evan Braswell,
John V. da Graça, and Perry Edwards
5 Plant Virus Diagnostics: Traditional to Recent and Emerging
Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
V. K. Baranwal, Sajad Un Nabi, and Manoj K. Yadav
6 Epidemiology and Management of Potato Virus Y . . . . . . . . . . . . . 113
Tyler D. B. MacKenzie, Xianzhou Nie, and Mathuresh Singh
7 Major Fungal French Bean Diseases: Epidemiology and
Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
S. K. Gupta and Guervinder Singh
8 Whitefly-Transmitted Plant Viruses and Their Management . . . . . 175
P. S. Soumia, G. Guru Pirasanna Pandi, Ram Krishna,
Waquar Akhter Ansari, Durgesh Kumar Jaiswal,
Jay Prakash Verma, and Major Singh
9 Recent Advances in Management of Bacterial Diseases of Crops . . . 197
M. R. Ravikumar, H. S. Mahesha, J. U. Vinay, and K. Dinesh
10 Resistance Breeding and Exploitation of Wild Relatives for New
Resistance Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
N. K. Singh, Anjali Joshi, Smrutishree Sahoo, and Birendra Prasad
xi
xii Contents
11 New-Generation Fungicides for Sustainable Production and

Disease Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Dele Omoyele Adeniyi, Deepshikha Kunwar,
Lelia Nkechinyere Dongo, David Adedayo Animasaun,
and T. Aravind
12 Toxicity of Organophosphate Pesticide on Soil Microorganism:
Risk Assessments Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Durgesh Kumar Jaiswal, Ram Krishna, Saurabh Singh, Tarun Belwal,
Jay Prakash Verma, and Janardan Yadav
13 ‘Cu-Chi-Tri’, a New Generation Combination for Knowledge-Based
Management of Oomycete Pathogen, Phytophthora infestans . . . . . . . 297
Nandani Shukla, P. Lemke, B. M. Moerschbacher, and J. Kumar
14 Seaweed and Associated Products: Natural Biostimulant for
Improvement of Plant Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Jai Singh Patel and Arpan Mukherjee
15 Secondary Metabolites from Microbes for Plant Disease
Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
U. V. A. Buddhika and S. Abeysinghe
16 Beneficial Root Microbiota: Transmogrifiers of Secondary
Metabolism in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Akanksha Singh, Rupesh Chaubey, Stuti Srivastava,
Sumit Kushwaha, and Rakesh Pandey
17 Microbial Consortia for Plant Disease Management
and Sustainable Productivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Shamarao Jahagirdar, Gurudatta Hegde, P. U. Krishnaraj,
and D. N. Kambrekar
18 Microbial Biofilm: Formation, Quorum Sensing,
and Its Applications in Plant Disease Management . . . . . . . . . . . . . 385
Pravallikasree Rayanoothala, M. Divya, Sunita Mahapatra,
and Srikanta Das
19 Role of Endophytes in Plant Disease Management . . . . . . . . . . . . . 399
Sunanda Chakraborty, Debanjana Debnath, Sunita Mahapatra,
and Srikanta Das
20 Bioprospecting of Diseases of Horticultural Crops in India . . . . . . . 425
V. Devappa, C. G. Sangeetha, and N. Jhansirani
21 Transgenerational Plant Immunity in Plant Disease Management . . 457
Md Mahtab Rashid, Raina Bajpai, Basavaraj Teli, Ankita Sarkar,
and Birinchi Kumar Sarma
Contents xiii
22 Concept of Effectors and Receptors in Improving Plant

Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
C. S. Karibasappa, Yogendra Singh, T. Aravind, and K. P. Singh
23 Transgenic Technology for Disease Resistance in Crop Plants . . . . . 499
T. Makeshkumar, K. Divya, and S. Asha
24 RNAi Technology: A Novel Platform in Crop Protection . . . . . . . . 561
Munmi Borah and Naga Charan Konakalla
25 Genome Editing for Plant Disease Resistance . . . . . . . . . . . . . . . . . 577
Rajeev Singh
26 Green Nanotechnology and Its Application in Plant Disease
Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
V. B. Nargund, J. U. Vinay, K. N. Basavesha, S. Chikkanna,
S. Jahagirdar, and R. R. Patil
27 Plant Disease Management in Organic Farming System:
Strategies and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Laxmi Rawat, T. S. Bisht, and Dinesh Chandra Naithani
28 Organic Agriculture for Plant Disease Management . . . . . . . . . . . . 643
D. K. Shahi, Sweta Kachhap, Arvind Kumar, and B. K. Agarwal
29 Pest Risk Analysis and Plant Quarantine Regulations . . . . . . . . . . . 663
V. Celia Chalam, Kavita Gupta, Ruchi Sharma, Vaishali Dutt Sharma,
and A. K. Maurya
30 Remote Sensing Technology and Its Applications
in Plant Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
Ghada A. Khdery
31 Decision-Making Tools for Integrated Disease Management . . . . . . 703
K. P. Singh, T. Aravind, Amit Kumar Srivastava,
and C. S. Karibasappa
32 Bioinformatics in Plant Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . 725
Aamir Khan, Sakshi Singh, and Vinay Kumar Singh
Editors and Contributors
About the Editors
Dr. Krishna P. Singh is a Professor in the Department of Plant Pathology, G. B.

Pant University of Agriculture and Technology, Pantnagar, India. He graduated from
Gorakhpur University and holds master’s and doctoral degrees in Mycology and
Plant Pathology from Banaras Hindu University, India. He was awarded UGC and
CSIR fellowships. He has been a member of the editorial board of Applied Microbi-
ology (UK), Plant Disease Research, Indian Journal of Plant Pathology and Indian
Phytopathology. He is also a fellow of Indian Phytopathological Society, Indian
Society of Mycology and Plant Pathology and the International College of Nutrition
(FICN). He specializes in plant disease epidemiology and forecasting and has
published 73 research papers in national and international journals.
Dr. Shamarao Jahagirdar is a Professor in the Department of Plant Pathology,

University of Agricultural Sciences, Dharwad, India. A specialist in host plant
resistance and biological control of crop diseases, he graduated from University of
Agricultural Sciences, Dharwad, and holds Master’s & Doctoral degrees from
University of Agricultural Sciences, Bengaluru. He received an Indian Council of
Agricultural Research JRF and SRF from the Government of Karnataka, as well as
four international fellowship awards from the governments of Korea, Israel and the
Netherlands. He is a Fellow of the Indian Phytopathological Society, International
Benevolent Research Forum and Society for Advancement of Biodiversity Science.
He has published 150 papers in national and international journals.
Dr. Birinchi Kumar Sarma is a Professor in the Department of Mycology and

Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University,
Varanasi, India. A specialist in legume pathology and genomics, he completed his
post-doctoral research at the University of California, Davis, USA. He was a Visiting
Researcher at Dalhousie University, Canada, under the Shastri Indo-Canadian
Institute – FTI Award. He was also made an Associate of the National Academy
of Agricultural Sciences, New Delhi. He has published over 85 research papers,
edited 3 books and filed 5 patents. He also serves as editor for the international
journals PLOS ONE and Frontiers in Microbiology.
xv
xvi Editors and Contributors
Contributors
S. Abeysinghe Department of Botany, University of Ruhuna, Matara, Sri Lanka

Dele Omoyele Adeniyi Department of Plant Biology, University of Ilorin, Ilorin,
Kwara State, Nigeria
B. K. Agarwal Department of Soil Science and Agricultural Chemistry, Birsa
Agricultural University, Ranchi, Jharkhand, India
Irfan Ahamad Department of Plant Protection, Aligarh Muslim University,
Aligarh, Uttar Pradesh, India
David Adedayo Animasau Department of Plant Biology, University of Ilorin,
Ilorin, Kwara State, Nigeria
Waquar Akhter Ansari ICAR- India Institute of Vegetable Research, Varanasi,
Uttar Pradesh, India
T. Aravind Department of Plant Pathology, College of Agriculture, G. B. Pant
University of Agriculture, Pantnagar, Uttarakhand, India
S. Asha ICAR-Central Tuber Crops Research Institute, Thiruvananthapuram,
Kerala, India
Raina Bajpai Department of Mycology and Plant Pathology, Institute of Agricul-
tural Sciences, Banaras Hindu University, Varanasi, India
V. K. Baranwal Advanced Center of Plant Virology, Division of Plant Pathology,
ICAR-Indian Agricultural Research institute, New Delhi, India
K. N. Basavesha Department of Plant Pathology, College of Agriculture, Univer-
sity of Agricultural Sciences, Dharwad, Karnataka, India
Tarun Belwal College of Biosystems Engineering and Food Science, Zhejiang
Key Laboratory for Agri-Food Processing, Zhejiang University, Hangzhou, People’s
Republic of China
T. S. Bisht Krishi Vigyan Kendra, Ranichauri, Tehri, Uttarakhand, India
Munmi Borah Plant Virology Laboratory, Department of Plant Pathology, Assam
Agricultural University, Jorhat, Assam, India
W. Evan Braswell USDA APHIS PPQ CPHST, Mission Laboratory, Edinburg,
TX, USA
U. V. A. Buddhika School of Agricultural and Wine Sciences, Charles Sturt
University, Australia
Sunanda Chakraborty Department of Plant Pathology, Bidhan Chandra Krishi
Viswavidyalaya, Mohanpur, West Bengal, India
Editors and Contributors xvii
V. Celia Chalam Division of Plant Quarantine, ICAR-National Bureau of Plant

Genetic Resources, New Delhi, India
Rupesh Chaubey Department of Microbial Technology and Nematology, CSIR-
Central Institute of Medicinal and Aromatic Plants, Lucknow, India
S. Chikkanna Department of Plant Pathology, College of Agriculture, University
of Agricultural Sciences, Dharwad, Karnataka, India
Srikanta Das Department of Plant Pathology, Bidhan Chandra Krishi
Debanjana Debnath Department of Plant Pathology, Bidhan Chandra Krishi
V. Devappa Department of Plant Pathology, College of Horticulture, Bengaluru,
India
K. Dinesh Department of Plant Pathology, UAS, Dharwad, Karnataka, India
K. Divya ICAR - Central Tuber Crops Research Institute, Thiruvananthapuram,
India
M. Divya Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya,
Mohanpur, West Bengal, India
Lelia Nkechinyere Dongo Plant Pathology Section, Cocoa Research Institute of
Nigeria, Oyo, Nigeria
Perry Edwards CROPTIX, PA, USA
John V. da Graça Texas A&M University Kingsville Citrus Center, Weslaco, TX,
USA
Kavita Gupta Division of Plant Quarantine, ICAR-National Bureau of Plant
S. K. Gupta School of Agriculture, Shoolini University, India
G. Guru Pirasanna Pandi ICAR-National Rice Research Institute, Cuttack,
Odisha, India
Mohammad Haniph Shah Department of Plant Protection, Aligarh Muslim Uni-
versity, Aligarh, Uttar Pradesh, India
Gurudatt M. Hegde Institute of Organic Farming, UAS, Dharwad, Karnataka,
India
Shamarao Jahagirdar University of Agricultural Sciences, Dharwad, Karnataka,
India
Durgesh Kumar Jaiswal Institute of Environment and Sustainable Development,
Banaras Hindu University, Varanasi, Uttar Pradesh, India
xviii Editors and Contributors
N. Jhansirani Department of Plant Pathology, College of Horticulture, Bengaluru,

India
Anjali Joshi Department of Genetics and Plant Breeding, G. B. Pant University of
Agriculture & Technology, Pantnagar, Uttarakhand, India
Sweta Kachhap Department of Soil Science and Agricultural Chemistry, Birsa
D. N. Kambrekar University of Agricultural Sciences, Dharwad, Karnataka, India
C. S. Karibasappa Department of Plant Pathology, GB Pant University of Agri-
culture and Technology, Pantnagar, India
Aamir Khan Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India
Mujeebur Rahman Khan Department of Plant Protection, Aligarh Muslim Uni-
versity, Aligarh, Uttar Pradesh, India
Ghada A. Khdery National Authority for Remote Sensing and Space Sciences
(NARSS), Cairo, Egypt
Naga Charan Konakalla Department of Plant Protection Biology, Swedish
University of Agricultural Sciences, Alnarp, Sweden
P. U. Krishnaraj University of Agricultural Sciences, Dharwad, Karnataka, India
Ram Krishna Department of Environment & Sustainable Development, Institute
of Environment and Sustainable Development, Banaras Hindu University, Varanasi,
Arvind Kumar Department of Soil Science and Agricultural Chemistry, Birsa
J. Kumar Department of plant pathology, G. B. panty University of Agriculture
and Technology, Pantnagar, Uttarakhand, India
Madhurababu Kunta Texas A&M University Kingsville Citrus Center, Weslaco,
TX, USA
Deepshikha Kunwar Department of Plant Pathology, College of Agriculture,
G. B. Pant University of Agriculture, Pantnagar, Uttarakhand, India
Sumit Kushwaha Department of Microbial Technology and Nematology, CSIR –
P. Lemke Institute for Biology and Biotechnology of Plants, University of
Munster, Munster, Germany
Tyler D. B. MacKenzie Agricultural Certification Services Inc., Fredericton, NB,
Canada
Editors and Contributors xix
Sunita Mahapatra Department of Plant Pathology, Bidhan Chandra Krishi

H. S. Mahesha Crop Improvement Division, ICAR-IGFRI, Jhansi, Uttar Pradesh,
India
T. Makeshkumar ICAR- Central Tuber Crops Research Institute,
Thiruvananthapuram, India
A. K. Maurya Division of Plant Quarantine, ICAR-National Bureau of Plant
Dipannita Mitra Department of Cellular Coordination, Leibniz Institute of Plant
Biochemistry, Halle (Saale), Germany
B. M. Moerschbacher Institute for Biology and Biotechnology of Plants, Univer-
sity of Munster, Munster, Germany
Arpan Mukherjee Institute of Environment and Sustainable Development,
Dinesh Chandra Naithani Plant Pathology Division, College of Forestry, Tehri,
India
V. B. Nargund College of Agriculture, University of Agricultural Sciences,
Dharwad, Karnataka, India
Xianzhou Nie Fredericton Research and Development Centre, Agriculture and
Agri-Food Canada, Fredericton, NB, Canada
Rakesh Pandey Department of Microbial Technology and Nematology, CSIR-
Jong-Won Park Texas A&M University Kingsville Citrus Center, Weslaco, TX,
USA
Jai Singh Patel Department of Plant Food and Environmental Sciences, Faculty of
Agriculture, Dalhousie University, Halifax, Canada
R. R. Patil Department of Plant Pathology, College of Agriculture, University of
Agricultural Sciences, Dharwad, Karnataka, India
Birendra Prasad Department of Genetics and Plant Breeding, G. B. Pant Univer-
sity of Agriculture & Technology, Pantnagar, India
Muhammad Priyadi Department of Pharmacy, Faculty of Health Sciences,
Universitas Muhammadiyah Palangkaraya, Indonesia
Md. Mahtab Rashid Department of Mycology and Plant Pathology, Institute of
Agricultural Sciences, Banaras Hindu University, Varanasi, India
M. R. Ravikumar College of Agriculture, Hanumanamatti, UAS, Dharwad,
Karnataka, India
xx Editors and Contributors
Laxmi Rawat Plant Pathology Division, College of Forestry, Tehri, India

Pravallikasree Rayanoothala Department of Plant Pathology, Bidhan Chandra
KrishiViswavidyalaya, Mohanpur, West Bengal, India
C. G. Sangeetha Department of Plant Pathology, College of Horticulture, UHS
Campus, GKVK Post, Bengaluru, Karnataka, India
Ankita Sarkar Department of Mycology and Plant Pathology, Institute of Agri-
cultural Sciences, Banaras Hindu University, Varanasi, India
Birinchi Kumar Sarma Department of Mycology and Plant Pathology, Institute of
Agricultural Sciences, Banaras Hindu University, Varanasi, India
D. K. Shahi Department of Soil Science and Agricultural Chemistry, Birsa Agri-
cultural University, Ranchi, Jharkhand, India
Smreetushree Shahoo Department of Genetics and Plant Breeding, G. B. Pant
University of Agriculture & Technology, Pantnagar, India
Ruchi Sharma Division of Plant Quarantine, ICAR-National Bureau of Plant
Vaishali Dutt Sharma Division of Plant Quarantine, ICAR-National Bureau of
Plant Genetic Resources, New Delhi, India
Nandani Shukla Department of Plant Pathology, G. B. panty University of Agri-
culture and Technology, Pantnagar, Uttarakhand, India
Akanksha Singh Department of Microbial Technology and Nematology, CSIR-
Guervinder Singh Department of Plant Pathology, Dr. Y.S. Parmar University of
Horticulture and Forestry, Solan, India
K. P. Singh Department of Plant Pathology, GB Pant University of Agriculture and
Technology, Pantnagar, India
Major Singh ICAR-Directorate of Onion and Garlic Research, Pune, Maharashtra,
India
Mathuresh Singh Agricultural Certification Services Inc., Fredericton, NB,
Canada
N. K. Singh Department of Genetics and Plant Breeding, G. B. Pant University of
Agriculture & Technology, Pantnagar, India
Rajeev Singh Center for Tumor Biology and Immunology, Philipps Universität
Marburg, Marburg, Germany
Sakshi Singh Department of Molecular and Human Genetics, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
Editors and Contributors xxi
Saurabh Singh Department of Environment & Sustainable Development, Institute

of Environment and Sustainable Development, Banaras Hindu University, Varanasi,
Vinay Kumar Singh Centre for Bioinformatics, School of Biotechnology, Institute
of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India
Yogendra Singh Department of Plant Pathology, GB Pant University of Agricul-
ture and Technology, Pantnagar, India
P. S. Soumia ICAR-Directorate of Onion and Garlic Research, Pune, Maharashtra,
India
Amit Kumar Srivastava Institute of crop Science and Resource Conservation,
crop Science Group, Katzenburgweg 5, University of Bonn, Bonn, Germany
Stuti Srivastava Department of Microbial Technology and Nematology, CSIR-
Basavaraj Teli Department of Mycology and Plant Pathology, Institute of Agri-
cultural Sciences, Banaras Hindu University, Varanasi, India
Sajad Un Nabi Advanced Center of Plant Virology, Division of Plant Pathology,
Pooja Upadhyay Department of Plant Pathology, G.B. Pant University of Agricul-
ture and Technology, Pantnagar, Uttarakhand, India
Jay Prakash Verma Department of Environment & Sustainable Development,
Institute of Environment and Sustainable Development, Banaras Hindu University,
J. U. Vinay Department of Plant Pathology, UAS, Dharwad, Karnataka, India
Janardan Yadav Department of Soil Science, Institute of Agricultural Sciences,
Manoj K. Yadav Advanced Center of Plant Virology, Division of Plant Pathology,
Emerging Plant Diseases: Research Status
and Challenges 1
Dipannita Mitra
Abstract
Plant diseases result in significant crop destruction thereby inadequate food

supply and lead to economic and post-harvest losses in the agricultural production
sector throughout the world. Early detection of plant diseases and pathogens is
important for maintaining sustainability for the economy of the agricultural
sector. The prevention of plant disease and pathogens during the early stages
aids in plant health control and yield improvement. It is also crucial to analyze the
disease spread in plants for overcoming the issues related to physiological and
biological states in crop protection. This chapter reviews the research status of the
various emerging plant diseases responsible for a large amount of crop destruc-
tion every year all over the world and the challenges that the agricultural sector
face to overcome this problem.
Keywords
Plant pathogen disease · Symptomatic stage · Remote sensing · Flow cytometry
1.1 Introduction
Agriculture plays a dominant part in the worldwide economy and is the main source
of food, fiber, fuel, timber, income, and employment, thus maintaining socioeco-
nomic stability. The major threat to agriculture is nationwide crop losses due to
pathogen-induced plant diseases, which is considered to be a primary challenge for
the whole scientific community. The branch of plant pathology thus largely focuses
D. Mitra (*)
Department of Cellular Coordination, Leibniz Institute of Plant Biochemistry, Halle (Saale),
Sachsen Anhalt, Germany
Bad Waldsee, Baden Württemberg, Germany
# Springer Nature Singapore Pte Ltd. 2021 1

K. P. Singh et al. (eds.), Emerging Trends in Plant Pathology,
https://doi.org/10.1007/978-981-15-6275-4_1
2 D. Mitra
on increasing the fundamental understanding of host-pathogen interactions to detect

plant diseases and mitigate crop losses and enhance the total agricultural yield.
It was estimated in 1994 that worldwide crop losses due to plant diseases ranged
from 9.7 to 14.2% of the total yield; the figure is different in modern days due to the
emergence of new pesticides with a varied target range (Zadoks 1996). But, the
ability of these target pathogens to gradually develop resistance to frequently used
pesticides has allowed diseases to remain persistent and is proving to be a big threat
in today’s world (Strange and Scott 2005). A recent article in the Food and
Agriculture Organization (FAO) of the United Nations reported that there has been
a loss of about 20 to 40% of the global crop production due to pest infestations.
“Each year, plant diseases cost the economy around $220 billion and the invasive
pathogen around US$70 billion” as per the FAO reports. This huge amount of losses
deprives more than 800 million of the total worldwide population of adequate
consumable food (http://www.fao.org/news/story/en/item/1187738/icode/).
The fact that plant pathologists should be concerned with the need to minimize
losses due to endemic diseases correlates with the introduction of new foreign
pathogens resulting from the globalization of plants and plant products (Mack
et al. 2000).These plant pathogens can rapidly spread from an infected or diseased
plant to a healthy plant. The microorganisms that cause plant diseases include fungi,
viruses, nematodes, bacteria, and mycoplasmas (Lucas et al. 1992).
Plant diseases can be defined as any deviation from their healthy state with
symptoms and disorders mainly on their shoots, leaves, flowers, fruits, stem, and
roots. Diseased plants produce poor yields in terms of both quantity and quality.
Losses in the total yield in a plant can occur from the seed to the harvesting stage.
Quality of a plant product is attenuated when diseased spots or blotches are present;
thus monitoring plant health and detecting the pathogens early in the plant life cycle
is essential to reduce the chances of disease spread and also to facilitate effective
management practices.
It is a well-known fact that vegetable crops represent an important economic
segment of the total global economic production. But, gradually reoccurrence of
crop diseases has played havoc toward the society and for total agronomical growth.
For example, the famous famine in Ireland which was started in Europe during 1845
as an epidemic was caused by the late blight of potato by Phytophthora infestans
(Mundt et al. 2009). Similarly, plant pathogens threaten other food crops globally
including citrus, banana, and grapes. In southwestern Europe, a region known for its
grape cultivation, a disease caused by phytoplasmas called Flavescence doree is
widespread and is a major cause of annual economic losses (Martinelli et al. 2016).
Similarly, ready-to-eat salads like bagged salads have gained popularity through-
out the world especially in Europe since their introduction in the early 1980s,
marking opportunities for the fresh food industry. As this industry is growing, the
number of new diseases is growing in parallel. The past review indicates that these
seasonal salads are grown massively in a highly dense region in five to six cycles
annually in the same farms (Fig. 1.1); thus it lacks crop rotation and also sometimes a
shortage of applicable fungicides causing the growth of many fungal diseases, e.g.,
downy mildew of basil and Fusarium wilt of lettuce (Farr and Rossman 2019).
1 Emerging Plant Diseases: Research Status and Challenges 3
Fig. 1.1 Multitunnel

cultivation of lettuce and wild
rocket in southern Italy with
five to six production cycles
per year. (Source: Gullino
et al. 2019)
Other examples of major economic losses due to plant diseases are soybean rust
which is mainly a fungal disease in soybeans, but it was reported that by removing
20% of the infection, the farmers could make a profit of $11 million (Roberts et al.
2006). It was also estimated that the crop losses due to pathogen infection in the
United States could be attributed to non-native or foreign plant pathogens, e.g.,
chestnut blight fungus, Dutch elm diseases, and Huanglongbing citrus diseases
(Pimentel et al. 2005; Sankaran et al. 2010).
Plant diseases can be spread over a larger area of cultivation land with time
through the accidental introduction of vectors or through plant materials. The other
route of the spread of plant pathogens can be through ornamental plants that act as
hosts. Ornamental plants are always in increasing demand and are sold worldwide
before these diseases could be detected.
Recent technology has made it possible to consume crops that are produced on
foreign land. Thus, international import-export has resulted in new kind of plant
diseases, where a very low level of seed contamination can result into rapid emer-
gence of new diseases in a totally new geographic areas, thus resulting into severe
crop losses; it can affect the biological equilibrium of that region and sometimes also
start an epidemic (Gullino et al. 2019). The globalization of agricultural products has
resulted in many new resistant strains of pathogens which causes diseases that are
tough to detect. If these new strains migrate to a healthy area of cultivation, the crops
may not be able to resist these new pathogen strain infestations, similarly like
P. infestans in the 1840s. The problem of plant diseases is a very challenging factor
in developing countries because of their limited resources to fight these pathogens
through scientific research. Lack of proper resources makes the developing nations
unable to efficiently identify the disease causal organisms and detect and mitigate the
symptoms for crop yield loss.
In this chapter, we have focused mainly on the emerging plant diseases and
strains, the risk they possess in the successful cultivation of crops worldwide, the
possibility to detect these strains at an early stage by using traditional and innovative
4 D. Mitra
Fig. 1.2 Aerial image of

olive groves in Puglia in Italy
showing olive trees infected
by Xylella fastidiosa (left).
(Source: https://www.
theguardian.com/world/2019/
sep/09/deadly-olive-tree-
disease-spreads-france)
detection methods, and lastly the challenges that plant disease management faces in
the modern era. These challenges need to be addressed through science-based
cooperation on a global scale at a scientific and political level (Fig. 1.2).
1.2 Emerging Plant Diseases and Their Research Status
Our knowledge of global crop losses due to plant-pathogen infestations is very

limited. There is an increase in emerging pathogen strains, and with that integrated
disease management should evolved as well. In recent years, there has been a great
spike in types of plant pathogens and strains especially in imported plants and plant
products. Changes in climate conditions have also resulted in various new plant
diseases. For example, according to recent news, the Animal and Plant Health
Agency (APHA) reports some new plant diseases and new pests with their
symptoms and also gave information about the countries where they can be found.
Figure 1.3 and Fig. 1.4 show some examples of emerging plant diseases and plant
pests.
Recent survey articles published by British Broadcasting Corporation (BBC) and
The Guardian report the massive outbreaks caused by Xylella fastidiosa in European
countries like Italy, France, Germany, and Spain, wiping out entire olive groves and
thus causing major economic losses in these countries. This disease is also called
olive quick decline syndrome and is believed to affect more than 350 plant species.
This disease has also spread in the vineyards in the north and south of America. It
was first detected in Puglia in Italy in the year 2013, but now it has spread almost in
every corner of Europe (https://www.theguardian.com/world/2019/sep/09/deadly-
olive-tree-disease-spreads-france).
The European Commission suspects it could threaten olive gardens throughout
the world and thus has sought crop protection actions against the spread of Xylella.
To help mitigate the problem, the Royal Horticultural Society of the United King-
dom has come up with some new principles for future-proofing UK gardens: (1) All
imported semi-mature trees will be held for 12 months before planting them,
(2) evaluation of plant health risk will be monitored according to the criteria
Fig. 1.3 Emerging new plant diseases, symptoms, and regions where they are present. (Source:
https://www.rhs.org.uk/science/plant-health-in-gardens/protect-your-garden/new-pd-risks)
provided by the Royal Horticultural Society, and (3) this society will generate a list
of suppliers who meet these specified criteria.
Other major factors in causing various new plant diseases are the climate changes
resulting from natural and human activities. Climate change is the result of the
increasing amount of global trade, agricultural system modifications, and changes in
the consumer lifestyle; the global circulation of the crop also affects the global
circulation of new pathogens and diseases.
6 D. Mitra
Fig. 1.4 Emerging new plant pests, symptoms, and region where they are present. (Source: https://
www.rhs.org.uk/science/plant-health-in-gardens/protect-your-garden/new-pd-risks)
Plants and pathogens not only interact in isolation but also with the environment
according to the “disease triangle” concept, for instance, for a disease to occur by a
pathogen, a specific favorable environment is required (Fig. 1.5). Various environ-
mental conditions affecting plant disease development include water, temperature,
light, soil quality, wind speed, CO2 concentration, and others. Though we know that
plants have evolved various sophisticated defense mechanisms like PAMP-triggered
immunity (PTI), effector-triggered immunity (ETI), RNA interference (RNAi), and
hormonal regulation via abscisic acid, jasmonic acid, and ethylene, but new studies
have shown that environmental conditions can gradually modulate these defense
Fig. 1.5 Disease triangle

showing four dimensions
responsible for plant diseases,
pathogen, host plant,
environment, and time.
(Source: http://www.ucanr.
org/blogs/blogcore/postdetail.
cfm?postnum¼28845)
mechanisms (Couto and Zipfel 2016; Wu et al. 2019). For example, high humidity
condition interferes with ETI-associated mechanism; thereby the response to
C. fulvum Avr4 and Avr9 effectors by tomato CfR proteins is reduced when air
humidity reaches more than 95% (Wang et al. 2005). Since tropical climates are
prevalent in most developing countries, plant diseases are more common, causing a
great part of economic loss. In contrast, cold temperate reduces the chances of rapid
disease spread.
To solve the problems related to the emerging plant diseases, pathogen exclusion
through the plant quarantine must be the first step to combat food security in both
developing and developed countries. Other methods that need to be implemented
should be intercropping and crop rotation methods, use of pesticides, adequate
knowledge of the molecular mechanisms of these pathogen-host interactions, and
knowledge about post-harvest protection. The classically accepted phenomenon of
host-pathogen interactions now will no longer be relevant for these emerging plant
diseases. Thus, we must thrive to improve the traditional detection methods and
focus more on new approaches for these new pathogen strains. During the last
100 years, accuracy and precision in the detection of plant diseases were based
solely on the traditional methods; however, these methods are too slow and ineffec-
tive and thus need to be improved and updated.
Maintaining genetic variability in crop plants is also of major importance for
better crop yield. Future breeding programs for new improved varieties of crops
must incorporate the growth and biotic and abiotic resistance variability, which
should favor plant immunity and not the pathogen virulence. These features are
found mostly in wild-type relatives of cultivated crops and possess combined abiotic
and biotic resistance over a long time. But this is not the case in the modern crop
plant variety. With the help of genome-wide association study (GWAS) analysis
method and various marker-assisted selection methods, these features can be
introduced into the cultivated variety of plants.
8 D. Mitra
1.3 Overview of Disease Detection Methods and Their Ability

to Combat Emerging Diseases
Plant pathologists define “plant disease monitoring as detection i.e. deviation from a
healthy state of plants to stressed state, identification i.e. diagnosis of symptoms for
various diseases, and quantification i.e. measurement of disease severity for
e.g. reduced chlorophyll content or reduced leaf area,” etc. (Mahlein et al. 2012).
After the onset of plant disease symptoms, there are many methods applied to
detect the presence of diseases, for example, two main methods used are enzyme-
linked immunosorbent assay (ELISA) which is based on proteins produced by the
pathogen and polymerase chain reaction (PCR), based on specific DNA sequences
of the plant pathogen (Prithiviraj et al. 2004; Das 2004; Li et al. 2006; Saponari et al.
2008; Ruiz-Ruiz et al. 2009; Yvon et al. 2009).
In spite of the availability of these techniques, there is always a demand for fast,
sensitive, and effective methods for the detection of plant diseases caused by varied
plant pathogens. According to Sankaran et al. (2010), disease detection techniques
can be broadly classified into two main groups: direct and indirect methods
(Fig. 1.6).
Among the direct approaches, molecular methods and serological methods pro-
vide essential tools for accurate plant disease detection. Although DNA-based
molecular methods and serological methods have improved plant disease detection,
they are sometimes not very reliable, especially at the asymptomatic stage.
Other modern methods based on nucleic acid and protein analysis have been
proven to be more efficient in plant disease detection (Martinelli et al. 2015). The
main conclusion from a review by Martinelli et al. (2015) states
Fig. 1.6 Methods of Plant disease detection. (Source: Sankaran et al. 2010)
(1) novel sensors based on the analysis of host responses, for example, differential mobility
spectrometer and lateral flow devices, deliver much more reliable and instantaneous results
and can effectively detect early infections directly in the field; (2) secondly, biosensors based
on phage display and biophotonics can also detect infections very fast although they can be
also integrated with other systems; and lastly (3) remote sensing techniques coupled with
spectroscopy-based methods allow high spatialization of the results, these techniques can
prove to be very effective as a very reliable, sensitive and rapid preliminary identification of
primary infections. These tools in the long run help plant disease management and comple-
ment serological and DNA-based molecular methods.
According to plant pathologists, serological and PCR-based methods are the

commonly used methods to confirm plant disease detection, but volatile and
biophotonic sensors provide rapid and more effective results and may be used to
identify infections at asymptomatic stages. Remote sensing technologies are also
very efficient tools to spatialize diagnostic results and thus provide agriculture more
sustainability and safety, avoiding expensive use of pesticides for crop protection
(Martinelli et al. 2015).
1.3.1 Molecular Methods for Disease Detection
Molecular methods for plant disease detection are well established. It was reported
that the sensitivity of the molecular techniques for detecting bacteria ranged from
10 to 106 colony-forming units/mL (Lopez et al. 2003). The most commonly used
molecular techniques for plant disease detection are fluorescence in situ
hybridization (FISH) and PCR. As shown in Fig. 1.6, the other most commonly
used methods include immunofluorescence, flow cytometry, FISH, and DNA
microarrays.
Another categorization of the molecular methods can be nucleic acid-based, i.e.,
(1) DNA-based methods like FISH and the PCR variants nestedPCR (nPCR),
cooperativePCR (Co-PCR), multiplex PCR (M-PCR), real-time PCR (RT-PCR),
and DNA fingerprinting. (2) RNA-based methods include reverse transcriptase PCR,
nucleic acid sequence-based amplification (NASBA), and AmpliDet RNA
(Martinelli et al. 2015; Lopez et al. 2003). In the PCR method, the DNA of the
pathogen is extracted, purified, and amplified. Then it is used for gel electrophoresis
which if shows a specific band confirms the presence of the plant pathogen. The
concept of molecular detection methods is based on the specific design of
oligonucleotides and probes. Target sequences for molecular detection methods
can be obtained from the National Center for Biotechnology Information (NCBI,
Bethesda, MD, USA).
DNA fingerprinting is another molecular genetic method for plant pathogen
detection, where unique patterns are identified in the DNA of the plant pathogen
samples also called polymorphisms. This method was first described by Alec
Jeffreys in 1984. The various DNA fingerprinting methods use either PCR or
restriction fragment length polymorphism (RFLP) and sometimes both to target
10 D. Mitra
specific areas of DNA. Apart from these methods, DNA diagnostic microarrays are
being used for plant pathogen detections.
1.3.2 Serological Assays
In the ELISA-based method of plant disease detection, the microbial protein

associated with a specific disease is injected into an animal that produces the
antibodies against these microbial proteins, better known as antigens. These
antibodies are then extracted from the animal and are tagged with a fluorescence
dye and enzymes and are used for the detection of host-target interactions. ELISA
method was first used in the 1970s and is so far the most widely used immunodiag-
nostic technique because of its efficiency and specificity. ELISA is also a highly
sensitive method, but its sensitivity depends on the samples and the volume of
samples, for example, bacteria can be detected only at 100 cfu mL – 1 (Schaad
et al. 2002, 2003).
Antibodies against many viruses and bacteria have been developed and are being
used in numerous ELISA methods for plant disease detections globally, but due to
the fact that they might show cross-reactivity, monoclonal antibodies using hybrid-
oma technology have been developed which are more specific to the target (Nolasco
et al. 2002; Holzloehner et al. 2013). To date, both polyclonal and monoclonal
antibodies are available and are being used for various plant disease detections by
ELISA. Pathogens like viruses, bacteria, and fungi can now be detected using these
specific antibody techniques such as western blots, immuno-binding assays, and
serologically specific electron microscopy (SSEM) (Alarcon et al. 1990; Caruso
et al. 2002; Serological methods for detection and identification of viral and bacterial
plant pathogens. A laboratory manual 1990).
However, these molecular and serological techniques have some limitations, for
instance, they are time-consuming, require an elaborate sample preparation proce-
dure, are labor-intensive, and require specific reagents to detect each specific patho-
gen. Also, sometimes the concentrations of seed samples, soil, water, and pathogen
are below the sensitivity limit, thus hindering efficient detection by these methods.
Another issue is the occurrence of false negatives and false positives due to the
degradation of target DNA sequence or poor quality of the reagents (Louws et al.
1999). Lastly, the cost of equipment, reagents, sample preparation, etc. used in
molecular detection methods are very high making it less popular for most
agriculture-based industries. Thus, spectroscopic techniques can be potential alter-
native methods for the rapid detection of new plant pathogens.
1.3.3 Spectroscopic and Imaging Techniques
Recent research developments focus on automated nondestructive methods of plant

disease detection that will act as an efficient tool for disease monitoring on a large
scale. With the advancement of spectroscopic methods, the detection of plant
diseases has simplified. Many spectroscopic and imaging techniques have been
studied for the detection of the early and late stages of plant diseases. Some of the
methods include fluorescence imaging, infrared spectroscopy, fluorescence spectros-
copy, visible spectroscopy, nuclear magnetic resonance spectroscopy, etc. Spectro-
scopic methods can either be based on imaging or non-imaging techniques and help
in crop disease monitoring because of their potential, flexible, and cost-effective role
as operational instruments.
The most common imaging-based spectroscopic approaches include fluorescence
spectroscopy where the fluorescence from the object of study is measured after being
excited with an ultraviolet spectrum. To monitor plant stress and physiological states
in plants and to monitor nutrient deficiency in plants, usage of laser-induced
fluorescence is very popular (Belasque et al. 2008; Cerovic et al. 1999). Imaging
spectroscopy was used and has proved to be very effective for wheat kernels for
Fusarium head blight disease and also for weed infestations (Delwiche and Kim
2000; Okamoto et al. 2007).
Non-imaging spectroscopy methods are based on optical properties of leaf
pigments, chemical components, and structural features. These spectra collected
are then used for various remote sensing detection methods; this method has been
used to detect winter wheat yellow rust, aphid infestation, curl mite infestation, etc.
(Jacquemoud and Ustin 2001; Stilwell et al. 2013; Yuan et al. 2014; Zhang et al.
2014).
1.3.4 Other Innovative Detection Methods
During the past few years, many novel approaches were developed which are rapid,
inexpensive, efficient, and reliable, for example, lateral flow microarrays (LFM)
using an easily visualized colorimetric signal (Carter and Cary 2007). Metabolomics
is used as well to detect plant metabolites from primary and secondary metabolism
for various plant pathogens (Ibanez et al. 2014; Martinelli et al. 2016).
Volatile compounds emitted by plants for their growth, defense, and survival
purposes can also be used as biomarkers to detect plant diseases in volatile com-
pound profiling using gas chromatography-mass spectrometry (GC-MS) (Cardoza
et al. 2002).
Biophotonics is also an emerging technique that has been developed for efficient
plant pathogen disease detection. The main concept of this technology is based on
the molecular detection of probe-target interactions based on specific peptide
sequence recognition where the probe-target complex is identified using ELISA.
This method uses proteins as probes, increasing the possibility of multiple epitopes
for a single target present resulting in a cross-reaction. To mitigate this problem,
probe size is reduced to obtain more specificity and sensitivity via various biosensors
(Goulart et al. 2010).
12 D. Mitra
1.3.5 Remote Sensing Method
Remote sensing method can be defined as tracking an object without any physical
contact but rather by measuring the electromagnetic energy, i.e., emitted or reflected
by the surface of the earth (De Jong and Meer 2004). This is an indirect detection
method in which vegetation conditions from a distance are monitored and the spatial
extent and patterns of vegetation characteristics and plant health are evaluated
(Martinelli et al. 2015).
Plant stress or infections caused by various pathogens can be monitored by
remote sensing by analyzing the change in radiation emitted and used by plants.
APAR is the absorbed photosynthetic active radiation which is the total energy
absorbed by the plant and can be calculated based on the plant’s total leaf area and by
the concentration of chlorophyll pigments since leaf chlorophyll content is reduced
due to necrotic and chlorotic lesions. APAR used by a healthy plant is primarily for
photochemical reactions (0–20%) and reflects the rest of the energy as heat
(75–90%) and fluorescence (2–5%). Both plant physiological processes under stress
conditions and plant parameters like leaf pigments, water content, and chlorophyll
content can be detected by remote sensing methods (Meroni et al. 2009).
To summarize this part, early detection of pathogen infection is pivotal to avoid
epidemics. Usually, primary infections begin in the growing season, and secondary
infections are mostly spread by vectors leading to symptomatic disease stage with a
severe loss in total yield. Some pathogens are exceptional and they remain latent and
only infect the plant later in their life cycle. Thus, various detection methods can
detect plant pathogens at various stages in their life cycle. Figure 1.7 obtained from
Martinelli et al. (2015) represents an overview of the features of innovative methods.
1.4 Challenges of Plant Disease Management
Increasing the population with an expectation to reach nine billion by the year 2050
(Godfray et al. 2010) and changing diets and consumption patterns in the modern
world suggest that the production of food must be more efficient to meet the ever-
increasing demand. Figure 1.8 depicts the ever-changing percent yield change per
year for maize, rice, wheat, and soybeans. However, the existence of pre- and post-
harvest losses is the major hindrance to achieve this goal. The challenges of plant
pathology are increasing with every passing day with depleting natural resources, a
decrease in agricultural production, and an increase in epidemics of plant diseases
globally (Ray et al. 2013).
Thus, in current times, greater emphasis must be given to sustainable plant
disease management strategies that ensure food security and societal development.
The three major components, i.e., (1) society, (2) economics, and (3) ecology, must
be considered in plant disease management strategies. These strategies must focus on
ensuring food security and social stability by increasing crop productivity and
providing a supply of diverse and reasonable priced crops.
ELISA
4
3
2
Volatile sensors qPCR
1
0
Remote sensing Biophotonics

Availability Detection Stage Rapidity Spatialization
Fig. 1.7 Comparison of methods for plant disease detection. (Source: Martinelli et al. 2015) The
qualitative scales indicate 1 poor, 2 fair, 3 good, and 4 very good. The categories evaluate individual
techniques with respect to (i) Availability—ease of use, availability of equipment, and cost;
(ii) detection stage—when infections can be detected (4 infected vectors present, 3 isolated infected
plants, 2 many infected plants, and 1 symptomatic stage disease has spread over the cultivated area);
(iii) speed—total time required between collection of field data and the delivery of results (thus
includes sample collection, preparation, and testing); (iv) spatialization—the potential to spatialize
results(4 input data already carried out in a spatialized dimension, 3 data easily spatializable, 2 data
difficult to spatialize, and 1 data not subject to spatialization); and (v) reliability—effective accuracy
of results
From the economic point of view, the ratio of input and output of plant disease
management approaches must focus on more effective evaluation of direct and
indirect economic benefits thus helping the agricultural and ecological sustainability
(He et al. 2016).
Plant pathogens are considered a weapon for global terrorism and are a major
issue of political challenges in all countries. Since the majority of the country’s
economy depends on agricultural yield, poverty, crop loss, and food security are
major concerns for plant pathologists. Throughout the world, the International
Agricultural Research Centers have initiated some programs in the management of
plant disease, and each of these centers is responsible to handle certain crops, for
example, WARDA (Africa Rice Center, CIAT (Centro Internacional de Agricultura
Tropical, ICARDA (International Center for Agricultural Research in the Dry
Areas), ICRISAT (International Crops Research Institute for the Semi-Arid Tropics,
and ICRAF (World Agroforestry Centre) are some of the few.
On the international level, the Food and Agriculture Organization (FAO) of the
United Nations along with ISPP, i.e., International Society for Plant Pathology, plays
a major role in addressing the challenges faced due to plant diseases. These
organizations mainly make the farmers aware of changing food security policy,
train plant pathologists in developing countries, train farmers of plant disease
management techniques, and aim to mitigate this major global issue with small-
scale improvements.
14
D. Mitra
Fig. 1.8 Maps of observed rates of percent yield change per year in (1) maize (2) rice (3) wheat and (4) soybean yields. Red areas show where yields are
declining whereas the fluorescent green areas show where rates of yield increase – if sustained – would double production by 2050. (Source: Ray et al. 2013)
A recent review argues the fact that to achieve sustainable plant disease manage-
ment, we must understand the plant disease infestation mechanisms and strive to
improve the disease management system. Global agricultural productivity and food
quality can be improved and will, as a result, boost the global economy. According
to He et al. (2016), to combat the recent plant disease challenges, plant pathologists
worldwide should follow some strategies which are, as quoted, “(i) epidemic and
evolutionary patterns of plant disease under changing climate and agricultural
production concept; (ii) the role of ecological considerations in agricultural produc-
tivity and crop health; (iii) social-economic analysis of plant disease epidemics and
management; and (iv) technology development for integrating management of major
crop diseases with ecological principles.” To conclude, our main aim should always
be maintaining food security for a stable society by maintaining good crop health by
regularly improving scientific approaches.
References
Alarcon B, Lopez MM, Cambra M, Gorris MT, Guerri J (1990) Differentiation of Erwinia
carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica isolated from potato
by Western blot and subsequent indirect ELISA. J Appl Bacteriol 69:17–24
Belasque J, Gasparoto MC, Marcassa LG (2008) Detection of mechanical and disease stresses in
citrus plants by fluorescence spectroscopy. Appl Opt 47:1922–1926
Cardoza YJ, Alborn HT, Tumlinson JH (2002) In vivo volatile emissions from peanut plants
induced by simultaneous fungal infection and insect damage. J Chem Ecol 28:161–174
Carter DJ, Cary RB (2007) Lateral flow microarrays: a novel platform for rapid nucleic acid
detection based on miniaturized lateral flow chromatography. Nucleic Acids Res 35:e74
Caruso P, Gorris MT, Cambra M, Palomo JL, Collar J, Lopez MM (2002) Enrichment double-
antibody sandwich indirect enzyme-linked immunosorbent assay that uses a specific monoclo-
nal antibody for sensitive detection of Ralstonia solanacearum in asymptomatic potato tubers.
Appl Environ Microbiol 68:3634–3638
Cerovic Z, Samson G, Iribas F, Tremblay N, Moya I (1999) Ultraviolet-induced fluorescence for
plant monitoring: present state and prospects. Agronomie 19:543–578
Couto D, Zipfel C (2016) Regulation of pattern recognition receptor signalling in plants. Nat Rev
Immunol 16:537–552
Das AK (2004) Rapid detection of Candidatus Liberibacter asiaticus, the bacterium associated with
citrus Huanglongbing (Greening) disease using PCR. Curr Sci 87:1183–1185
De Jong S, Meer F (2004) Remote sensing image analysis: including the spatial domain. Springer,
Dordrecht
Delwiche S, Kim M (2000) Hyperspectral imaging for detection of scab in wheat. Environmental
and Industrial Sensing: SPIE, p. OE
Farr DF, Rossman AY (2019) Fungal databases U.S. national fungus collections, ARS, USDA.
https://nt.ars-grin.gov/fungaldatabases/. Accessed February 2019
Godfray HCJ, Beddington JR, Crute IR, Haddad L, Lawrence D, Muir JF, Pretty J, Robinson S,
Thomas SM, Toulmin C (2010) Food security: the challenge of feeding 9 billion people. Science
327:812
Goulart LR, Vieira CU, Freschi AP, Capparelli FE, Fujimura PT, Almeida JF, Ferreira LF, Goulart
IM, Brito-Madurro AG, Madurro JM (2010) Biomarkers for serum diagnosis of infectious
diseases and their potential application in novel sensor platforms. Crit Rev Immunol
30:201–222
16 D. Mitra
Gullino ML, Gilardi G, Garibaldi A (2019) Ready-to-eat salad crops: a plant Pathogen’s heaven.
Plant Dis 103(9):2153–2170. https://doi.org/10.1094/PDIS-03-19-0472-FE
He D, Zhan J, Xie L (2016) Problems, challenges and future of plant disease management: from an
ecological point of view. J Integr Agric 15:705–715
Holzloehner P, Schliebs E, Maier N, Füner J, Micheel B, Heilmann K (2013) Production of
monoclonal camelid antibodies by means of hybridoma technology (P3376). J Immunol 190
(1 Supplement):135.14
Ibanez AM, Martinelli F, Reagan RL, Uratsu SL, Vo A, Tinoco MA, Phu ML, Chen Y, Rocke DM,
Dandekar AM (2014) Transcriptome and metabolome analysis of citrus fruit to elucidate puffing
disorder. Plant Sci 217–218:87–98
Jacquemoud S, Ustin S (2001) Leaf optical properties: a state of the art. Proceedings 8th interna-
tional symposium physical measurements & signatures in remote sensing (CNES).
Li W, Hartung JS, Levy L (2006) Quantitative real-time PCR for detection and identification of
Candidatus Liberibacter species associated with citrus huanglongbing. J Microbiol Methods
66:104–115
Lopez MM, Bertolini E, Olmos A, Caruso P, Gorris MT, Llop P, Penyalver R, Cambra M (2003)
Innovative tools for detection of plant pathogenic viruses and bacteria. Int Microbiol 6:233–243
Louws F, Rademaker J, de Bruijn F (1999) The three Ds of PCR-based genomic analysis of
phytobacteria: diversity, detection, and disease diagnosis. Annu Rev Phytopathol 37:81–125
Lucas GB, Campbell CL, Lucas LT (1992) Causes of plant diseases. In: Lucas GB, Campbell CL,
Lucas LT (eds) Introduction to plant diseases: identification and management. Springer US,
Boston, pp 9–14
Mack RN, Simberloff D, Lonsdale WM, Evans H, Clout M, Bazzaz FA (2000) Biotic invasions:
causes, epidemiology, global consequences, and control. Ecol Appl 10:689–710
Mahlein AK, Oerke EC, Steiner U, Dehne HW (2012) Recent advances in sensing plant diseases for
precision crop protection. Eur J Plant Pathol 133:197–209
Martinelli F, Scalenghe R, Davino S, Panno S, Scuderi G, Ruisi P, Villa P, Stroppiana D,
Boschetti M, Goulart LR, Davis CE, Dandekar AM (2015) Advanced methods of plant disease
detection. A review. Agron Sustain Dev 35:1–25
Martinelli F, Scalenghe R, Giovino A, Marino P, Aksenov AA, Pasamontes A, Peirano DJ, Davis
CE, Dandekar A (2016) Proposal of a Citrus translational genomic approach for early and infield
detection of Flavescence dorée in Vitis. Plant Biosystems – An International Journal Dealing
with all Aspects of Plant Biology 150:43–53
Meroni M, Panigada C, Rossini M, Picchi V, Cogliati S, Colombo R (2009) Using optical remote
sensing techniques to track the development of ozone-induced stress. Environ Pollut
157:1413–1420
Mundt CC, Sackett KE, Wallace LD, Cowger C, Dudley JP (2009) Long-distance dispersal and
accelerating waves of disease: empirical relationships. Am Nat 173:456–466
Nolasco G, Sequeira Z, Soares C, Mansinho A, Bailey AM, Niblett CL (2002) Asymmetric PCR
ELISA: increased sensitivity and reduced costs for the detection of plant viruses. Eur J Plant
Pathol 108:293–298
Okamoto H, Murata T, Kataoka T, Hata SI (2007) Plant classification for weed detection using
hyperspectral imaging with wavelet analysis. Weed Biol Manag 7:31–37
Pimentel D, Zuniga R, Morrison D (2005) Update on the environmental and economic costs
associated with alien-invasive species in the United States. Ecol Econ 52:273–288
Prithiviraj B, Vikram A, Kushalappa AC, Yaylayan V (2004) Volatile metabolite profiling for the
discrimination of onion bulbs infected by Erwinia carotovora ssp. carotovora, Fusarium
oxysporum and Botrytis allii. Eur J Plant Pathol 110:371–377
Ray DK, Mueller ND, West PC, Foley JA (2013) Yield trends are insufficient to double global crop
production by 2050. PLoS One 8:e66428
Roberts M, Schimmelpfennig D, Ashley E, Livingston M, Ash M, Vasavada U (2006) The value of
plant disease early-warning systems: a case study of USDA’s soybean rust coordinated frame-
work. U.S. Dept. of Agriculture, Economic Research Service, Washington, DC
Ruiz-Ruiz S, Ambrós S, Vives MDC, Navarro L, Moreno P, Guerri J (2009) Detection and
quantitation of Citrus leaf blotch virus by TaqMan real-time RT-PCR. J Virol Methods
160:57–62
Sankaran S, Mishra A, Ehsani R, Davis C (2010) A review of advanced techniques for detecting
plant diseases. Comput Electron Agric 72:1–13
Saponari M, Manjunath K, Yokomi RK (2008) Quantitative detection of Citrus tristeza virus in
citrus and aphids by real-time reverse transcription-PCR (TaqMan). J Virol Methods 147:43–53
Schaad NW, Opgenorth D, Gaush P (2002) Real-time polymerase chain reaction for one-hour
on-site diagnosis of Pierce’s disease of grape in early season asymptomatic vines. Phytopathol-
ogy 92:721–728
Schaad NW, Frederick RD, Shaw J, Schneider WL, Hickson R, Petrillo MD, Luster DG (2003)
Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues.
Annu Rev Phytopathol 41:305–324
Serological methods for detection and identification of viral and bacterial plant pathogens. A
laboratory manual (1990) St. Paul, Minnesota: APS Press
Stilwell AR, Hein GL, Zygielbaum AI, Rundquist DC (2013) Proximal sensing to detect symptoms
associated with wheat curl mite-vectored viruses. Int J Remote Sens 34:4951–4966
Strange RN, Scott PR (2005) Plant disease: a threat to global food security. Annu Rev Phytopathol
43:83–116
Wang C, Cai X, Zheng Z (2005) High humidity represses Cf-4/Avr4- and Cf-9/Avr9-dependent
hypersensitive cell death and defense gene expression. Planta 222:947–956
Wu X, Valli A, Garcia JA, Zhou X, Cheng X (2019) The tug-of-war between plants and viruses:
great progress and many remaining questions. Viruses 11(3)
Yuan L, Huang Y, Loraamm RW, Nie C, Wang J, Zhang J (2014) Spectral analysis of winter wheat
leaves for detection and differentiation of diseases and insects. Field Crops Res 156:199–207
Yvon M, Thébaud G, Alary R, Labonne G (2009) Specific detection and quantification of the
phytopathogenic agent ‘Candidatus Phytoplasma prunorum’. Mol Cell Probes 23:227–234
Zadoks JC (1996) Crop production and crop protection: Estimated losses in major food and cash
crops: E.-C. Oerke, H.-W. Dehne, F. Schonbeck, and A. Weber. Elsevier Science B.V.,
Amsterdam, 1994. 808 pp. Price: Dfl 290, US$ 165.75 (hardback). ISBN 0 444 82095
7. Agric Syst 51:493–495
Zhang J, Yuan L, Pu R, Loraamm RW, Yang G, Wang J (2014) Comparison between wavelet
spectral features and conventional spectral features in detecting yellow rust for winter wheat.
Comput Electron Agric 100:79–87
Emerging Plant Diseases Under Changing
Climate Scenario 2
Muhammad Priyadi and Pooja Upadhyay
Abstract
The effect of changing climate on plant diseases has been a point of debate since
long time. This changing climate may cause imbalance in the ecosystem and directly
contribute to the disease development in various crops. Different climatic
conditions, e.g., change in sunlight including UV light, temperature, air, rainfall,
soil nutrients, carbon dioxide, ozone gas, greenhouse gas emission, and other
factors, are affecting the interaction of host plant and pathogens, e.g., fungi, bacteria,
virus, nematode, viroid, phytoplasma, and spiroplasma, which are opening doors for
the emergence of new diseases and pathogens worldwide. These newly emerged
diseases may turn out to be an epidemic under favorable conditions if not regulated
wisely as changing climatic conditions are providing favorable environment to the
spread and establishment of novel pathogens into new and non-native areas. By
keeping all these points into consideration, this chapter focuses on correlation
between climatic conditions and disease development and impact of changing
climatic conditions on disease development and emergence of new pathogens
around the globe. It also puts emphasis on factors responsible for emergence of
novel pathogens as well as their possible management tactic to regulate their adverse
outcome on agriculture and human to sustain food security in future.
Keywords
Climate change · Emerging plant disease · Pathogen · Environment
M. Priyadi (*)
Department of Pharmacy, Faculty of Health Sciences, Universitas Muhammadiyah Palangkaraya,
Palangka Raya City, Central Kalimantan, Indonesia
e-mail: muhammad.priyadi@umpalangkaraya.ac.id
P. Upadhyay
Department of Plant Pathology, G.B. Pant University of Agriculture and Technology, Pantnagar,
Uttarakhand, India

https://doi.org/10.1007/978-981-15-6275-4_2
20 M. Priyadi and P. Upadhyay
2.1 Introduction
Plant diseases are ubiquitous in nature and found in all the parts of the world
wherever plants grow. Diseases are responsible for losses of at least 10% of global
food production, representing a threat to food security (Strange and Scott 2005).
Though plant diseases are found in each and every climate, primarily hot and humid
condition is most favorable for development and dissemination of plant diseases
caused by various pathogens, e.g., fungi, bacteria, nematode, phytoplasma,
spiroplasma and virus and viroid.
Plant pathologists are considering the impact of environment influences on plant
disease development since long time. The classic disease triangle emphasizes the
interactions among host, pathogen, and environment for causing disease (Grulke
2011). In this triangle susceptible host, virulent pathogen and favorable environmen-
tal conditions are the essential components for disease development. If this interac-
tion persists for a certain period of time, the disease development takes place and the
triangle gets converted into disease tetrahedron. So the role of environment in
pathogenesis is very important as it affects host, pathogen, as well as host pathogen
complex. Thus, temperature, moisture, wind velocity, light, soil PH, soil structure,
etc. are environmental factors that have considerable influence on plant diseases. The
close relationship between the environment and diseases suggests that climate
change may cause the emergence of new plant pathogens and new disease epidemics
may take place under favorable conditions. Climate change has become a serious
concern around the world in recent years. We have seen the consequences of global
warming, such as melting of glaciers and raising water level, disturbed climate
cycles, and extreme environmental conditions in some parts of the world. Apart
from this, greenhouse gas level in the atmosphere is increasing due to various human
activities (Elad and Pertot 2014). It is assumed that global temperature has increased
by 0.8 C and will increase from 0.9 C to 3.5 C in the next 10 years (Das et al.
2016). Changing weather affects temperature, humidity, rainfall, wind, and ecosys-
tem balance, causing several problems such as floods, droughts, forest fires, etc.
Besides, it will directly affect the support of plant ecosystems that exist in nature.
Climate change can have direct or indirect impacts on plants through complex
mechanisms at different places (Pautasso et al. 2012). Usually, hot weather along
with high humid conditions can increase the risk of various types of plant diseases
that threaten many crop commodities in every region. Thus, change in environmen-
tal conditions as increasing global temperature and disturbed rain pattern at different
regions will affect the defensive ability of plants against pathogenic attack. Climate
change can affect the distribution of plant diseases in large geographical areas,
resistance and tolerance of plants against diseases, and the severity of plant diseases
(López et al. 2012; Nazir et al. 2018; Ziska et al. 2018).
Emergence of plant pathogens in new regions can be a possible outcome of
changing climate due to favorable conditions for pathogen establishment in new
region. Environmental changes in a short time or a long time will have an impact on
the growth, productivity, and population of microorganisms that also live around
2 Emerging Plant Diseases Under Changing Climate Scenario 21
plants, and they change the microclimate of plants permanently leading to suscepti-
bility against new pathogens (Nurhayati 2013).
2.2 Historical Impact of Emerging Diseases
Emergence of new disease or a new strain of pathogen in a non-native place is a

phenomenon which has been witnessed by humans since ages. An emerging disease
is an original case or group of cases that are newly recognized or newly appeared in
an area and can increase at a very fast rate in incidence and severity (Daszak et al.
2003). It represents the initial presence of a disease in a crop and, if left unchecked,
can result in disease epidemics of disastrous proportions. The introduction, or
arrival, of potentially hazardous plant pathogens to a new cropping area generates
risks in food production. Indeed, a large number of registered plant epidemics have
reduced the production of various crops in the world throughout history (Strange and
Scott 2005).
There are a number of examples of epidemics witnessed by history which
impacted the human life like never before. Among the most devastating cases
affecting humanity were the famines in Ireland and Bengal in which more than
three million people died and got migrated to different countries. The arrival of a
new strain of Phytophthora infestans (Mont.) de Bary in Ireland and the practice of a
potato monoculture caused the death of one million people (Andrivon 1996; Forbes
2004; Agrios 2005). Similarly, the shocking epidemic of leaf blight of rice caused by
the fungus Cochliobolus miyabeanus brought hunger and serious damage on the
population of Bengal state of India. Because of the epidemic, more than two million
people died, especially in the cities of Calcutta and Dhaka (Padmanabhan 1973).
There are many other cases about the arrival of new pathogens to a non-native area,
by the addition of monoculture practices and favorable environmental conditions,
which facilitated the occurrence of devastating diseases, causing human deaths
because of starvation and economic losses (Ullstrup 1972; Strange and Scot 2005).
Continuous evolution of pathogens may lead to the emergence of new, dangerous
strains of such plant pathogens which may spread immensely under favorable
conditions as new crop species with higher qualities are constantly introduced in
different countries and typically grown in large monoculture fields. Further, many
varieties and cultivars are selected mainly by their productivity, without consider-
ation for their susceptibility to pathogens which might be the major threat for the
huge spread of disease. These favorable conditions suggest some plausible reasons
why new diseases might emerge and flourish (Oliver and Solomon 2008).
2.3 Impact of Changing Climatic Components on Plant Disease

Development
Climate change is generally called as a long-term shift in the statistics of the weather.
It has been observed that last decade of the twentieth century and the beginning of
the twenty-first have been the warmest period in the entire global instrumental
temperature record.
2.3.1 Temperature
Increase in the globally averaged temperature is very likely due to the observed
increase in greenhouse gas concentrations. This greenhouse gas concentration in the
atmosphere is supposed to be increased by human activities, thus causing climate
change in the form of global warming. These activities intensified after the industrial
revolution at the end of the eighteenth century possibly by the intense use of natural
resources such as fossil fuel burning, deforestation, and other land use activities.
Temperature affects the disease cycle of any pathogen, starting from survival,
spread, penetration, development, and reproduction rates of pathogens and their
vectors. Temperature plays a very important role in spread of viral diseases as this
is one of the important environmental parameters in regulating the biology of insects
which can act as vector or carriers of viral pathogens. Generally, behavior, distribu-
tion, reproduction, and development of insects are largely affected by climate
differences in any region (Ghini et al. 2008). Therefore, it is important to maintain
environmental conditions, especially temperature to avoid the establishment and
dissemination of plant pathogens.
Besides, due to changes in temperature growth stage, development rate and
pathogenicity of infectious agents and the physiology and resistance of the host
plant may alter (Chakraborty et al. 1998; Chakraborty and Datta 2003). Change in
temperature can directly affect the secondary spread of disease by affecting the
survival of pathogen between two seasons. In some cases, change in temperature
may favor the development of different inactive pathogens, which could induce an
epidemic if it gets established and finds favorable conditions for longer period.
Temperature change may impact plant diseases by combining with other factors.
For example, increase in temperatures with sufficient soil moisture cause humid
microclimate in crop by increasing evapotranspiration and may lead to the incidence
of diseases favored under such conditions (Mina and Sinha 2008).
Temperature is one of the important factors affecting the occurrence of bacterial
diseases such as Ralstonia solanacearum, Acidovorax avenae, Burkholderia
glumea, etc. Thus, bacteria can proliferate in those areas where temperature-
dependent diseases have not been observed before (Kudela 2009). With the increase
in temperature, winter duration, growth rate, and reproduction of pathogen may be
modified (Ladányi and Horváth 2010). Similarly, the incidence of vector-borne
diseases will get altered up to some extent as climate can substantially influence
the development and distribution of vectors of viral diseases. Changes may result in
geographical distribution, increased overwintering, changes in population growth

rates, increases in the number of generations, extension of the development season,
changes in interspecific interactions, and increased risk of invasion by migrant pests
(Gregory et al. 2005). Because of the short life cycles of insects, mobility, reproduc-
tive potential, physiological sensitivity to temperature, and even modest climate
change will have rapid impacts on the distribution and abundance of vectors. Thus,
increase in temperature may result in high rate of development of insect, obtaining a
greater number of insect generations per cycle. Furthermore increase in temperature
could determine the distribution of areas favorable for overwintering (Garrett et al.
2006) or even more lethal zones where the insect cannot survive.
2.3.2 Carbon Dioxide Gas (CO2)
Increased CO2 gas in the air by climate change can encourage greater plant biomass
production which is also influenced by the availability of water and nutrients from
the soil (López et al. 2012). Different studies showed the impact of increased CO2
concentration on pathogen and disease development. The effect of elevated
concentrations of CO2 on the infection of barley by Erysiphe graminis was observed
as the percentage of conidia that progressed to produce colonies was lower in such
plants grown in higher CO2 concentration (Hibberd et al. 1996). Increase in the
amount of CO2 gas can increase the production of pathogenic fungal spores (Das
et al. 2016) and have impact on the severity of plant diseases. Similarly, high
concentration of carbohydrates or biomass in the host tissue promotes the develop-
ment of biotrophic fungi such as rust (Chakraborty et al. 2000). Increase of CO2
concentration relate to severity level of plant disease (Debela and Tola 2018).
According to the research and experiments, elevated levels of CO2 can directly
affect the growth of pathogen. Growth of the germ tube, appressorium, and conidium
of C. gloeosporioides fungi is slower at high concentrations of CO2 (700 ppm)
according to Chakraborty et al. (2000), However, once the pathogen infects the
plant, the fungus quickly develops and achieves sporulation. In contrast, the rate of
sporulation was greater at high concentrations of CO2 (700 ppm). In another study,
Hibberd et al. (1996) evaluated powdery mildew in barley and found that an
acclimation of photosynthesis at elevated CO2 caused larger reductions in plant
growth also, and the percentage of conidia that progressed to produce colonies was
lower in plants grown in high CO2 (700 ppm) than in low CO2 (350 ppm). Thus
change in CO2 concentration affects the ability of the pathogen to cause disease on
its host.
2.3.3 Light and Ultraviolet (UV)
Light has immense impact not only on the growth of plant but on diseases also.
Intensity and duration of light may either increase or decrease the susceptibility of
plants for infection and also the severity of disease. Light mainly causes production
of etiolated plants due to reduced light intensity which in turn increases the suscep-
tibility of plants to non-obligate parasites but decreases the susceptibility of plants to
obligate parasites. It also enhances the susceptibility of plants toward viral
infections.
Ultraviolet light is part of the sun with a variety of benefits for plants. Ultraviolet
has a role in inhibiting disease infections in plants because ultraviolet can increase
the accumulation of plant protection pigments (López et al. 2012) and inhibit spore
production from pathogens.
2.3.4 Ozone Gas
Ozone gas along with elevated carbon dioxide gas affects the plant pathogens and
their activity. Tiedemann and Firsching (2000) studied the increase in the ozone (O3)
concentration in combination with CO2 increase, for spring wheat plants infected or
not with leaf rust disease (Puccinia recondita f. sp. tritici). They observed that the
leaf rust disease was strongly inhibited by O3, but unaffected by elevated CO2. They
also observed that elevated CO2 largely equalized the negative effects of ozone gas
on rate of photosynthesis, growth, and yield parameters, but was not capable of
compensating the detrimental effects of fungal infection. Thus alteration in the
amount of ozone may impact the pathogen as well as disease development.
2.3.5 Rainfall and Humidity
High rainfall increases the humidity of the air and soil which can be helpful factor in
causing plant diseases. Besides, humidity is an ideal condition for some pathogens to
emerge and develop because hot weather also increases the humidity of the environ-
ment (Das et al. 2016). Rainfall plays an important role in plant disease develop-
ment. It has been observed that occurrence and severity of many diseases is directly
related with amount and frequency of rainfall in that area. For example, in case of
apple scab disease caused by Venturia inaequalis, at least 9 h continuous wetting of
leaves with temperature of 18 to 23 C is required causing primary infection by the
pathogen. In powdery mildew disease, rainfall adversely impacts the infection of
pathogen as presence of free moisture on the plant surface lowers the spore germi-
nation of the pathogen.
Relative humidity is very critical in fungal spore germination and the develop-
ment of storage rots. Relative humidity plays a very important role in development
of disease in some biotroph pathogens as well. As in case of white rust disease
caused by Albugo candida, germination of fungal sporangia and release of zoospores
take place at 16–18 C temperature along with 80–90% relative humidity for 72 h.
In storage pathogens, Rhizopus stolonifer, causing soft rot of sweet potato does
not cause infection if relative humidity is maintained at 85–90%, even if the storage
temperature is optimum for the growth of the pathogen. Under these conditions, the
sweet potato root produces corky tissues that wall off the Rhizopus fungus. Moisture
is generally needed for fungal spore germination, multiplication and penetration of

bacteria, and initiation of infection, e.g., germination of powdery mildew spores
occurs at 90–95% relative humidity.
2.3.6 Effect of Soil Moisture
Soil moisture influences the development of diseases by affecting the survival and
spread of the pathogen. Soil moisture may be a limiting factor in the development of
certain root rot diseases, e.g., high soil moisture levels favor development of
destructive water mold fungi, such as species of Aphanomyces, Pythium, and
Phytophthora. Clogging of the soil particles with moisture decreases the amount
of oxygen and raises carbon dioxide levels in the soil which makes the roots more
susceptible to root rotting organisms. Many plant diseases are more severe in low
soil moisture condition, e.g., charcoal rot of corn, sorghum, and soya bean
(Macrophomina phaseolina), take-all of cereals (Gaeumannomyces graminis), com-
mon scab of potato (Streptomyces scabies), and onion white rot (Sclerotium
cepivorum).
2.3.7 Effect of Wind
Wind plays a very important role in dissemination and spread of pathogens in large
areas sometimes from one continent to the other which is a common cause for
epidemics in plant diseases. Major epidemic diseases caused by fungi, bacteria,
and viruses spread either directly by wind or indirectly by insects which can travel
long distances with the wind. These spread, establishment, and development of
pathogen in new areas are the primary reasons of evolution of new pathogenic
races of the pathogen which turn into a challenge to disease management due to
their high adaptability in the new area. In case of rust diseases, fungal spores as
uredospores and many conidia are transported to many kilometers by wind. Wind if
accompanied with rain splashes becomes more devastating in case of bacterial
diseases as it helps in spread of bacteria from the infected tissues to the healthy one.
2.3.8 Drought
Climate change causes uncertain seasons such as drought and even forest fires.
Drought affects the physiology of plant species by weakening their defense system
and increasing the resistance of some plant pathogens through the process of
adaptation (Elad and Pertot 2014). When plants are stressed due to lack of moisture
or excessive heat, they become more susceptible to those diseases which are favored
by dry conditions such as charcoal rot disease of field crops, Aspergillus ear rot of
corn, etc.
2.4 Emergence of New Diseases and Pathogens Due to Climate

Change
Climate change may cause favorable environment to the emergence of new

pathogens so that they may reach and survive in new host at new region as emerging
pathogens have ability to infect a broad number of plants as well as new hosts
altogether. The emergence of diseases is thought to be the result of various factors
such as interactions among other pathogenic organisms, plant-pathogen interaction,
plant-insect-pathogen interaction, and adverse environmental conditions (i.e., irreg-
ular water regime and prolonged droughts). Many authors mention that adverse
factors can interact and “help” to subsequently cause complex diseases. For exam-
ple, Deberdt et al. (2014) proposed that climatic factors could change the nature of
microorganisms turning them into opportunistic pathogens which may lead to the
emergence of new plant pathogens.
Climate change, reflected by changes in average temperatures, reduction of
annual rainfall, irregular distribution of rainfall, and extended drought periods,
may modify the growth or quality of crops and potentially cause plant mortality
(Carnicer et al. 2011). Some authors agree that when plants become weakened or
stressed by environmental factors, microorganisms can easily colonize plants
thereby causing plant death (Moricca and Ragazzi 2008; Moricca et al. 2016).
Indeed, global warming has contributed to the decline of trees and plants worldwide
(Allen et al. 2010).
In this context, some human pathogens have been recorded affecting plants.
Enterobacter cloacae (Jordan) is a clear example of a human pathogen exploring
new hosts as plants. The bacterium has been linked to nosocomial outbreaks (Gaston
1988; Van den Berg et al. 2000) but was reported as a plant pathogen causing disease
on onion (Allium cepa L.) in the USA. Later, this bacterium was reported to be
affecting many other plant hosts including mulberry (Morus L.) in China (Wang
et al. 2010), dragon fruit (Hylocereus spp.) in Malaysia (Masyahit et al. 2009),
macadamia (Macadamia integrifolia Maiden & Betche) in Hawaii (Nishijima et al.
2007), lucerne (Medicago sativa L.) seeds in China, cassava (Manihot esculenta
Crantz) in Venezuela, and chili pepper (Capsicum annuum L.). These events indicate
that this bacterium has emerged as a plant pathogen in three continents, North
America, South America, and Asia by switching its original hosts due to pathogen
evolution.
In another example, bacterium Xylella fastidiosa which typically affects grape
(Vitis vinifera L.) with Pierce’s disease started affecting a new plant host, causing
mulberry leaf scorch in California (Hernandez et al. 2006). There may be two key
factors, natural host plant of X. fastidiosa and insect, which can possibly aid to the
introduction and spread of this pathogen in to the new host and region causing new
diseases. Natural host plant can serve as reservoirs for the bacterium and insects
feeding upon these plants can become transmitting agent of the disease after getting
infected (Hopkins and Purcell 2002).
Another example is Tomato leaf curl New Delhi virus (ToLCNDV), which was
first described on tomatoes in 1995 in India (EPPO 2016), then other countries in
Asia found this virus on a wide range of crops. ToLCNDV was observed on
courgette (Cucurbita pepo var. giromontiina) in 2012 in Spain (San Ambrosio and
Fernández 2014). After Spain, it was detected in Tunisia in January 2015, causing
high severity on cucumber (Cucumis sativus L.), melon (Cucumis melo L.), and
courgette (C. pepo var. giromontiina) (Mnari-Hattab et al. 2015). The virus was
transmitted in a persistent mode by the whitefly Bemisia tabaci (San Ambrosio and
Fernández 2014) to other parts of the world. The insect vector migration could be the
reason for the spread of the disease in Spain, then in Tunisia, and in Italy. As
conditions became favorable for whitefly in different countries due to climate
change, they could possibly spread the disease in newer regions.
One classic example of pathogen evolution and emergence in more virulent form
is Puccinia graminis f. sp. tritici Ug99 which is present in Uganda, Kenya, Ethiopia,
Sudan, Yemen, Iran, Tanzania, Eritrea, Rwanda, Egypt, South Africa, Zimbabwe,
and Mozambique. It affects wheat causing losses up to 70% or more by causing
wheat stem rust. A new virulent strain was identified in wheat fields in Uganda, and
in 1999, it was designated as Ug99. This new race broke the resistance conferred by
the gene Sr31 present in wheat stem rust-resistant varieties. This is a new global
threat to wheat cultivation as its transmission takes place by wind or by the
movement of people which spreads it immensely (Singh et al. 2011).
2.5 Possible Causes of Emergence of Plant Pathogens
There are numerous hypotheses for possible causes for emergence of new patho-
genic organisms, e.g., bacteria, fungi, and virus.
1. The organism may be endemic in the crop regions but the new host discovered
recently so pathogen successfully infected new host for survival.
2. After being endemic the organism became pathogenic, due to an increase in the
organism’s virulence or due to a decrease in the defense ability of host.
3. The organism may have been recently introduced into a new area and previously
unexposed hosts, and the organism is pathogenic to novel plants (e.g., chili
pepper).
4. Insect vectors exploit new plants, harboring the pathogenic organism and trans-
mitting the organism to subsequent plants.
Besides all these factors, alterations in the host-pathogen interaction process can
be a possible cause of occurrence of emerging pathogens. It is well known that
pathogens use specialized secretion systems to produce proteins to infect plants or
produce specialized structures or secrete toxins to invade plant cells (Doehlemann
et al. 2009). To respond to this infection, plants modulate for its defense against the
pathogen and pathogen may evolve in the process. This scenario of alterations in the
host-pathogen relationship can be seen in the interaction between avocado (Persea
americana Mill.) fruit and the fungus Colletotrichum gloeosporioides (Penz.) where
the flavonoid epicatechin is synthesized by the avocado fruit to protect itself from the
laccase protein produced by C. gloeosporioides. The influence of geography is

evident where the variability in the pathogenicity of genes within the same species
is more evident in specific geographical areas. Isolates of C. gloeosporioides from
Mexico showed increasing capabilities to metabolize epicatechin, when compared
with isolates from Israel (Guetsky et al. 2005).
In case of viral diseases, the first opportunity for virus emergence is the exposure
to new susceptible host plant. Successful initial infection of the host plant by a
pathogenic organism is critical in establishment of the organism as an emerging
pathogen or virus. The virus can remain in its initial form but is capable of geneti-
cally modifying itself to aid in exploitation of the susceptible host plant.
2.6 Actions for Mitigating Emergence of New Pathogens
Climate change is a nature’s phenomenon which cannot be regulated or managed by

human, but impact of climate change can be mitigated by managing the intense
human activities to avoid possibility of new pathogen emergence. Some climate
change mitigation strategies can be as follows:
• Renewable energy – Use of renewable energy such as wind, solar, or tidal energy
can reduce our dependency on fossil fuels such as coal, oil, and natural gas.
Therefore, this will reduce carbon dioxide emissions in the atmosphere.
• Carbon capture – It involves the capturing of greenhouse gases especially carbon
dioxide gas from waste gases released from power stations and then storing it
underground in old coal mines or gas fields. This also reduces emissions of such
gases in the atmosphere.
• Afforestation – Plantation of more number of trees so that more carbon dioxide
will be absorbed from the atmosphere during photosynthesis process.
These strategies may mitigate the effect of climate change up to some extent, but
to avoid the impact of emerged plant pathogens, strict regulation and phytosanitary
measures need to be imposed to avoid possible epidemics. Thus countries are
making efforts to minimize threats by establishing regulatory measures to prevent,
control, or eradicate diseases caused by pathogens potentially dangerous to crops.
National and international organizations for free information about dissemination
among scientists, governments, and the public would be crucial to minimize the
threat of emerging pathogens.
Phytosanitary regulations are performed worldwide to prevent, combat, and
eradicate pests affecting plants. For example, quarantine is an example to prevent
the entry of pathogen or any material which may harbor the pathogen to the area
where these pathogens do not exist. Quarantine also enables delayed introduction of
pathogens into new areas until risks have been evaluated. Foreign quarantines
prevent the introduction and presence of exotic pests, and domestic quarantines
slow the spread and control or eradicate any pest that has been introduced to a certain
country.
Phytosanitary programs include surveillance at ports, airports, and borders to

avoid entry of any suspected material to avoid introduction of pathogen. These
programs have generated successful results to stop the introduction of diseases
from abroad. Despite similar programs and regulations, pathogens manage to arrive
in new crop areas via vectors, humans, or environmental factors which may cause
novel pathogen emergence.
2.7 Conclusion
Climate change is an important phenomenon that affects agricultural production

significantly. By anticipating the future, we can prepare ourselves for problems
caused by climate change, especially those related to agricultural activities, which
generate the greatest amount of food consumed by humans. For several centuries,
pests and plant diseases have played an important role in agricultural production, but
global warming may modify areas affected by pests and diseases. So studies must be
performed to assess pest and disease stages under the effects of climate change,
determination of the magnitude of disease, and identification of the measures to
minimize the risk of infection needed. Along with emergence of new pathogens and
diseases, countries need to focus on minimizing the potential factors of such
emergence, and because of the global distribution of some plant pathogens,
researchers need to build international networks in coordination with competent
authorities, to address the public policies for managing the most destructive diseases,
those that are classified as quarantine diseases. So all the countries need to focus on
major food threats and collaborate to design phytosanitary regulations and establish
diagnostic protocols which would thereby strengthen government decisions. The
protocols which are able to make accurate detection of the pathogen are the need of
the hour. Protocols such as Morphological analyses, coupled with DNA sequence
data, facilitate the identification of new pathogens or variants of pathogen. These
diagnostic tools contribute to a rapid and accurate detection of new pathogens and
should be consulted for the development of proper diagnostic protocols to manage
the adverse results of newly emerged pathogen which may cause epidemic if not
managed timely and carefully. Some mitigating plant need to apply for prevent,
characterise and minimize the adverse of climate change.
References
Agrios GN (2005) Plant pathology, 5th edn. Elsevier, New York, p 922
Allen CD, Macalady AK, Chenchouni H, Bachelet D, McDowell N, Vennetier M, Kitzberger T,
Rigling A, Breshears DD, Hogg EHT, Gonzalez P, Fensham R, Zhang Z, Castro J, Demidova N,
Lim JH, Allard G, Running SW, Semerci A, Cobb N (2010) A global overview of drought and
heat-induced tree mortality reveals emerging climate change risks for forests. For Ecol Manag
259:660–684
Andrivon D (1996) The origin of Phytophthora infestans populations present in Europe in the
1840s: a critical review of historical and scientific evidence. Plant Path 45:1027–1035
Carnicer J, Coll M, Ninyerola M, Pons X, Sánchez G, Peñuelas J (2011) Widespread crown

condition decline, food web disruption, and amplified tree mortality with increased climate
change-type drought. PNAS 108:1474–1478
Chakraborty S, Datta S (2003) How will plant pathogens adapt to host plant resistance at elevated
CO2 under a changing climate. New Pathologist 159:733–742
Chakraborty S, Murray GM, Magarey PA, Yonow T, O’Brien R, Croft BJ, Barbetti MJ,
Sivasithamparam K, Old KM, Dudzinski MJ, Sutherst RW, Penrose LJ, Archer C, Emmentt
RW (1998) Potential impact of climate change on plant diseases of economic significance to
Australia. Australas Plant Pathol 27:15–35
Chakraborty S, Pangga IB, Lupton J, Hart L, Room PM, Yates D (2000) Production and dispersal of
Colletotrichum gloeosporioides spores on Stylosanthes scabra under elevated CO2. Environ
Pollut 108:381–387
Das T, Majumdar M, Devi RK, Rajesh T (2016) Climate change impacts on plant diseases. SAARC
J Agric 14L:200–209
Daszak P, Cunningham AA, Hyatt AD (2003) Infectious disease and amphibian population
declines. Divers Distrib 9:141–150
Debela C, Tola M (2018) Effect of elevated CO2 and temperature on crop-disease interactions
under rapid climate change. Int J Environ Sci Nat Resour 13(1):555851
Deberdt P, Guyot J, Coranson-Beaudu R, Launay J, Noreskal M, Rivière P, Vigné F, Laplace D,
Lebreton L, Wicker E (2014) Diversity of ralstonia solanacearum in French Guiana expands
knowledge of the “emerging ecotype”. Phytopathology 104:586–596
Doehlemann G, Van Der Linde K, Abmann D, Schwammbach D, Mohanty A, Kahmann R (2009)
Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant
cells. PLoS Pathog 5:e1000290
Elad Y, Pertot I (2014) Climate change impacts on plant pathogens and plant diseases. J Crop
Improv 28:99–139
EPPO (European and Mediterranean Plant Protection Organization) (2016) PQR-EPPO database on
quarantine pest. Available from: http://www.eppo.int/DATABASES/pqr/pqr.htm
Forbes G (2004) Global overview of late blight. In: Lizarraga C (ed) Proceedings regional
workshop potato late blight East and Southeast Asia and the Pacific, Yezin, pp 3–10
Garrett KA, Dendy SP, Frank EE, Rouse MN, Travers SE (2006) Climate change effects on plant
disease: genomes to ecosystems. Annu Rev Phytopathol 44:489–509
Gaston MA (1988) Enterobacter: an emerging nosocomial pathogen. J Hosp Infect 11:197–208
Ghini R, Hamada E, Bettiol W (2008) Climate change and plant disease. Sci Agric 65:98–107
Gregory PJ, Ingram JS, Brklacich M (2005) Climate change and food security. Philos Trans R Soc
Lond Ser B Biol Sci 360(1463):2139–2148
Grulke NE (2011) The nexus of host and pathogen phenology: understanding the disease triangle
with climate change. New Phytol 189:8–11
Guetsky R, Kobiler I, Wang X, Perlman N, Gollop N, Quezada GA, Hadar I, Prusky D (2005)
Metabolism of the flavonoid epicatechin by laccase of colletotrichum gloeosporioides and its
effect on pathogenicity on avocado fruits. Phytopathology 95(11):1341–1348
Hernandez MR, Costa HS, Dumenyo CK, and Cooksey DA (2006) Differentiation of strains of
Xylella fastidiosa infecting grape, almonds, and oleander using a multiprimer PCR assay. Plant
Dis 90:1382–1388
Hibberd JM, Whitebread R, Faraar JF (1996) Effect of 700 μmol mol¯1 CO2 and infection with
powdery mildew on the growth and carbon partitioning of barley. New Phytol 134:309–315
Hopkins DL, Purcell AH (2002) Xylella fastidiosa: cause of pierce’s disease of grapevine and other
emergent disease. Plant Dis 86(10):1056–1066
Kudela V (2009) Potential impact of climate change on geographic distribution of plant pathogenic
bacteria in Central Europe. Plant Prot Sci 45:S27–S32
Ladányi M, and Horvath L (2010) A REVIEW OF THE POTENTIAL CLIMATE CHANGE
IMPACT ON INSECT POPULATIONS – GENERAL AND AGRICULTURAL ASPECTS.
Applied Ecology and Environmental Research 8 (2):143–151
López RY, Pacheco IT, GonzÃlez RGG, Zul MHI, Carranza JAQ, and GarcÚa ER (2012) The
effect of climate change on plant diseases. AFRICAN JOURNAL OF BIOTECHNOLOGY, 11
(10), 2417–2428
Masyahit M, Sijam K, Awang Y, Ghazali M (2009) First report on bacterial soft rot disease on
dragon fruit (Hylocereus spp.) caused by Enterobacter cloacae in peninsular Malaysia. Int J
Agric Biol 11:659–666
Mina U, Sinha P (2008) Effects of climate change on plant pathogens. Environ Forensic 14(4):6–10
Mnari-Hattab M, Zammouri S, Belkadhi MS, Doña DB, Nahia EB, Hajlaoui MR (2015) First report
of Tomato leaf curl New Delhi virus infecting cucurbits in Tunisia. New Dis Rep 31:21
Moricca S, Linaldeddu BT, Ginetti B, Scanu B, Franceschini A, Ragazzi A (2016) Endemic and
emerging pathogens threatening cork oak trees: management options for conserving a unique
forest ecosystem. Plant Dis 100(11):2184–2193
Moricca S, Ragazzi A (2008) Fungal endophytes in Mediterranean oak forests: a lesson from
Discula quercina. Phytopathology 98:380–386
Nazir N, Bhat K, Shah TA, Badri Z, Bhat F, Wani T et al (2018) Effect of climate change on plant
diseases. Int J Curr Microbiol App Sci 7(6):250–256
Nishijima KA, Wall MM, Siderhurst MS (2007) Demonstrating pathogenicity of Enterobacter
cloacae on macadamia and identifying associated volatiles of gray kernel of macadamia in
Hawaii. Plant Dis 91:1221–1228
Nurhayati. (2013). The Effects of Climate Change on Plant Diseases and Possible Means for Their
Mitigation. Paper presented at the Proceeding international seminar on climate change and food
security, Palembang.
Oliver RP, Solomon PS (2008) Recent fungal diseases of crop plants: is lateral gene transfer a
common theme. Mol Plant-Microbe Interact 21:287–293
Padmanabhan SY (1973) The great Bengal famine. Annu Rev Phytopathol 11:11–24
Pautasso M, Döring T, Garbelotto M, Pellis L, Jeger M (2012) Impacts of climate change on plant
disease – opinions and trend. Eur J Plant Pathol:1–18
San Ambrosio MIF, Fernández AOA (2014) Sintomatología del virus del rizado de tomate de
Nueva Delhi (Tomato leaf curl New Delhi virus, ToLCNDV) en los cultivos españoles.
Phytoma España: La Revista Profes Sanidad Veg 257:36–41
Singh RP, Hodson DP, Huerta-Espino J, Jin Y, Bhavani S, Njau P, Herrera-Foessel S, Singh PK,
Singh S, Govindan V (2011) The emergence of Ug99 races of the stem rust fungus is a threat to
world wheat production. Annu Rev Phytopathol 49:465–481
43:83–116
Tiedemann A, Firsching KH (2000) Interactive effects of elevated ozone and carbon dioxide on
growth and yield of leaf rust-infected versus non-infected wheat. Environ Pollut 108:357–363
Ullstrup AJ (1972) The impact of the southern corn leaf blight epidemics of 1970–71. Annu Rev
Phytopathol 10:37–50
Van den Berg RWA, Claahsen HL, Niessen M, Muytjens H, Liem K, Voss A (2000) Enterobacter
cloacae outbreak in the NICU related to disinfected thermometers. J Hosp Infect 45:29–34
Wang H, Meng B, Han H (2010) The discussion on mulberry as a green afforestation tree species.
North Sericulture 31(1):45–47
Ziska HL, Bradley AB, Wallace DR, Bargeron TC, LaForest HJ, Choudhury AR et al (2018)
Climate Change, Carbon Dioxide, and Pest Biology, Managing the Future: Coffee as a Case
Study. Agronomy 8(152):1–21
Emerging Important Nematode Problems
in Field Crops and Their Management 3
Mujeebur Rahman Khan, Irfan Ahamad,
and Mohammad Haniph Shah
Abstract
Plant diseases are one of the major limiting factors in the production of food crops
and in attaining food security and food safety. Around 40–50% of the crop
produce is eaten away or destroyed by pests and pathogens, and its one fourth
is attributed to plant diseases. Among various groups of plant pathogens,
phytonematodes cause 5–20% yield decline in food crops, which may account
to a net loss of 2–7% in crop-based food. Rice, wheat, and maize are most
important food crops and are frequently attacked by plant nematodes. Generally,
the crop damage caused by nematodes remains hidden to farmers because of
nonappearance of discernable symptoms. In addition to direct damage,
nematodes aggravate the infection of soilborne pathogens or act as vector leading
to development of disease complexes. To prevent yield losses and to improve
crop productivity and yield quality, it is essential to make growers realize the
economic significance of plant nematodes. Adequate extension programs are
needed to be implemented to demonstrate and advise appropriate nematode
management methods to the crop growers. With regard to food crops, especially
cereals, primary emphasis may be given to some of the most important nematode
genera such as Meloidogyne, Pratylenchus, Ditylenchus, and Heterodera as these
nematodes are widely distributed in agricultural fields and cause tremendous
damage to food crops. Common cultural practices, viz., deep plowing, flooding,
fallowing during summer, removal and burning of weeds and remnants of
previous crops, use of certified and disinfested planting materials, and cultivation
of non-host, resistant, or tolerant crops, may substantially prevent crop losses in
cereals caused by nematodes. Further, seed priming with biopesticides of
M. R. Khan (*) · I. Ahamad · M. H. Shah

Department of Plant Protection, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

https://doi.org/10.1007/978-981-15-6275-4_3
34 M. R. Khan et al.
Trichoderma spp. or Pseudomonas fluorescens as a general treatment may prove

quite effective in growing healthy crop with considerably higher yields.
Keywords
Plant diseases · Cereals · Phytonematodes · Disease management · Biocontrol
3.1 Introduction
The Indian agriculture after facing several challenges has achieved self-sufficiency
for the last two to three decades in numerous agricultural products particularly
cereals, pulses, oilseeds, etc. (Khan and Jairajpuri 2012). During the 1950s, the
indigenous production was insufficient to meet the food requirement of the people
equivalent to one third of the today’s population, and the country had to rely on 8–10
million tons of food import annually. The continued positive planning and policy of
the governments on promotion of agriculture since independence has ultimately led
to self-sufficiency in food crops and also an increase of around 20% in the net
national area under crop cultivation in comparison to 1950 (Indiastat 2018). With the
advancement of technology of food production and disease management, the net
national productivity has increased by around 3.5 times in food grains, 1.75 times in
fruits, and 2.25 times in vegetables. Presently India is self-sufficient in food grains
and is able to offer food to the existing population with ease and also able to store
two buffers each of 454.10 lakh MT in 2018 and 711.18 lakh MT in 2019 (FCI
2019).
The food grain cultivation in India occupies a major share in the food production.
The food crops, primarily cereals, are cultivated in around 150 million hectares
(mha), which is 72.2% of the total area under cultivation (Fig. 3.1). Important food
crops are rice, wheat, sorghum, maize, etc., and among them rice occupies the
greatest area under cultivation (43.7 mha), followed by wheat (28.15 mha
Fig. 3.1). The total annual production of food crops in India is 267.5 million tons
(Fig. 3.1). Rice and wheat contribute around 36 and 29.3% of the total production of
food crops adding 96.43 and 78.4 million tons annually to the national food basket,
respectively (Fig. 3.1).
India, after attaining self-sufficiency and food security, has focused to ensure
food quality to meet the challenges of malnutrition and hunger as well as to compete
in the global trade for export incentives. Pest and pathogens are important constraints
in improving the crop productivity as well as the quality. In developing countries
including India, around 50% of the total produce is lost quantitatively or qualita-
tively due to pests and diseases at pre- and post-harvest stages (Khan and Jairajpuri
2010; Fig. 3.2). Diseases caused by various pathogenic fungi, bacteria, viruses,
nematodes, etc. have been found to be responsible for 25% of the total losses
inflicted by pests and pathogens. Among the diseases, greatest losses are inflicted
by fungi (42%) followed by bacteria (27%), viruses (18%), and nematodes (13%) of
the total losses caused by the pathogens (Fig. 3.2).
3 Emerging Important Nematode Problems in Field Crops and Their Management 35
Total crop Area (in hac.) in 2017-18 Total area of food grain (in hac.) in 2017-18
Barley, 660.8
Oilseeds, e,
24507.9 Baj Maiz
0
Pulses,
ra,
7 938
481
29813.16 Rice, 43774.07
Food grains, 127524.29
Wheat, 29650.59
Total crop production (Thusand tonn) Total food grain prodution (Thousand tonn)
in 2017-18 in 2017-18
Oilseeds: Barley, 1780.81

Puls Baj Maize,
es: 31459.26 ra, 3
254 9202875
16.2 9
2 Rice, 112757.61
Food grains: 285013.5 Wheat, 99869.52
Fig. 3.1 Pie chart showing cultivation area and production of food crops in India
Crop losses by pests and pathogens Crop loses by plant pathogens
Stored grain Nematodes,

pest, 13%
Rod Diseases, 25% 13%
ents, 8
%
Others, 6% Viruses, 18% Fungi, 42%
Insects, 23%
Weeds, 28% Bacteria, 27%
Fig. 3.2 Pie chart showing crop losses caused by pests and pathogens
3.2 Plant Parasitic Nematodes
Nematodes are vermiform or threadlike animals, and their body is thin, flexible,
generally elongated (0.3–11 mm), and unsegmented and tapers at both the ends
(Khan 2008; Fig. 3.3). Basically, the nematodes are aquatic animals thriving best in
water, but they have adapted to terrestrial habitats. Nematodes constitute 80–90% of
Fig. 3.3 Plant parasitic

nematodes in water isolated
from a crop field in the faculty
farm at Aligarh Muslim
University, India
all the multicellular animals; fortunately, only a fraction of this number possesses
ability to parasitize plants, and the rest are free-living surviving on various substrates
(Khan 2008).
Plant parasitic nematodes are considered important pathogens of numerous agri-
cultural crops. Nematodes cause damage to plants by injuring and feeding on the root
hairs, epidermal cells, cortical cells, and/or stealer cells (Khan and Jairajpuri 2012).
A large number of nematodes are ectoparasites feeding on root surface, e.g.,
Tylenchus, Rotylenchus, Tylenchorhynchus, Belonolaimus, Hoplolaimus,
Trichodorus, Longidorus, etc. However, a considerable number of nematodes fully
enter inside the host roots and are called endoparasites, such as root-knot nematodes
(Meloidogyne spp.), cyst-forming nematodes (Heterodera spp.), and root-lesion
nematode (Pratylenchus spp.). Whereas, some nematodes such as citrus nematode
(Tylenchulus semipenetrans) and reniform nematode (Rotylenchulus reniformis) are
semi-endoparasites as they partially enter the host tissue (Siddiqui 2005).
The most common effect of nematode parasitism is debilitation of the plant even
without appearance of any symptom (Perry and Moeus 2013). In addition to direct
damage, nematodes often aid or aggravate the diseases caused by fungi, bacteria, and
viruses or may break resistance of cultivars to pathogens (Khan 1993). Hairy root of
roses caused by Agrobacterium rhizogenes is of minor importance, but in the
presence of Pratylenchus vulnus the disease becomes severe (Sitaramaiah and
Pathak 1993). The Fusarium wilt-resistant cultivars of cotton become susceptible
in the presence of root-knot nematodes (Atkinson 1982). Plant nematodes may also
act as vectors for bacteria, fungi, and viruses. Anguina tritici carries Clavibacter
tritici and Dilophospora alopecuri to shoot meristem of wheat (Khan and Dasgupta
1993). Ringspot viruses (NEPO viruses), e.g., tobacco ring spot virus, are transmit-
ted by Xiphinema and Longidorus species. Trichodorus and Paratrichodorus spe-
cies act as vector for certain tobraviruses such as tobacco rattle and pea early
browning viruses (Taylor and Brown 1997).
3.3 Nematode Infestation in Food Crops and Its Management
Phytonematodes are potential pests of all kinds of food crops including cereal crops
(Perry and Moens 2013). They attack root, stem, leaves, crown, inflorescence,
flowers, and developing grains (Southey 1986). The crop damage depends on the
plant species or cultivar, nematode species, level of soil infestation, and the
prevailing environmental conditions. Nematodes usually cause severe reduction in
the plant growth and yield, both quantitatively and qualitatively (Khan 2008). Molya
of wheat (Heterodera avenae), ufra of rice (Ditylenchus angustus), root rot of maize
(Pratylenchus zeae), root-knot (Meloidogyne spp.) etc. are some of the diseases
which cause serious economic loss to cereal crops (Khan and Jairajpuri 2010).
Despite a significant impact on agriculture, nematodes have not been recognized
as major pests of crops in particular the cereal crops. This is probably because of the
fact that the damage caused by nematodes is less obvious than that caused by fungi
or other pathogens and remains hidden from the sight of farmers. Moreover, foliage
of cereal crops dries at maturity, and the plants are harvested from the ground level
leaving behind the roots in soil on which the nematodes cause some recognizable
symptoms.
The stunting of plants and mild yellowing of foliage are the debilitation
symptoms generally caused by phytonematodes and resemble with nutritional defi-
ciency (Fig. 3.4). As a result, fertilizer in place of a nematicide is applied, which
proves ineffective and noneconomic. These nonspecific or general symptoms of
nematode infestation appear in the patches of plants irregularly distributed in a field
and show stunted growth and sparse and dull green or pale yellow foliage (Luc et al.
2005). The infested plants show incipient wilting despite adequate moisture avail-
able in the soil during sunny days, but recover at night. Further, roots so weakened
and damaged by nematodes are easily invaded by many bacteria and fungi, leading
to accelerated root decay (Khan 1993). This secondary damage also does not draw
immediate attention, and an incurable stage is soon reached leading to severe yield
loss. However, in heavily infested fields, characteristic symptoms appear on roots or
shoots. Symptoms develop more frequently on roots because mostly nematodes are
root feeders (Khan 2008). Specific symptoms are root lesions, root rot, root pruning,
root galls, leaf tip whitening, seed galls, cessation of panicle growth, etc. (Fig. 3.5).
Fig. 3.4 General symptoms

of nematode attack in a field.
Plants in patch showing
stunted growth with chlorotic
foliage
Fig. 3.5 Specific symptoms caused by nematodes on plants. Root knot by Meloidogyne on tomato
(a), sponge gourd (b), and rice (c); dirty root by Rotylenchulus reniformis on vegetable (d); seed gall
by Anguina tritici on wheat (e); and panicle chaffiness by Aphelenchoides besseyi (f)
Phytonematodes may cause about 5–20% yield loss with an average of around
13% when various crops are considered. The yield losses vary greatly depending on
inoculum level and host species (Khan et al. 2009). The severe infection may result
to as much as 80–90% yield decline in an individual field, and sometimes plants fail
to give yield of any economic value. Crop losses due to nematodes are greater in the
developing countries than the developed countries. It is probably due to unplanned
agricultural practices, unawareness of the farmers about nematodes, and nonavail-
ability of nematicides. In the developed countries where management practices are
properly implemented, relatively lesser crop damage due to nematodes occurs. In the
USA alone, annual monetary loss due to nematodes has been estimated above $ 6.0
billion (Agrios 2005). In India, about 10–20% crop losses occur due to nematode
infestations. At high population levels, much greater losses may occur in susceptible
crops. Cereal crops are considerably susceptible to nematode attack and exhibit yield
decline of economic value (Table 3.1).
The researches and field-based data have shown that nematodes act as a potential
factor in limiting the productivity of cereals crops in India as well as in other parts of
the world. From an Indian point, the important nematode diseases in cereals are
described under. These diseases cause significant yield decline in cereals; hence their
management is essentially required to improve cereal productivity in the country.
Table 3.1 Crop losses Crop Yield loss (%)

caused by nematodes to
Cereals 7–22
food crops
Wheat 7–19
Rice 10–22
Maize 6–18
Barley 7–27
Oat 3–12
Fig. 3.6 Root symptoms of Heterodera avenae on wheat: Roots of a maturing plant with white
females and infected root showing bushy rootlets
3.3.1 Wheat
3.3.1.1 Molya Disease Caused by Heterodera avenae

The disease is prevalent in almost all wheat-growing regions in India. The affected
plants are stunted with smaller, yellow, and fewer leaves. Tillering and development
of spikelets are reduced and emergence of earheads is delayed. The infected roots
become elongated and have tuft of rootlets at the distal end (Fig. 3.6). The molya
may lead to 15–100% reduction in the grain yield of wheat depending on the
nematode population and wheat variety.
The cereal cyst nematode H. avenae has a narrow host range; hence, the nematode
can be successfully managed by crop rotation for 2–3 years with non-hosts in heavily
infested areas. The rotation with non-hosts or keeping the field fallow during Rabi
reduced the cyst population by 50 and 75% and increased the wheat yield by 83 and
135% after 1 and 2 years, respectively (Smiley and Nicol 2009). Singh (1985)
reported that cultivation of resistant barley and mustard crops reduced the nematode
population by 57–58% and 50%, respectively. A decline of 87–100% in the popula-
tion of H. avenae may be achieved by growing Brassica campestris var. dichotoma,
B. campestris var. sarson, B. campestris var. toria, B. juncea, and Eruca sativa for
one season (Singh et al. 1987). Further, the cultivation of resistant barley cvs. BH393
and fenugreek (Trigonellafoenum-graceum) reduced cyst population by 63 and
68%, respectively, and subsequent crop of wheat was better after cultivation of
fenugreek. Plant resistance is an effective method of nematode control which

prevents nematode reproduction and shortens the rotation period without involving
extra cost to the growers.
The genes imparting resistance to different populations of H. avenae in wheat
have been identified in different countries (Montes et al. 2003; de Majnik et al. 2003;
Martin et al. 2004). The genes resistant to CCN from Aegilops variabilis and
A. triuncialis have been transferred to wheat (Jahier et al. 1998; Romero et al.
1998). Montes et al. (2008) reported higher level of resistance against Spanish
pathotype Ha71 in wheat lines carrying Cre1, Cre4, or Cre7 genes than carrying
Cre8 or Cre3 genes. The cvs. Katyil and AUS 10894 showing resistance in Australia
were found susceptible to H. avenae in India (Dhawan 1988; Bishnoi and Bajaj
2004). The sources of resistance in wheat to Indian population of H. avenae have
also been identified (Kanwar et al. 2001, Kaur et al. 2008) and are being incorporated
in breeding programs. A resistant cv. Raj Molya Rodhak1 (Raj MR1) was released
for cultivation in Rajasthan in 2002 (Mathur et al. 2004; Sharma et al. 2004).
Resistance in this variety is governed by single dominant gene, which offers both
pre and post-infection resistance (Kanwar and Bajaj 2009). Nevertheless, some of
the H. avenae-resistant wheat and barley varieties are susceptible to H. filipjevi
(Bishnoi and Bajaj 2002; Kanwar et al. 2004) suggesting separate breeding programs
for the two species.
The nematicides should be used against CCN when other options of control have
been exhausted. Aldicarb and carbofuran at 1.0–1.5 kg a. i./ha have been widely
used against cereal cyst nematode in India, but after Bhopal gas tragedy in 1984,
carbofuran is the only nematicide available in Indian market. Metam sodium has
been found quite effective in suppressing the soil population of CCN (Kanwar and
Bajaj 2010). Depending upon availability and feasibility, two or more control
practices can be integrated for achieving effective control of H. avenae. For exam-
ple, a combined application of nitrogenous fertilizer and nematicides significantly
increased the yield of wheat and barley (Handa et al. 1978; Sakhuja et al. 1978).
Bhatti et al. (1980) reported that integration of early sowing date (November) with
aldicarb/Furadan at 2 kg a.i. /ha resulted in a significant enhancement in the wheat
yield and reduction in cyst population. Application of neem seed powder, FYM, and
Trichoderma viride with nematicides effectively controlled H. avenae infection in
wheat (Pankaj et al. 2008). Soil application of Purpureocillium lilacinum or
T. harzianum during the field preparation may also suppress nematode population
in soil (Khan 2016).
3.3.1.2 Ear Cockle or Seed Gall Disease Caused by Anguina tritici

The seed gall caused by Anguina tritici is an important disease in several underde-
veloped and developing countries including India, Pakistan, Ethiopia, Romania,
Iraq, Syria, Yugoslavia, etc. In India, the ear cockle disease is reported to occur in
Bihar, Haryana, Himachal Pradesh, Jammu and Kashmir, Madhya Pradesh,
Rajasthan, and Uttar Pradesh. Its incidence is much higher in socially backward
and tribal areas where certified seeds are not used (Nandal et al. 2010).
Fig. 3.7 Anguina tritici symptoms on wheat plant (a), seed galls (b), and healthy seeds (c)
The diseased plants show basal swelling of stem; crinkling, curling, twisting, and
rolling of the leaves; profused tillering; and stunted growth. The diseased earheads
are smaller and broader than the normal ones with or without awns on them, and in
such earheads instead of formation of grains, cockles (seed galls) are formed. The
cockles are brown to black at maturity and contain large number of second-stage
juveniles of Anguina tritici in a quiescent state (Fig. 3.7). The nematode also aids a
bacterium, Clavibacter tritici, and a fungus, Dilophospora alopicrozi, causing
yellow slime (tundu) and twist diseases, respectively. Average incidence of the
diseases and decline in the yield of wheat caused by this nematode in India has
been reported as up to 50%.
Since A. tritici is a strict seed borne in nature, use of cockle-free seeds is the best
approach to control the seed gall disease as nematode cannot survive in soil or in
seed galls lying in the field (Popov et al. 2006; Paruthi et al. 2009). Seeds can be
cleaned by separating the cockles from the healthy seeds using the mechanical and
physical methods. Winnowing the contaminated seeds by winnowing basket in an
open area is a common practice in several parts of India to separate the cockles from
the wheat grains. Sieving the contaminated wheat seeds with sieves of definite mesh
has been a common method to achieve gall eradication in several countries (Nandal
et al. 2010). But complete removal of galls is not achieved by this method. Putting
the nematode-infested wheat seeds into a drum containing water and stirring it well
with some wooden stick or hand for some time help in removing cockles from the
infested seed lot. The galls because of lighter in weight float on the surface of water
and are removed by ordinary sieves and buried deep or burnt. This method is
relatively more effective but still 10–15% cockles remain with healthy seeds. Use
of brine by putting the seed in 10–30% concentrations of salt solution is another
method of removing the cockles from the grains. Addition of salt increases the range
and duration of floating of cockles, and 100% recovery of cockles is attainable with
this method (Paruthi and Bhatti 1992). The seeds are required to be washed in water
several times before sowing to avoid injurious effect of salt on germination of seeds
(Nandal et al. 2010).
3.3.2 Rice
Rice is a susceptible host to a number of nematodes, viz., Ditylenchus angustus,

Hirschmanniella oryzae, H. gracilis, H. mucronata, M. incognita, M. javanica,
M. arenaria, M. graminicola, M. triticoryzae, M. oryzae, and M. salasi, which
cause following disease in paddy.
3.3.2.1 Root-Knot Disease Caused by Meloidogyne graminicola

Among Meloidogyne spp., M. graminicola is the most serious and emerging threat to
the rice cultivation in various rice-growing regions of the country. Earlier this
nematode was prevalent in upland rice, but during the last 25–30 years, the nematode
has rapidly spread to deepwater and irrigated rice in India. Unlike other Meloidogyne
spp., M. graminicola incites galls on the tip of lateral roots (terminal galls) which are
spiral or hook-like (Fig. 3.7; Khan and Anwer 2011; Salalia et al. 2017). The
nematode attacks rice in the nursery as well as in the main field and causes
substantial yield loss to the crop. The rice root-knot nematode is found on rice
mainly in South and Southeast Asia, but also in South Africa, the USA, Colombia,
and Brazil. Now it is a potential pest of upland, lowland, irrigated, and deepwater
rice throughout the world including India (Haque et al. 2019). In India, the nematode
has been reported from all states where rice is cultivated (Prasad et al. 2010; Khan
and Ahamad 2019). Common weeds of rice fields and rotation crops such as wheat,
onion, cabbage, and tomato are also susceptible to the nematode. MacGowan and
Langdon (1989) reported 100 host plants of M. graminicola, which includes food,
fodder, fruits, ornamentals, and weeds. The symptoms produced due to the infection
of M. graminicola are manifested in the form of characteristic hook- or spindle-like
terminal galls on the roots (Fig. 3.8). Due to formations of galls, the absorption
capacity of roots is impaired, leading to stunting of plant growth and yellowing of
leaves. These symptoms appear on the plants in patches within a nursery or main
field (Fig. 3.8).
The rice root-knot nematode causes significant yield decline in upland rice and
rainfed lowland rice (Prasad et al. 2010). It has frequently been observed in irrigated
Fig. 3.8 Root-knot nematode, Meloidogyne graminicola infecting rice. Root galls on a plant (a),
yellowing of nursery (b), and severely suppressed growth of young plants in a field (c)
rice (Prot and Matias 1995; Khan and Jairajpuri 2010). The yield losses are greater
under flooded conditions (Kinh et al. 1982; Prot et al. 1994) than in upland rice and
are responsible for the poor plant growth and yield in aerobic rice (Krreye et al.
2009). The ability of M. graminicola to attack oats and cereals in addition to rice in
Southeast Asia makes this nematode a potential threat to small grain production
(Dutta et al. 2012). The impact of M. graminicola on rice yield has been well
established, with yield losses up to 20–90%. In India, M. graminicola is responsible
for 20–35% yield decline in rice due to poorly filled kernels (Prasad et al. 2010;
Dutta et al. 2012; Khan et al. 2012, 2014; Khan and Ahamad 2019). Khan et al.
(2012) have reported 20–31% yield decline at 1000 J2/kg soil. The basmati rice has
been found highly susceptible to the rice root nematode, posing a major economic
threat to the growers. The root-knot in rice can be managed using the following
methods depending on the circumstances.
Sesbania rostrata is a very good host for M. graminicola when grown in
non-flooded soils. Hence its cultivation as a green manure trap crop before rice in
non-flooded soils infested by M. graminicola may reduce nematode population in
the field. However, care in the time of plowing should be taken as a delay may lead to
increase in the population. However, under rainfed conditions S. rostrata should not
be used. Growing Tagetes sp. and Crotalaria spp. (C. incana and C. mucronata)
may lead to reduction in M. graminicola.
Crop rotation with non-host crops like jute, mustard, and chickpea and rice-
resistant varieties (TKM-6, Patna 6, Dumai, Ch 47, Hamsa) has been found effective
in reducing M. graminicola infestation. Soil amendments with water hyacinth
compost (300 or 600 g/4.5 kg soil) reduced root-knot nematode infestation and
increased plant growth (Roy 1976). Rice-mustard-rice crop sequence, followed by
rice-maize-rice, and rice-fallow-rice were reported to be effective in reducing nema-
tode development (Kalita and Phukan 1990; Reddy 2018). Crop rotation with
non-host crops, viz., sweet potato, cowpea, sesamum, castor, sunflower, soybean,
onion, turnip, and cauliflower, may inhibit nematode development (Rao et al. 1984;
Rao 1985; Soriano and Reversat 2003; Mantelin et al. 2017).
Good puddling before transplanting helps in improving water retention in soil
which reduces aeration and also reduces nematode movement and invasion of fresh
rice roots by infective juveniles of M. graminicola and M. triticoryzae (Garg et al.
1995). The population of Meloidogyne spp. decreased drastically when the field was
puddled every year over a 10-year period (Gaur 2003). In Bangladesh widespread
infestation of M. graminicola was found when direct-seeded rice was grown instead
of puddled rice (Padgham et al. 2004).
Biocontrol agents especially Trichoderma harzianum (Bokhari and Fardos 2009),
Purpureocillium lilacinum (¼Paecilomyces lilacinus; Kiewnick and Sikora 2006),
Pochonia chlamydosporia (Khan et al. 2005; Niu et al. 2010), Aspergillus niger
(Khan and Anwer 2008; Ashoub et al. 2009), Pseudomonas fluorescens (Khan and
Haque 2011), P. putida (Haque et al. 2019), and Bacillus subtilis (Dawar et al. 2008)
have shown great potential in suppressing the infection of Meloidogyne spp. (Khan
2016; Khan et al. 2019) on different crops. The BCAs can be applied through seed
treatment (Santos et al. 1992), root-dip (Khan et al. 2005), or soil application (Zeinat
et al. 2010) depending on the crop and convenience of the grower. However, the
degree of effectiveness of the BCA may vary with the method of application (Khan
2007).
The soil solarization in nursery bed may effectively reduce the nematode popula-
tion. Prior to sowing, the nursery bed is covered with a plastic polyethylene tarp for
3–4 weeks in summer, and nursery bed treatment with carbofuran or phorate at 2 kg
a.i./ha was found very effective in reducing root-knot nematode infestation in rice
nursery (Das et al. 2018). Carbofuran at 2.4, 4.8, and 7.2 g a.i./kg seed reduced galls
of M. oryzae by 57, 79, and 80%, respectively (Segeren et al. 1985). Seed soaking
with 0.1–0.2% carbofuran for 12 h was found to be effective in reducing
M. graminicola population (Rahman and Das 1994). Basamid at 40 g/m2 was
found to be most effective in reducing rice root-knot nematode population (Singh
2017). Soil application of Furadan, phorate, isazophos, cartap, carbosulfan, or
quinalphos at 0.5, 1.0, and 2.0 kg/ha reduced 82% galling of M. graminicola at
2 kg (Khan et al. 2016). The nursery bed treatment with carbofuran (3G) at 0.3 g a.i./
m2 followed by the main field treatment with carbofuran (3G) at 1 kg a.i./ha 40 days
after transplanting is effective to reduce nematode population (Walia and Khan
2018). A combination of P. fluorescens at 20 g/m2 + T. harzianum at 20 g/
m2 + carbofuran effectively controlled rice root knot in rice (Narasimhamurthy
et al. 2017). Combined application of neem cake + vermicompost + Trichoderma
spp. in field resulted in almost gall-free plants (1–2 galls/root systems, Kumar et al.
2017). Das et al. (2018) reported that an application of carbofuran at 0.3 g a.i/m2 in
the nursery bed combined with field application of Trichoderma viride at 2.5 kg /ha
pre-incubated with 25 kg FYM at 45 days after transplanting proved quite effective
and economical for the management of M. graminicola in the field. Field application
of neem cake+vermicompost+Trichoderma spp. was also found effective against
M. graminicola (Kumar et al. 2017).
3.3.2.2 Ufra Disease Caused by Ditylenchus angustus

The ufra is an important prevalent disease of rice in Malaya, Malaysia, Burma, the
Philippines, Egypt, Southern Thailand, Madagascar, parts of West Coast, Vietnam
(Bora and Rahman 2010), and India. The nematode attacks rice in Assam, West
Bengal, Maharashtra, Andhra Pradesh, and U.P. The ufra is characterized by
mosaic-like discoloration arranged in a splash pattern or chlorosis or white streaks
on young leaves and sheaths. The entire leaf may become twisted or severely
malformed, or the basal portion of the young leaf becomes wrinkled and whitish
green (Fig. 3.9). Under severe infestation, the leaf margin crinkles and leaf tip gets
twisted. The emerging panicle becomes distorted, bears sterile or empty grains at the
base, and produces normal grains only near the tip (Bora and Rahman 2010). Yield
losses due to ufra have been reported as 5–100% depending on the rice cultivar and
nematode population. Management of D. angustus is difficult because of the nature
of the deepwater rice ecosystem. However, following methods may prove effective
against the nematode.
Burning of diseased stubbles, followed by plowing may substantially reduce the
incidence of ufra disease in the next crop (Ou 1972; Walia and Khan 2018). Stubble
Fig. 3.9 The deformed and

underdeveloped panicles due
to infection by Ditylenchus
angustus
burning should be carried out in an organized way to avoid remaining of infested

patches unburnt, but this may cause air pollution. Instead of burning the stubble may
be collected in a big pit, and some Trichoderma formulation be sprayed on the
stubble and be allowed to decompose to use this as a biopesticide compost for next
crop. This may give additional earning to the farmer as the decomposed stubble
produced from 1 ha may be sufficient for 4–5 ha. For root stubbles, plowing should
be carried out soon after harvest of the crop so as to give sufficient time to
decompose the stubbles and to expose the nematodes to the sun. The nematode
does not survive in the quiescent state for longer period of exposure to sun and
desiccation. The field should be kept free from weeds and ratoons. Removal of
infected leaves along with upper portion of rice plant also reduces the nematode
population in the field. Rotation of deepwater rice with non-host crop such as jute
(Oletorious spp.) and mustard (Brassica sp.) reduces the ufra disease in the next
crop. In areas with good irrigation facilities, cultivation of autumn rice (boro rice)
followed by summer rice (ahu rice) will result in control of this disease, as this
nematode will find no host during its active period. However, this practice may not
be feasible in most of the deepwater rice-growing areas, where floodwater rises to a
high level, in which cultivation of ahu rice may not be possible. Cultivation of late
cultivars (Padmapani) before the flood and earlier harvesting shall escape the
nematode attack (Mac Geachie and Rahman 1983). The rice cvs. Bazail-65,
Jalamagna, Basudev, AR-9(C), IR 13437-20-4E-PI, IR 17643-4, Lakhi, Karkati,
BR 308-3-3-2, Rayada 16-011, Rayada 16-013, Rayada 16-05, Rayada 16-06,
Rayada 16-07, Ba Tuc, etc. are quite effective in tolerating the nematode attack
(Plowright et al. 2002; Bora and Rahman 2010). The wild rice Oryza subulata is also
resistant to D. angustus (Miah and Bakr 1977); Walia and Khan (2018). Rahman and
MacGeachie (1982) found three promising rice entries, viz., CN 540, NC 493, and
TCA 55, completely free from ufra infection. Two rice entries, namely, Fukuhonami
and Matsuhonami, were found highly resistant and, seven, namely, Rayeda, Bazail,
Hunenwase, Shinanokogane, Kinonishiki, Aokazi, and Koshinihsini, were resistant
against ufra (Haque and Latif 2011).
Chemical control is also an option, difficult to apply in deepwater rice. The
disease generally manifests under flooded condition, making almost impossible to
implement curative measure. This necessitates higher dose of chemical which may
cause serious health and environmental hazards and harm the fish fauna and other
aquatic resources. Among the pesticides, carbofuran has been found most effective
in controlling the disease (Bora and Rahman 2010). When carbofuran was applied at
the time of transplanting, maximum yield and minimum ufra infestation were
recorded (Rahman 1993; Islam et al. 2013). Latif et al. (2011) reported that the
ufra infestation was significantly reduced when 1 kg ai/ha Furadan 5G was applied
20 days before transplanting of infested seedlings in the field. Walia and Khan
(2018) reported that the seed treatment with carbosulfan (25EC) at 3% a.i. (w/w) and
foliar spray with carbosulfan 20.2% a.i. at 40 ml and 120 days after transplanting is
helpful in reducing disease incidence. Haque et al. (2013) reported that application
of neem seed kernel extract, Furadan 5G, and Bavistin 85WP was applied singly and
in different combinations to develop an integrated management package against
ufra. Single spray of neem seed kernel extract at 10% concentration in combination
with application of Furadan 5G at 10 kg/ha or Bavistin 85 WP at 1.5 kg/ha ensured
lower ufra incidence and maximized rice yield.
3.3.2.3 Root Yellowing and Rotting Disease Caused by

Hirschmanniella spp
Another important nematode disease in rice is caused by Hirschmanniella oryzae,
which has been reported from countries like Thailand, India, China, Pakistan, the
Philippines, Bangladesh, Cuba, Madagascar, Sri Lanka, the USA, East Asia,
Taiwan, New Zealand, Nigeria, Vietnam, Europe, and Japan. In India, the nematode
attacks rice in Andhra Pradesh, Kerala, and Tamil Nadu (Ghosh and Manna
2010a, b). The infested plants are stunted with fewer and shorter tillers. Foliar
growth is retarded and flowering is delayed. Necrotic areas develop on the roots
(Fig. 3.10). Infestation with Hirschmanniella spp. may result in 10–70% yield loss.
Application of neem cake at 1 ton/ha and press mud at 10 ton/ha (Johnathan and
Pandiarajan 1991) and castor oil cake and mustard oil cake (Khan and Shaukat 1998;
Lakshmy 2014) significantly reduced Hirschmanniella oryzae populations.
Incorporation of T. erecta whole plant ranked first among the organic amendments
Fig. 3.10 Symptoms caused by Hirschmanniella spp. in rice (a) aboveground and (b) under-
ground. (https://images.app.goo.gl/nQqeM6UjysLS9pwq6, https://images.app.goo.gl/
R2xpQLDpJVX59AdB6)
(Lakshmy 2014). Rotations of rice with cabbage and tobacco reduced populations of
H. oryzae by 83–88% in paddy fields (Gao Xue Biao et al. 1998; Sikora et al. 2018).
The cropping sequences following Pankoj Paddy!Jalmasta jute (Hibiscus
sabdariffa L.) ! 2-month fallow ! Pankoj Paddy or Pankoj Paddy ! 1-month
fallow ! wheat (Triticum aestivum) ! Disimasta jute (Corchorus olitorius) ! 1-
month fallow ! Pankaj Paddy have been found effective against the nematode
(Ghosh 2001; Ghosh and Buddhadeb 2008). During cultivation of Jalmasta jute, the
population of H. oryzae decreased more than 50% (Chen et al. 2012).
Rice cultivars belonging to the Japonica group are found to be more susceptible to
H. oryzae than the other groups (Youssef 1999). Significantly higher populations of
H. oryzae were found in Pakistan, Basmati and Basmati 370, while Basmati
385 supported the lowest population (Randhawa et al. 1992). Walia and Khan
(2018) reported that the rice cultivars TKM 9, CR 142-3-2, CR 52, N 136, and W
136 were resistant to H. oryzae. Wild rice species, Porteresia coarctata, showed the
highest degree of resistance to H. mucronata (Panigrahi and Mishra 1995). Oryza
collina and O. nivara were moderately resistant to H. oryzae.
Application of phosphamidon and chlorpyrifos as root dip at 0.02% for 20 min
before planting reduced Hirschmanniella oryzae population to 0.83/2 g root 30 days
after transplanting (Ramakrishnan et al. 1984). Bare root dip treatment with
carbosulfan and phosphamidon at 0.3% reduced H. oryzae population by 46.2 and
40% and increased grain yield by 35.1 and 34.7 q/ha, respectively, over untreated
control (Lahan et al. 1999). Nursery bed treatment with carbofuran at 0.3 g a.i./m2
followed by field application of carbofuran at 1 kg a.i./ha 40 days after transplanting
in endemic spots at farmers’ field has been recommended for management of root-
knot nematode (Meloidogyne graminicola) and rice root nematode
(Hirschmanniella oryzae) in rice (Khan et al. 2010).
3.3.2.4 White Tip Caused by Aphelenchoides besseyi

This disease is known by different names, viz., white tip of rice, “black grain”
disease of rice, “ear blight” of Italian millet, and “heart blight” of rice. A. besseyi is
widely distributed in Andhra Pradesh, Assam, Bihar, Himachal Pradesh, Kerala,
Maharashtra, Meghalaya, Orissa, Punjab, Tamil Nadu, Tripura, and West Bengal
causing chaffiness of panicles and whitening of leaf tip (Fig. 3.11; Khan 2010). The
nematode also plays an important role in the development of rice disease complex,
synergizing Acrocylindrium (¼Sarocladium) oryzae, Curvularia spp., Fusarium
spp., etc. (Rao and Prakash 2002). Yield loss varies from 10 to 71% in nematode-
affected rice fields.
The infested plants show white tip or whiplike malformation of the top third of
the leaf blade (Fig. 3.9). At the flowering stage, chaffiness and abnormal elongation
of glumes in some spikelets, rachii, and rachillae occur. Infected plants show
reduced vigor, height, and weight of spikelets and number of grains. Abnormal
elongation of the panicles (Rao 1978) and chaffiness or scattered chaffiness in the
florets also occur in case of severe infestations (Fig. 3.9; Prasad et al. 2007). In some
rice cultivars, A. besseyi may produce only the symptoms of small grains and
Fig. 3.11 White tip nematode damage in leaves, ovary, and spikelets. (Source: https://images.app.
goo.gl/GPbhQkZjWCCcf4sSA)
erect panicles, but not the typical leaf white tip. The disease can be controlled using
the following methods.
Physical methods such as storage of A. besseyi-infested seeds in regulated gas
medium (97.5% nitrogen and 2.5% oxygen) for 10 days at 25 C kill the quiescent
larvae. The treatments with hot water at 53–54 C for 15 min may also eliminate the
infestation in seeds (Youssef 2014; Pashi et al. 2017a, b). A combination of seed
treatment (0.3% by seed weight) and spraying with benomyl (2.5 g/dm3 at 1 or
15 days after transplanting) may fully protect rice plants from seed-borne infestation
of A. besseyi. The seed treatment with hot water or chemical treatment if adopted at
community level, infestation of A. besseyi in rice, can be effectively controlled.
Amin and Al-Shalaby (2005) found that soaking of rice seeds infected with
A. besseyi in hot water at 70 C for 15 minutes and hot air-drying treatment at
70 C for 24 hours showed best results in controlling white tip nematode without any
effect on sprouting.
Growing of tolerant or resistant cultivars may reduce A. besseyi population in rice
and thus prevents yield losses. A few important rice varieties tolerant/resistant to
A. besseyi are Bluebonnet, Bluebonnet 50, and Starbonnet (Papova et al. 1994), IR
841, IAC 435, IAC 120, IAC 899 (Rao et al. 1986; Sivakumar 1988), AUS 15854
(Baheti and Verma 2001), Binam, Domsiah, Khazar and Hassansarayi, Sepidroud,
Kadus, Hassani and Ramezani, Hashemi, Deilamani (Jamali and Mousanejad 2011),
and Asahi (Tulek et al. 2015).
Seed treatment with carbofuran, aldicarb, sulfone, or methomyl at 0.1% WP has
been found highly effective in controlling A. besseyi, but seed germination may get
11–29% low (Kuriyan 1995). Wang et al. (2006) reported soaking of seeds in 16%
cartap and prochloraz cartap solution for 48–60 h before sowing satisfactorily
controlled white tip in rice. Application of carbofuran 3G treatment before
transplanting or seed disinfection followed by carbofuran 3G water surface treatment
at the early stage of injury provided effective control of the disease (Khan et al.
2006). Application of monocrotophos 36 SL at 0.075% through seed treatment
before sowing and foliar spray monocrotophos at 20 and 50 DAS (days after sowing)
was found to the most effective and reduced the foliar distortion and boot leaf stage
nematode infestation (Prasad and Varaprasad 1992; Pathak and Khan 2010; Pashi
et al. 2017a, b).
Islam et al. (2015) reported that the integrated treatment with brine solution, hot
water, and Furadan 3G significantly enhanced different parameters of the plant and
yield and reduced the disease incidence. Khan et al. (2006) reported that the
integration of NeemAzal and pongamia oil with chemicals as well as hot water
treatment was found more effective in managing whit tip disease. Hot water treat-
ment of rice seeds for 10 min at 50–55 C followed by foliar spray with carbosulfan
(25 EC) at 0.1% 40 days after transplanting reduced the infestation of white tip
nematode (Khan et al. 2010).
3.3.2.5 Leaf Curl and Wilt Disease Caused by Hoplolaimus indicus

The disease is caused by Hoplolaimus indicus which frequently occurs in all the rice-
growing states in the country. The general symptoms are stunting, chlorosis, curling
of young leaves, and wilting of tip of old leaves in early stages of crop growth
(Fig. 3.12; Rahman 2010). The nematode damages the roots internally leading to
distortion in the arrangement of vascular bundles. Crop loss depends on the nema-
tode populations. Infestation with H. indicus is common in upland areas where other
cereals are grown in a rice-based cropping system, causing up to 18% losses in rice.
Following measures may help in suppressing the nematode attack.
Crop rotation may effectively reduce the H. indicus population in soil. The
paddy-gram rotation for 2 consecutive years followed by paddy-mustard or paddy-
garlic significantly decreased the H. indicus population in the field (Haider et al.
2001). The okra-cowpea-cabbage, okra-brinjal-okra, and okra-cucumber-mustard
sequences were found to be most effective and reduced the population of
H. indicus (Chandra and Khan 2011). The nematode population was considerably
lower in plots under mustard, sunhemp, and Tagetes cultivation (Haque and Prasad
1983). In another crop rotation schedule, the fallowing proved effective against most
of the plant parasitic nematodes. A fallow period of 3 months greatly reduced the
Fig. 3.12 Leaf curling and wilt caused by Hoplolaimus indicus. (Source: https://images.app.goo.
gl/GPbhQkZjWCCcf4sSA)
population of H. indicus (Khan and Chawla 1975). Deep plowing may prove more
suppressive to the nematode than the normal plowing. Deep plowing before the
wheat-chili followed by fallow and thereafter lentil-cowpea-mung cropping
sequence significantly reduced the total population of H. indicus (Wani 2005).
Soil application with diazinon at 250 ppm, dimethoate at 500 ppm, or DBCP at
1000 ppm has been recorded to be quite effective against H. indicus in rice (Biswas
and Rao 1971). The root dip treatment of rice nursery at 500 ppm diazinon and
DBCP for 10 min reduced the invasion and development of the nematode inside the
rice roots. Nemaphos and Terracur P were found highly effective against endopara-
sitic stage of H. indicus. The phorate, fensulfothion, and dimethoate were also found
highly effective in reducing the population of H. indicus and other nematodes and
increasing plant weight and yield (Alam et al. 1981). About 82% reduction in
population of H. indicus was recorded due to application of phenomiphos at
10.0 kg/ha in soil (Sethi and Meher 1991).
3.3.3 Maize
Numerous nematode species, viz., Heterodera zeae, H. avenae, Pratylenchus zeae,

and Meloidogyne spp. attack the maize crop, among them. Heterodera spp. are of
immense economic importance. In India, H. zeae occurs in major maize-growing
regions in Himachal Pradesh, Rajasthan, Gujarat, Punjab, Haryana, Uttar Pradesh,
Uttaranchal, Bihar, Jharkhand, Madhya Pradesh, and Maharashtra (Parihar and
Siddiqui 2010). Aboveground symptoms are nonspecific. The infected plants tassel
earlier but bear smaller cobs with relatively fewer grains. The presence of cysts of
H. zeae on the root surface is the most important and characteristic symptom of the
nematode infection in maize (Fig.3.13). Root system of the infected maize plant
becomes bushy and is poorly developed. The yield loss due to H. zeae and H. avenae
may be up to 40%, and the nematode infestation in maize can be minimized by
adopting the following strategies.
There is good scope of using crop rotation as a control strategy for H. zeae, since
the nematode has a limited host range. Monoculture of maize and other host crops in
the same field should be avoided (Parihar and Siddiqi 2010). Two-year rotation with
non-host crops like vegetables, pulses, and oilseeds can be fruitful, as it would bring
Fig. 3.13 Nematode damage

showing in maize field by
H. zeae. (Source: https://
images.app.goo.gl/
zFm5CkoU18mzJy5YA)
down the nematode populations below economic threshold levels (Srivastava and
Chawla 2005). Two to three deep plowings at 10–15 days interval during April–May
in hot summer may also reduce the nematode population densities to a considerable
extent (Srivastava and Chawla 2005). The maize cultivars Ageti-76 and Karnal-1
have been found to be moderately resistant to H. zeae in India (Kali Ram 1989).
Chemicals offer effective control of H. zeae in maize. Carbofuran (3G) and
phorate (10G) at 2 kg a.i./ha have been found to reduce the nematode population
and subsequent increase in the maize yield. Carbosulfan (25 ST) used as seed
treatment at 2–3% (w/w) also provided drastic decline in the soil population of
H. zeae (Srivastava and Chawla 2005; Kaushal et al. 2007). Soil application of
cadusafos at 1 kg ai/ha at the time of sowing or in the split dose significantly
enhanced the plant growth and reduced cyst population (Srivastava and Lal 2007).
The seed treatment of acephate (2%w/w) suppressed the soil population of H. zeae
and enhanced the plant growth and yield of maize (Baheti et al. 2015).
Seed treatment and soil application on maize with carbosulfan 25 ST 2% in
combination with carbofuran at 1 kg a.i./ha increased cob weight, stalk weight, and
grain yield of maize and reduced the final cyst population by 80% (Singh et al.
1997). Biocontrol agents have also been found effective against H. zeae. The seed
treatment with biocontrol agents, Purpureocillium lilacinum, Pochonia
chlamydosporia, and Trichoderma viride (1, 2, and 4% w/w), suppressed the cyst
nematode and enhanced the yield. The treatment with P. lilacinum (4%)was found
most effective in reducing the infection of H. zeae,followed by P. chlamydosporia
(4; Bahetiet al. 2015). Baheti et al. reported that the seed treatment (2% w/w) with
P. lilacinum, P. chlamydosporia, and T. viride and soil application (2 g/plant) of leaf
powder of neem, karanj, and lantana effectively controlled the maize cyst nematode.
3.3.4 Barley
The nematodes of economic importance that infest barley include Heterodera spp.,
Pratylenchus spp., Meloidogyne spp., Anguina tritici, and Ditylenchus dipsaci.
Heterodera avenae is the principal species on temperate cereals and has been
detected in India (Bishnoi and Pankaj 2010). The disease is characterized by patchy
growth of stunted and yellow plants (Fig.3.14). Infested plants bear thin and narrow
leaves, reduced number of tillers, delayed emergence of ears, and reduced number of
spikelets and grains, ears, if formed, and have very few grains. Root system becomes
bushy, bunchy, and shallow due to its proliferation and has slight swelling near the
root tip. Such plants can be easily pulled out of the ground.
Cultural methods have been successful in managing the H. avenae population,
since this nematode possesses narrow host range being specific to cereals. Growing
non-host crops may prove much effective (Bishnoi and Pankaj 2010). Handa (1983)
found that nematode population decreased by 70% with continued rotation of
mustard, carrot, fenugreek, and gram or by fallowing. This led to 87% increase in
the barley yield. Singh et al. (2009a, b) and Shekhawat et al. (2017) observed
Fig. 3.14 Aboveground

symptoms caused by
Heterodera avenae on barley
decrease in the population of cereal cyst nematode with summer plowings and
subsequent increase in the yield of cereals. Growing of non-hosts and resistant
varieties such as barley cv. Morocco, Maroccaine, PI 253826, PL 101, C-164, and
Raj Kiran (Walia and Khan 2018) offers resistance against a wide range of
pathotypes of H. avenae s.s in India, but not effective against H. filipjevi of Punjab
and Ambala populations. Out of hundreds of lines showing resistance, one line
having local number RD 387 was found agronomically superior in yield and showed
complete resistance to prevailing pathotypes of CCN except Ambala population
(Bishnoi and Bajaj 2004).
A wide range of chemicals have been evaluated to control CCN in barley. Soil
application of DD, DBCP (EC), aldicarb, and carbofuran has shown high degree of
effectiveness against H. avenae in barley (Mathur et al. 1986; Handa et al. 1985;
Sharma 2003). Soil drill of carbofuran and aldicarb at 1.0 kg a.i/ha effectively
reduced the nematode population even when Pi was as high as >5 juveniles/g soil.
Indra and Sharma (2000) reported that soil application of Sebufos (cadusafos) 10G
(1 kg a.i./ha), Padan (cartap) 10G (2.0 kg/ha), and carbofuran 3G and seed treatment
with neem-based Achook (10 and 20%) suppressed the H. avenae attack on barley.
All treatments significantly increased grain and fodder yields and reduced the
number of cysts per plant.
Catenaria vermicola has been found parasitizing cyst, juveniles, and egg of
H. avenae, H. cajani, H. zeae, H. graminis, H. mothi, and H. sorghi in India (Bhatti
and Paruthi 2009), while Verticillium uniseptum parsitized the eggs of H. avenae
(Choudhary and Kaushal 1984). Several other fungi and like Catenaria auxiliaris,
Nematopthora gynophila, Verticillium chlamydosporium, Tarichium auxiliare, and
Cylindrocarpon destructans are also known to parasitize H. avenae in Britain (Bhatti
and Paruthi 2009). Bhattacharya and Swarup (1988) reported that Bacillus
penetrans, Glomus fasciculatum, and Pasteuria penetrans were found to be the
most destructive against H. avenae. Walia and Khan (2018) reported that seed
treatment with Azotobactor chrococcum (strain HT 54) may effectively reduce the
soil population of cereal cyst nematode and improve the grain yield.
3.3.5 Sorghum
Sorghum cyst nematode, Heterodera sorghi, Tylenchorhynchus vulgaris,

Hoplolaimus spp., Meloidogyne spp., and Rotylenchus spp. are important
phytonematodes associated with sorghum crop in India. Cyst nematode disease of
sorghum (H. sorghi) is quite prevalent in Haryana, Punjab, Delhi, Uttarakhand,
Madhya Pradesh, Himachal Pradesh, Jammu and Kashmir, Andhra Pradesh, and
Maharashtra. Most common symptoms of the disease are presence of light to dark
brown cysts on roots. The root-knot nematode, M. incognita, is another important
disease in sorghum (Srivastava and Chowla 2010). The nematode causes chlorosis
and stunting of foliage and galls on the roots. Root lesion disease caused by P. zeae
inflicts considerable yield loss to sorghum in India. The infested plants are stunted
and become yellowish. On roots, necrotic lesions are formed. Tylenchorhynchus
vulgaris is also an important nematode that feeds on the roots of sorghum. The
nematode causes decay and tip swelling of feeder roots.
Crop rotation with sugar beet, peas, beans, crucifers, and potatoes may suppress
the population of Pratylenchus crenatus in soil (Srivastava and Chawla 2010). Two
to three deep summers plowing in hot months of May/June followed by fallowing
can reduce the soil population of Pratylenchus and Meloidogyne spp. Some maize
cultivars like Nab Elgamal, Early American, Giza, and Balady have shown moderate
level of tolerance to P. zeae and can be cultivated in the areas/field with high
nematode infestation level. The maize cv. Kanchan, P128, possesses remarkable
degree of resistance against M. incognita, M. javanica, and M. arenaria (Khan et al.
1994) and may be recommended for cultivation in hot zones. Growing antagonistic
plants such as mustard, marigold, Asparagus sp., Crotalaria sp., and sesame can
reduce the population of the nematodes (Srivastava and Chawla 2010).
Soil application of Temik and Furadan at 2.5 kg/ha controlled P. delattrei.
Bergeson (1978) observed that application of carbofuran at 4.5 kg a.i./ha reduced
Pratylenchus population up to 84% and significantly increased the maize yield.
Kutche and Rossner (1978) recorded up to 90% control of Pratylenchus sp. with
different formulations of carbofuran. Walia and Khan (2018) recommended soil
application of carbofuran (3G) at 2 kg a.i./ha to control P. thornei.
Biocontrol strategy may also prove effective against nematode infection in
sorghum. Trichoderma harzianum at 25 kg/ha along with Pochonia chlamydosporia
at 10 kg/ha 1 week prior to sowing effectively managed lesion nematode,
Pratylenchus thornei (Abd-Elgawad and Askary 2018). Dias-Arieira et al. (2018)
evaluated the effect of Purpureocillium lilacinum and T. harzianum, either alone or
in combination, along with a bioactivator (mass) to control Pratylenchus
brachyurus. Both fungi were efficient in controlling the nematode when they were
applied alone, whereas the combination of the two did not improve nematode
control. However, the addition of moss to the combination of fungi (P. lilacinum + T.
harzianum) generally provided better control of the nematode and increased the
plant yield.
3.4 Conclusion and Future Prospects
The crop losses inflicted by phytonematodes to agriculture on a worldwide basis

appear enormous, as the data generated on this aspect has covered relatively limited
crops as well as countries neglecting by and large the Asian and African countries. In
this situation, the estimate of $100 billion annually appears to be unrealistic (Khan
2008). Hence to develop efficient management technology, it becomes mandatory to
undertake extensive survey and extension work to assess the actual enormity of the
problem. Although India is self-sufficient in food grain production, the national
average productivity is considerably lower than the global average productivity in
most of the crops. Hence, there is a lot of scope for improvising food grain
production from the area under present cultivation in India. This can be achieved
by implementing pest and disease management programs including plant nematodes
at the grassroot level.
The literature has revealed that several nematode species infest cereal crops in
India, but relatively a much smaller proportion of nematode community constitutes
potential plant parasitic genera and species that cause economic damage and yield
decline to food crops. Hence, special attention in a phase manner should be given to
reduce the population of five most economically important nematode genera, viz.,
Meloidogyne, Pratylenchus, Heterodera, Ditylenchus, and Aphelenchoides. The
most simple way of controlling the nematodes is crop rotation by growing
non-host resistant or tolerant crops. Since chemical application (nematicides) creates
several hazards, eco-friendly methods should be preferred. Deep plowing and
fallowing coupled with solarization should be taken as an integral strategy of disease
management in Indian farming system. Field sanitation especially the removal of
root stubs which support nematode survival and multiplication after harvesting the
cereals may prove effective in reducing the soil population of nematodes for
subsequent crops. Overflooding of water from the adjoining infested fields should
be avoided. In addition, certified and disinfested seeds/planting material coupled
with seed dressing with biocontrol agents such as Trichoderma spp. or Pseudomonas
fluorescens should be used. This package of practices shall help in reducing the
nematode populations in the field and shall prevent spread and introduction of initial
population densities of phytonematode inocula.
References
Abd-Elgawad MM, Askary TH (2018) Fungal and bacterial nematicides in integrated nematode
management strategies. Egypt J Biol Pest Control 28:74
Agrios GN (2005) Plant pathology, V edn. Elsevier Academic Press, San Diego, p 922
Alam MM, Khan AB, Saxena SK (1981) Efficacy of nematicides for field control of nematodes
infesting certain vegetable crops. Ind J Nematol 11:5–10
Amin WA, Al-Shalaby MEM (2005) Effect of soaking seed in hot water and nematicide on survival
of Aphelenchoides besseyi white tip nematode in rice. Pak J Nematol 22:163–172
Ashoub AH, Montasser SA, Mourad MH, Gamal M (2009) Impact of some fungi species as
biocontrol agent against the root-knot nematode, Meloidogyne. Aust J Basic App Sci
3:3617–3624
Atkinson GF (1982) Some disease of cotton. Bull Alabama Agric Exp Station 41:61–65
Baheti BL, Verma MK (2001) Varietal response against wheat seed gall nematode. National
Congress on Centenary Nematology in India. Appraisal and Future Plans Organised by IARI,
New Delhi, 5–7 Dec 2001, pp 130–131
Baheti BL, Dodwadia M, Rathore BS, Bhati SS (2015) Management of maize cyst nematode,
Heterodera zeae on sweet corn (Zea mays L. saccharata) through soil amendment. Ecoscan
7:305–309
Bergeson GN (1978) Control of the lesion nematode (Pratylenchus spp.) in corn with carbofuran. Pl
Dis Reptr 62:295–297
Bhattacharya D, Swarup G (1988) Pasteuria penetrans, a pathogen of the genus Heterodera, its
effect on nematode biology and control. Ind J Nematol 18:61–70
Bhatti DS, Paruthi IJ (2009) Nematode problems of wheat and barley and their management. In:
Upadhyay KMRH, Chamola OB, Dueby P (eds) Integrated pest and disease management. APH
Publishing
Bhatti DS, Dalal MR, Dahiya RS, Dhawan SC (1980) Integrated control of Heterodera avenae in
wheat. J Nematol 12:214–215
Bishnoi SP, Bajaj HK (2002) Response of barley cultivars to the Indian populations of Heterodera
avenae complex. Ind J Nematol 32:125–128
Bishnoi SP, Bajaj HK (2004) On the species and pathotypes of Heterodera avenae complex of
India. Ind J Nematol 34:147–152
Bishnoi SP, Pankaj (2010) Nematode infestation in barley and oats. In: Khan MR, Jairajpuri MS
(eds) Nematode infestation part I: food crops. National Academy of Sciences, Allahabad, pp
242–266
Biswas H, Rao YS (1971) Toxicological assays of nematicides with rice nematodes. Indian
Phytopath 24:159–165
Bokhari, Fardos (2009) Efficacy of some Trichoderma species in the control of Rotylenchulus
reniformis and Meloidogyne javanica. Arch Phytopath Plant Protec 42:361–369
Bora BC, Rahman MFB (2010) Stem nematode infestation in rice. In: Khan MR, Jairajpuri MS
123–139
Chandra B, Khan M (2011) Dynamics of soil nematodes in vegetable-based crop sequences in West
Bengal. Indian J Plant Protect Res 51:7–13
Chen S, Zheng X, Wang D, Chen L, Xu C, Zhang X (2012) Effect of long-term paddy-upland
yearly rotations on rice (Oryza sativa) yield, soil properties, and bacteria community diversity.
Sci World J 11
Choudhary PN, Kaushal KK (1984) Cylindrocarpon uniseptatum sp Nov-A new fungus from India.
Curr Sci 53:205–207
Das N, Behera SK, Sahoo S (2018) Fungal bio-agent for management of root-knot nematode
Meloidogyne graminicola in rice. Int J Curr Microbiol App Sci 6:2017–2020
Dawar S, Sattar A, Zaki MJ (2008) Seed dressing with biocontrol agents and nematicides for the
control of root-knot nematode on sunflower and okra. Pak J Bot 40:2683–2691
Dhawan SC (1988) Katyil, a resistant wheat cultivar susceptible to Indian population of cereal cyst
nematode, Heterodera avenae. Ind J Nematol 18:140
Dias-Arieira CR, de Araújo FG, Kaneko L, Santiago DC (2018) Biological control of Pratylenchus
brachyurus in soya bean crops. J Phytopathol 166:722–728
Dutta TK, Ganguly AK, Gaur HS (2012) Global status of rice root-knot nematode, Meloidogyne
graminicola. Afr J Microbiol Res 6:6016–6021
FCI (2019) Food grains Stock in Central Pool for current year 2018. http://fci.gov.in/stocks.php?
view¼46
Gao HB, Zhou H, Feng Z (1998) Effect of cultivation measures on Hirschmanniella oryzae. Chin
Rice Res Newslett 6:8–9
Garg RN, Gaur HS, Singh GC (1995) Effect of tillage practices and water regimes on the soil
physical properties and plant parasitic nematodes in rice. Ann P Protec Sci 3:121–126
Gaur HS (2003) Root-knot disease of rice and wheat: problem and management, Tech Bull 23(TB –
ICN: 1/2003). IARI, New Delhi
Ghosh SC (2001) Studies on nematode parasites associated with paddy crops of West Bengal, India.
Ph.D. thesis awarded from University of Calcutta Kolkata, India
Ghosh SC and Buddhadeb M (2008) Studies on nematode parasites associated with paddy crop of
West Bengal, India. Rec Zo Sur Ind, Occasional paper No. 287 pp 1–144
Ghosh SC, Manna B (2010a) Rice nematode infestation in rice. In: Khan MR, Jairajpuri MS (eds)
Nematode infestation part I: food crops. National Academy of Sciences, Allahabad, pp 192–217
Ghosh SC, Manna B (2010b) Rice nematode infestation in paddy. In: Khan MR, Jairajpuri MS (eds)
Haider MG, Askary TH, Nath RP (2001) Nematode population as influenced by paddy based
cropping sequences. Ind J Nematol 31:68–71
Handa DK (1983) Studies on ‘Molya’ disease (Heterodera avenae) of wheat and barley with special
emphasis on its control. Ph D thesis, University of Rajasthan, Jaipur, p 340
Handa DK, Mathur RL, Mathur BN (1978) Studies of the integrated control of Molya disease of
wheat and barley caused by Heterodera avenae in Rajasthan. Ind J Myco Pl Patho 8:70
Handa DK, Mathur RL, Mathur BN, Yadav BD (1985) Estimation of losses in barley due to cereal
cyst nematode in sandy and sandy loam soils. Indian J Nematol 15:163–166
Haque A, Latif MA (2011) Screening of ufra (Ditylenchus angustus) resistant rice genotypes.
Eco-friendly Agril J 4:527–530, 126:109–116
Haque MM, Prasad D (1983) Studies on crop rotation as a method of nematode control, Third
Nematology Symposium (Abstract), Nematological Society of India, Himachal Pradesh Krishi
Viswa Vidyalaya, Solan, May 24–26. 1983:29–30
Haque A, Bhuiyan MR, Islam S, Zaman MAU, Latif MA, Ali MA (2013) Integrated management
of Ufra (Ditylenchus angustus) disease of rice. Eco-friendly Agril J 6:285–290
Haque Z, Khan MR, Ahamad F (2019) Rice root-knot nematode and its management through
non-chemical approaches. In: Khan MR, Mukhopadhyay AN, Pandey RN, Thakur MP,
Singh D, Siddiqui MA, Akram M, Haque Z (eds) . Today and Tomorrow Publishes, New
Delhi, pp 225–250
Indiastat (2018). https://www.indiastat.com/agriculture-data/2/agricultural-area-land-use/152/area-
under-food-crops/448934/stats.aspx
Indra R, Sharma GL (2000) Management of cereal cyst nematode, Heterodera avenae in barley
through chemical. Curr Nematol 11:35–37
Islam A, Haque MR, Bhuiyan S, Zaman MAU, Latif MA, Ali MA (2013) Integrated management
of ufra (Ditylenchus angustus), Disease of rice. Eco-friendly Agril J 6:285–290
Islam MS, Rahman MH, Farazi MM, Hossain ATMS, Sultana A (2015) Integrated management of
seed borne nematode (Aphelenchoides besseyi) in T. Aman rice (Oryza sativa L.). Agriculturists
13(1):79–86
Jamali S, Mousanejad S (2011) Resistance of rice cultivars to white tip disease caused by
Aphelenchoides besseyi Christie. J Agric Technol 7:441–447
Jahier J, Rivoal R, Yu MQ, Abelard P, Tanguy AM, Barloy D (1998) Transfer of genes for
resistance to cereal cyst nematode from Aegilops variabilis Eig to wheat. J Genet Breed
52:253–257
Johnathan EI, Pandiarajan P (1991) Effect of soil amendments in controlling rice root nematodes.
Int Rice Res Newsl 16:25
Kali Ram (1989) Ph.D thesis, Haryana Agricultural University, Hisar, India
Kalita M, Phukan PN (1990) Reactions of some rice cultivars to Meloidogyne graminicola. Indian J
Nematol 20:215–216
Kanwar RS, Bajaj HK (2009) Mechanism of resistance in wheat varieties Raj MR1 and AUS 15854
to cereal cyst nematode, Heterodera avenae Woll. Indian J Nematol 39:104–105
Kanwar RS, Bajaj HK (2010) Cyst nematode infestation in wheat. In: Khan MR, Jairajpuri MS
192–217
Kanwar RS, Paruthi IJ, Yunus M (2001) Evaluation of wheat and triticale germplasm for resistance
to cereal cyst nematode Heterodera avenae Woll National Congress on Centenary of Nematol-
ogy in India : Appraisal and Future Plans, Dec 5–7, 2001. IARI, New Delhi, p 129
Kanwar RS, Bajaj HK, Vats R (2004) Development of Heterodera filipjevi on wheat varieties
resistant to Heterodera avenae. Indian J Nematol 34:205–206
Kaur DJ, Sharma I, Sohu VS, Bains NS (2008) Reaction of wheat genotypes to a population of
Heterodera avenae from Punjab. Nematol Medit 36:157–160
Kaushal KK, Srivastava AN, Pankaj CG, Khajan S (2007) Cyst forming nematodes in India – a
review. Ind J Nematol 37(1):75–79
Khan MR (1993) Interaction of coal-smoke pollution and root-knot nematode on eggplant. National
Symposium on Nematology, Hissar, p 82
Khan MR (2007) Prospects of microbial control of root-knot nematodes infecting vegetable crops.
In: Sharma N, Singh HB (eds) Biotechnology: Plant Health Management. International Book
Distributing Co, Lucknow, pp 643–665
Khan (2008) Plant nematodes, methodology, morphology, systematics. Biology and Ecolgy
Sicence Publishers, New Hampsphire, p 360
Khan MR (2010) White tip nematode infestation in rice. In: Khan MR, Jairajpuri MS (eds)
Khan MR (2016) Nematode biocontrol agents: diversity and effectiveness against phytonematodes
in sustainable crop protection. Indian Phytopathol 69:453–463
Khan MR, Ahamad F (2019) Incidence of root-knot nematode (Meloidogyne graminicola) and
resulting crop losses in paddy rice in northern India. Plant Dis. https://doi.org/10.1094/PDIS-12-
18-2154-RE
Khan E, Chawla ML (1975) CIH description of plant parasitic nematodes Set 5, No 66. Common-
wealth Institute of Helminthology, St Albans
Khan MR (1993) Interaction of coal-smoke pollution and root-knot nematode on eggplant. National
Symposium on Nematology, Hissar, p 82
Khan MR, Haque Z (2011) Soil application of Trichoderma harzianum and Pseudomonas
fluorescens reduces root knot of tobacco. Phytopathol Mediterr 50:257–266
Khan MR, Jairajpuri S (2010) Nematode infestations part I: food crops. National Academy of
Sciences, Allahabad, p 325. ISBN: 978-81-905548-2-4
Khan MR, Jairajpuri S (2012) Nematode infestation in horticultural crops, national scenario. In:
Khan MR, Jairajpuri MS (eds) Nematode infestation part III: horticultural crops. National
Academy of Sciences, Allahabad, pp 1–30
Khan A, Shaukat SS (1998) Effect of three organic amendments and carbofuran on population of
Hirschmanniella oryzae and Tylenchorhynchus annulatus and growth parameters of rice
Internat. J Nematol 8:94–96
Khan AA, Singh K, Khan MR, Khan MW (1994) Resistance of maize cultivars to root-knot
nematodes. Ann Appl Biol (SupplimentTAC) (UK) 124:102–103
Khan MR, Mohiddin FA, Khan SM, Khan B (2005) Effect of seed treatment with certain
biopesticides on root knot of chickpea. Nematol Medit 2:107–112
Khan MR, Shit S, Pal AK, Biswas B (2006) Management of foliar nematode, Aphelenchoides
besseyi infecting tuberose in West Bengal, India. Ind J Nematol 36(1):44–47
Khan MR, Anwer MA (2008) DNA and some laboratory test of nematode suppressing efficient soil
isolates of Aspergillus niger. Indian Phytopathol 61(2):59–73
Khan MR, Altaf S, Mohiddin FA, Khan U, Anwer A (2009) Biological control of plant nematodes
with phosphate solubilizing microorganism. In: Khan MS, Zaidi A (eds) Phosphate solubilizing
microbes for crop improvement. Nova Science Publishers, Inc, New York, pp 395–426
Khan MR, Jain RK, Singh RV, Pramanik A, Lin T (2010) Economically important plant parasitic
nematodes distribution ATLAS. Directorate of Information and Publications of Agriculture,
New Delhi
Khan MR, Anwer MA (2011) Occurrence of rice root-knot nematode and yield loss assessment in
Aligarh and Hathras districts of Uttar Pradesh, India. Indian J Nematol 41(1):34–40
Khan MR, Ashraf T, Shahid S (2012) Evaluation for relative susceptibility of rice against field
population of Meloidogyne graminicola. Indian J Nematol 42:46–52
Khan MR, Haque Z, Kausar Z (2014) Management of the root-knot nematode Meloidogyne
graminicola infesting rice in the nursery and crop field by integrating seed priming and soil
application treatments of pesticides. Crop Protect 63:15–25
Khan MR, Mohidin FA, Khan U, Ahamad F (2016) Native Pseudomonas spp suppressed the root-
knot nematode in in-vitro and in-vivo, and promoted the nodulation and grain yield in the field
grown mungbean. Biol Control (UK) 101:159–168
Khan MR, Haque Z, Rasool F, Salati A, Khan U, Mohidin FA, Zuhaib M (2019) Management of
root-rot disease complex of mungbean caused by Macrophomina phaseolina and Rhizoctonia
solani through soil application of Trichodermaspp. Crop Protect 119:24–29
Kiewnick S, Sikora RA (2006) Biological control of the root-knot nematode Meloidogyne incognita
by Paecilomyces lilacinus strain 251. Biol Control 38:179–187
Kinh D, Huong NM, Ut NV (1982) Root-knot disease of rice in the Mekong Delta Vietnam. Int
Rice Res Newslett 7:6–7
Krreye C, Bouman BAM, Faronilo JE, Llorca L (2009) Causes for soil sickness affecting early plant
growth in aerobic rice. Field Crop Res 114:182–187
Kumar D, Khilari K, Kumar N, Jain SK (2017) Integrated disease management of rice root knot
nematode (Meloidogyne graminicola) through organic amendments, Trichoderma spp. and
Carbofuran. J Pharmacognosy Phytochem 6:2509–2515
Kuriyan J (1995) Nematodes problems of rice crop in India II: Rice cyst nematode and white tip
nematode. In: Swarup G, Dasgupta DR, Gill JS (eds) Nematode pest management – an appraisal
of ecofriendly approaches. Nematological Society of India, New Delhi, p 300
Kutche K, Rossner J (1978) Anz Fur Schadlings K Pflan, Zenschutr. Umweltschutz 51:102–107
Lahan KK, Sinha AK, Das P (1999) Evaluation as chemicals as bare root-dip against rice root
nematode, Hirschmanniella oryzae. Ind J Nematol 29:8–12
Lakshmy KM (2014) Management of root-knot nematode, Meloidogyne sp (Kofoid and White) in
Coleus, Solenostemon rotundifolius (Poir) morton. Doctoral dissertation, College of Horticul-
ture, Vellanikkara
Latif MA, Ullah MW, Rafii MY, Tajul MI (2011) Management of ufra disease of rice caused by
Ditylenchus angustus with nematicides and resistance. Afr J Microbiol Res 5:660–1667
Luc M, Bridge J, Sikora RA (2005) Reflections on nematology in subtropical and tropical agricul-
ture. In: Plant parasitic nematodes in subtropical and tropical agriculture, pp 1–10
MacGeachie I, Rahman L (1983) Ufra disease: a review and a new approach to control. Trop Pest
Manag 29:325–333
MacGowan JB, Langdonm KR (1989) Hosts of the rice root-knot nematode Meloidogyne
graminicola. Fla Dept of Agric and consumer Serv Div Plant Ind. Nematology Circular No 172
Mantelin S, Bellafiore S, Kyndt T (2017) Meloidogyne graminicola: a major threat to rice agricul-
ture. Mol Plant Pathol 18:3
de Majnik J, Ogbonnaya FC, Moullet O and Lagudah ES (2003) The Cre1 and Cre 3 nematode
resistance genes are located at homeologous loci in the wheat genome. Mol Plant-Microbe
Interact 16: 1129–1134
Martin EM, Eastwood RF, Ogbonnaya FC (2004) Identification of microsatellite marker associated
with cereal cyst nematode resistance gene Cre3 in wheat. Aust J Agric Res 55:1205–1211
Mathur BN, Handa DK, Swarup G, Sethi CL, Sharma GL, Yadav BD (1986) On the loss estimation
and chemical control of ‘Molya’ disease of wheat caused by Heterodera avenae in India. Ind J
Nematol 16:152
Mathur BN, Sharma SN, Bhatnagar VK, Sharma GL, Sain RS, Bhatnagar SM, Singh S, Mishra A,
Nagpal CP, Midha RL (2004) Raj MolyaRodhak 1, a first commercial unique wheat variety
resistant to cereal cyst nematode in India. Wheat Inf Serv 98:25–27
Miah SA, Bakr MA (1977) Source of resistance to ufra disease of rice in Bangladesh. Int Rice Res
Newsl 2:8
Montes MJ, Lopez-Brana I, Romero MD, Sin E, Andres MF, Martin-Sanchez JA, Delibes A (2003)
Biochemical and genetic studies of two Heterodera avenae resistance genes transferred from
Aegilops ventricosa to wheat. Theor Appl Gen 107:611–618
Montes MJ, Andres MF, Sin E, Lopez-Brana I, Martin-Sanchez JA, Romero MD, Delibes A (2008)
Cereal cyst nematode resistance conferred by the Cre7 gene from Aegilops triuncialis and its
relationships with Cre genes from Australian wheat cultivars. Genome 51:315–319
Nandal SN, Paruthi IJ, Khan MR (2010) Seed gall nematode infestation in wheat. In: Khan MR,
Jairajpuri MS (eds) Nematode infestation part I: food crops. National Academy of Sciences,
Allahabad, pp 171–191
Narasimhamurthy HB, Ravindra H, Sehgal M (2017) Management of rice root-knot nematode,
Meloidogyne graminicola. Int J Pure Appl Biosci 5:268–276
Niu X, Wang YL, Chu YS, Xue HX, Li N, Wei LX, Mo MH, Zhang KQ (2010) Nematode toxic
aurovertin-type metabolites from a root-knot nematode parasitic fungus Pochonia
chlamydosporia. J Agric Food Chem 58:828–834
Ou SH (1972) Rice diseases. Commonwealth Mycological Institute, Kew, England
Padgham JL, Abawi GS, Duxbury JM, Mazid MA (2004) Impact of wheat on Meloidogyne
graminicola populations in the rice–wheat system of Bangladesh. Nematropica 34:183–190
Pankaj, Rohtagi D, Shakil NA, Sirohi A, Kumar J, Gaurav SS, Gaur HS, Mehar HC, Singh C,
Kishor V, Bishnoi SP (2008) Biochemical prediction of resistance in wheat to cereal cyst
nematode Heterodera avenae. Intian J Nematol 18:13–20
Parihar A, Siddiqui AU (2010) Nematode infestation in maize. In: Khan MR, Jairajpuri MS (eds)
Paruthi IJ, Bhatti DS (1992) Efficacy of different practices for the control of ear cockle in wheat
caused by Anguina tritici. Curr Nematol 3:1–4
Paruthi IJ, Nandal SN, Kumar A (2009) Wheat Seed Gall Nematode (Anguina tritici), its status and
management. In: Sharma GL (ed) Phytonematode management in field crops. Oxford Book
Company, Jaipur, pp 1–20
Pashi R, Maity A, Khan MR, Chakrabarty G (2017a) Management of white tip nematode
(Aphelenchoides besseyi) in rice in West Bengal. J Entomol Zool Stud 5:269–272
Pashi R, Maity A, Khan MR, Mondal S, Mukherjee A (2017b) Incidence of white tip nematode
(Aphelenchoides besseyi) in rice in West Bengal. J Entomol Zool Stud 5:200–208
Pathak B, Khan MR (2010) Comparative field efficacy of chemical, botanical and biological agents
against foliar nematode, Aphelenchoides besseyi infecting tuberose. Indian J Nematol 40:83
Perry RN, Moens M (eds) (2013) Plant nematology. CABI, Wallingford
Plowright RA, Caubel G, Mizen KA (2002) Ditylenchus species. In: Starr JL, Cook R, Bridge J
(eds) Plant resistance to parasitic nematode. CABI Publishing, New York, pp 107–139
Popov C, Raancius S, Cana L (2006) Technological sequences recommended at small grains,
established in autumn 2006, in prevention and control of seed and soil pests and diseases.
Probleme- de-protectia-plantelor 34:97–108
Popova MB, Zelenskii GL, Subbotin SA (1994) An assessment of resistance in cultivars of Oryza
sativa L to Aphelenchoides besseyi Christie, 1942. Russian J Nematol 2:41–44
Prasad JS, Varaprasad KS (1992) Elimination of white-tip nematode, aphelenchoides besseyi, from
rice seed. Fundam Appl Nematol 15(4):305–308
Prasad JS, Sharma OP, Gururaj K, Pasalu IC, Singh A, Sankar M (2007) White tip nematode
associated with chaffiness of rice in Haryana. Ind J Pt Protec 35(1):153
Prasad JS, Somasekhar N, Varaprasad KS (2010) Nematode infestation in Paddy. In: Khan MR,
Jairajpuri MS (eds) Nematode infestations, part I- food crop. Indian Academy of Sciences,
Bengaluru, pp 17–71
Prot JC, Matias DM (1995) Effects of water regime on the distribution of Meloidogyne graminicola
and other root-parasitic nematodes in a rice field toposequence and pathogenicity of M
graminicola on rice cultivar UPL R15. Nematologica 41:219–228
Prot JC, Soriano IRS, Matias DM (1994) Major root-parasitic nematodes associated with irrigated
rice in the Philippines. Fund App Nematol 17:75–78
Rahman ML (1993) Effect of time of nematicides application to hibiscus. Phytopathology
53:478–479
Rahman MF (2010) Lance nematode infestation in rice. In: Khan MR, Jairajpuri MS (eds)
Rahman ML, Das P (1994) Seed soaking with chemicals for reducing infestation of Meloidogyne
graminicola in rice. J agri Sc Soc North east India 7:107–108
Rahman ML, MacGeachie I (1982) Screening of resistance of ufra disease (Ditylenchus angustus)
in Deepwater rice. Proc of 1981 Int Deepwater rice Workshop, IRRI, Manila, Philippines
Ramakrishnan S, Varadharajan G, Sutharsan PD (1984) Insecticides root dip controls rice nema-
tode. Int Rice Res Newslett 9:20
Randhawa N, Singh I, sakhuja PK, Malhi SS (1992) Effect of date of transplanting of Basmati
cultivars and spacing in the population buildup of rice root-knot nematode, Hirschmanniella
oryzae. Indian J Nematol 21:4–8
Rao YS (1978) ICAR Symp on increasing yields of rice in Kharif. CRRI, Cuttack, pp 401–411
Rao YS (1985) Research on rice nematodes. In: Padmanabhan SY (ed) Rice in India. ICAR
Monograph, pp 591–615
Rao J, Prakash A (2002) Rice white-tip nematode. AZRA Publications, Cuttack, p 94
Rao YS, Prasad JS, Yadava CP, Padalia CR (1984) Influence of rotation crops in rice soils on
dynamics of plant parasitic nematode populations. Biol Agric Hort 2:69–78
Rao YS, Prasad JS, Panwar MS (1986) Nematode pests of rice in India. In: Non-insect pests and
predators. All India Scientific Writers Society, New Delhi, pp 65–71
Reddy PP (2018) Emerging crop pest problems: redefining management strategies. Scientific
Publishers, Jodhpur, p 179
Romero MD, Montes MJ, Sin E, Lopez-Brane I, Duce A, Martin-Sanchez JA, Andres MF, Delibes
A (1998) A cereal cyst nematode (Heterodera avenae Woll) resistance gene transferred from
Aegilops triuncialis to hexaploid wheat. TheorApl Gen 96:1135–1140
Roy AK (1976) Pathological effects of Meloidogyne graminicola on rice and histopathological
studies on rice and maize. Ind Phytopathol 29:359–362
Sakhuja PK, Singh I, Sharma SK (1978) Effect of nitrogen levels alone and in combination with
some nematicides on the population of cereal cyst nematode, Heterodera avenae and yield of
wheat. Indian J Nematol 8:159–162
Salalia R, Walia RK, Somvanshi VS, Kumar P, Kumar A (2017) Morphological, morphometric,
and molecular characterization of intraspecific variations within Indian populations of
Meloidogyne graminicola. J Nematol 49:254–267
Santos DMA, Ferraz S, Muchovej JJ (1992) Evaluation of 20 species of fungi from Brazil for
biocontrol of Meloidogyne incognita race-3. Nematropica 22:183–192
Segeren et al (1985) Meloidogyne oryzae, a pest of irrigated rice in Surinam. Int Rice Res Newslett
10:27
Sethi CL, Meher HC (1991) Effect of phenamiphos on soil nematodes and yield of cowpea, pea and
okra. Indian J Nematol 19:89–94
Sharma, GL (2003) Cereal cyst nematode (Heterodera avenae) in Rajasthan. Advances in nematol-
ogy, p 73
Sharma SN, Sain RS, Singh H (2004) Breeding for resistance to wheat nematodes (Heterodera
avenae). Acta Agro Hunga 52:189–197
Shekhawat PS, Bishnoi SP, Ghasolia RP (2017) Disease spectrum on barley in Rajasthan and
integrated management strategies. In: Management of wheat and barley diseases. Apple Aca-
demic Press, pp 467–503
Siddiqui MA (2005) Population changes of nematodes associated with Citrus reticulata and Citrus
aurantifolia. Arch Phytopathol Plant Protec 38:165–173
Sikora RA, Coyne D, Hallmann J, Timper P (2018) Plant parasitic nematodes in subtropical and
tropical agriculture. In: Cabi (ed) Plant resistance to parasitic nematode. CABI Publishing,
New York, pp 107–139
Singh H (1985) Effect of crop rotation on the population of cereal cyst nematode. Ind J mycol Pl
Pathol 15:263–264
Singh G (2017) Prevalence and management of rice root-knot nematode (Meloidogyne
graminicola) in rice. Doctoral dissertation, Punjab Agricultural University, Ludhiana
Singh et al (1987) Influence of some crucifers on Heterodera avenae, the cereal cyst nematode. Pl
Dis Res 2:93–94
Singh D, Parihar A, Siddiqui AU (1997) Effect of application of urea and carbofuran on maize in a
Heterodera zeae infested field. J Mycol Pl Pathol 27:319–320
Singh AK, Sharma AK, Jag S (2009a) Heterodera avenae and its management on wheat in India.
In: Cereal cyst nematodes: status, research and outlook. Proceedings of the First Workshop of
the International Cereal Cyst Nematode Initiative, Antalya, Turkey, 21–23 October 2009
(17–22), International Maize and Wheat Improvement Centre (CIMMYT)
Singh R, Benival MS, Karwasara RS, Saharan HS (2009b) Effect of data on the incidence of loose
smut flag smut and seed gall of wheat Bangladesh. Aust J Agric Res 34:1–4
Sitaramaiah K, Pathak KN (1993) Nematode bacterial disease interaction. In: Khan MW
(ed) Nematode interaction. Chapman and New Delhi, p 50
Sivakumar CV (1988) White tip nematode, Aphelenchoides besseyi. Indian J Nematol 18:342–344
Smiley RW, Nicol JM (2009) Nematodes which challenge global wheat production. In: Wheat
science and trade, pp 171–187
Soriano IR, Reversat G (2003) Management of Meloidogyne graminicola and yield of upland rice
in South-Luzon, Philippines. Nematology 5:879–884
Southey JF (1986) Plant nematology. Her Majesty’s Stationery, London, p 446
Srivastava AN, Chawla G (2005) Maize cyst nematode, Heterodera zeae a key nematode pest of
maize and its management. IARI, New Delhi-18
Srivastava AN, Lal J (2007) Effect of split application of cadusafos and carbofuran on reproduction
and fecundity of maize cyst nematode, Heterodera zeae and maize growth. Indian J Nematol 37
(1):78–80
Srivastava AN, Chawla G (2010) Nematode infestation in coarse cereals. In: Khan MR, Jairajpuri
MS (eds) Nematode infestation part I: food crops. National Academy of Sciences, Allahabad, pp
267–287
Taylor CE, Brown DJF (1997) Nematode vectors of plant viruses. CABI, Wallingford
Tulek A, Kepenekci I, Çiftcigil TH, Surek H, Akin K, Kaya R (2015) Reaction of some rice
cultivars to the white tip nematode, Aphelenchoides besseyi, under field conditions in the thrace
region of Turkey. Turkish J Agric Forest 39:958–966
Walia RK, Khan MR (2018) A compendium of nematode diseases of crop plants. ICAR-all India
Coordinated Research Projects on Nematodes in Cropping Systems, New Delhi, p 110
Wang ZY, Yang-Hong F, Ji-Mu X (2006) The small grains and erect panicle of rice caused by
nematode and control the disease in Jiangsu Province. J Nanjing Agric Univ 29:54–56
Wani AH (2005) Effect of cropping sequences and ploughing on plant parasitic nematodes and
plant growth in field. Indian J Nematol 35:63–67
Youssef MMA (1999) Population dynamics of Hirschmanniella oryzae, the rice root nematode in
relation to rice cultivar, soil temperature and nematode control. Pak J Nematol 17:39–46
Youssef MMA (2014) The leaf and bud nematode, Aphelenchoides besseyi, its identification,
economic importance and control measures. A Rev Middle East J 3:461–464
Zeinat KM, Nagwa MA, El-Sayed SA, Abd El-Wahab GS (2010) Optimization of microbial
biomass production as biocontrol agents against root knot nematode on faba plants. J Am Sci
6:245–255
Modern Tools for Detection and Diagnosis
of Plant Pathogens 4
Madhurababu Kunta, Jong-Won Park, W. Evan Braswell, John V. da
Graça, and Perry Edwards
Abstract
Plant diseases contribute to an estimated annual crop loss of $60 billion world-
wide, threatening the food security and thereby the survival of humankind. Plant
pathogens are an important component of the widely accepted disease triangle
concept where they cause diseases in susceptible hosts under favorable environ-
mental conditions. Under such favorable environmental conditions, pathogens
often spread rapidly to new hosts. Therefore, early and accurate detection of a
disease and diagnosis of its causal agent is highly important for disease control
and sustainable agricultural production. This chapter provides information on an
array of methods actively being used to detect and diagnose diseases. We include
discussion of established methods such as microscopy and serology, indexing,
and PCR as well as methods that are actively under development. An elaborate
account of information is provided on promising diagnostic methods based on
programmable nucleic acid-binding proteins such as clustered regularly
interspaced palindromic repeat (CRISPR)-associated (CAS) proteins, zinc finger
(ZnF) proteins, and transcription activator-like effector (TALE) proteins. Finally,
we discuss indirect pathogen detection technologies that utilize optical sensors to
identify changes in the host plant properties due to a disease.
M. Kunta (*) · J.-W. Park · J. V. da Graça

Texas A&M University Kingsville Citrus Center, Weslaco, TX, USA
e-mail: madhura.kunta@tamuk.edu; jongwon.park@tamuk.edu; john.dagraca@tamuk.edu
W. E. Braswell
USDA APHIS PPQ CPHST, Mission Laboratory, Edinburg, TX, USA
e-mail: evan.braswell@usda.gov
P. Edwards
CROPTIX, State College, PA, USA
e-mail: perry@croptix.solutions

https://doi.org/10.1007/978-981-15-6275-4_4
64 M. Kunta et al.
Keywords
Polymerase chain reaction · Plant pathogen · DNA · Detection · Antibodies ·

CRISPR-Cas · Biosensor
4.1 Introduction
The impact of exogenous forces on plant phenotype has long been observed among
both wild and cultivated plants. These exogenous forces include both biotic and
abiotic causes, but the importance of biotic factors was first identified by Robert
Hooke (1665) who illustrated in a book, Micrographia, that a plant pathogenic
microfungus, Phragmidium mucronatum, causes rose rust disease.
Although plant disease had been observed previously, plant pathology truly
began as a field of inquiry in the wake of the Irish potato famine of 1845. With the
death of over 1 million people and the migration of 1 million more, late blight of
potato altered world history. However, when Anton de Bary (1861) identified the
cause as Phytophthora infestans, he opened the door to routes of treatment, control,
and management previously unavailable.
Mirroring history, the first steps of any modern phytopathological investigation
are the detection and diagnosis of the causative pathogen. Moreover, given the
impact of plant pathogens there is substantial motivation to detect and diagnose
quickly, sensitively, and specifically.
To address these needs, methods have developed from simple observation of
disease symptoms on plants to rather sophisticated methods to detect portions of the
pathogen, by-products, or impacts of the pathogen. In this chapter, we review
established diagnostic methods such as microscopy, indexing, enzyme-linked immu-
nosorbent assay (ELISA), polymerase chain reaction (PCR), real-time PCR, and
other nucleic acid amplification methods. Further, we review progress on newer
methods actively under development. Specifically, we review the detection of
nucleic acids with nucleic acid-binding proteins, as well as indirect signatures of
pathogen presence using metabolomic, volatile organic compound (VOC) data, and
optical biosensors.
4.2 Established Methods
4.2.1 Microscopy and Serology
Beyond visual inspection of disease symptoms, microscopy was the first approach
utilized to identify the presence and identify of plant pathogens (Hooke 1665).
Technological advances in microscopy led to ever-increasing sensitivity and speci-
ficity. Ultimately, electron microscopy has allowed plant pathologists to determine
the shape and dimensions of different viruses, and the effects on host cells. For
4 Modern Tools for Detection and Diagnosis of Plant Pathogens 65
example, potyvirus-infected plants develop characteristic pinwheel inclusion bodies

only observable by electron microscopy (Edwardson 1974).
Some pathogens evaded early microscopy efforts. Early on plant pathologists
identified transmissible agents of disease that were smaller than bacteria. Beale
(1928) first identified something of their chemical nature—tobacco mosaic-infected
plants possessed an antigen (Beale 1928). Gratia (1933) then showed that plants
infected with different viruses possessed different specific antigens. Stanley (1935)
reported that the virus could be isolated in a crystalline state, and Bawden et al.
(1936) detected the presence of a nucleic acid. X-ray crystallography indicated that
TMV is rod-shaped (Bernal and Fankuchen 1937). This was soon confirmed by early
electron microscopy (Kausche et al. 1939).
Through the 1950s research showed that the nucleic acid component was more
important than initially thought (Mathews 1970). Nevertheless, it was the protein
components that received attention from a diagnostic standpoint. Several assays
were developed to help identify viruses with antisera raised in various mammals, but
especially rabbits. These included agglutination assays and gel precipitation assays,
especially the double diffusion test which could detect strain differences
(Ouchterlony 1962).
Eventually, serology and microscopy were brought together. One technique is to
apply antiserum to the particles on the grids; in the presence of the capsid protein, the
antiserum adheres and gives the particles a fuzzy appearance (Derrick 1973).
Another technique uses gold-labeled antisera (van Lent and Verduin 1985) or
fluorescent labeling (Nagaraj 1965) and has been useful in identifying the location
of the virus particles in ultrathin sections of plant cells. Antisera produced by
injecting purified virus into a mammal are composed of a mixture of antibodies,
each produced in response to a different site on the protein. Köhler and Milstein
(1975) developed a technique to produce antibodies specific to one antigenic site
(monoclonal antibodies) that proved useful in the detection of specific strains of
viruses.
Enzyme-linked immunosorbent assays (ELISA), developed by Clark and Adams
(1977), are done in microplates coated with antisera to trap any virus particles in the
sample. Trapped viruses are detected by adding alkaline phosphatase-linked antisera
along with the enzyme’s colorless substrate which the enzyme catalyzes into a
visible yellow product. This visible signal can be quantified with a
spectrophotometer.
Different variations of ELISA have been developed to improve speed, accuracy,
and quantification such as lateral flow devices that have been used for diagnostics of
bacterial pathogens (Alvarez 2004). Alternative serological approaches involve
separating viral proteins by polyacrylamide gel electrophoresis, transferring the
proteins to a cellulose membrane (a technique known as western blotting), and
then detecting the protein bands with nonspecific staining or specific immunoassay
(O’Donnell et al. 1982; Rybicki and von Wechmar 1982).
66 M. Kunta et al.
4.2.2 Pathogen Indexing
While there are several early illustrations and descriptions of viral infections of
plants (e.g., tulip color breaking), it was not until the demonstration that tobacco
mosaic could be mechanically transmitted to healthy plants (Mayer 1886), and that
the agent was able to pass through a bacterial filter (Iwanowski 1892), that a
pathogen other than a fungus or a bacterium could cause a plant disease. The first
diagnostic assays to be used for plant viruses were biological indicator plants.
Holmes (1929) was able to quantify Tobacco mosaic virus (TMV) by inoculating
a local lesion host. Smith (1931) was able to show that some potato diseases were
caused by mixtures of viruses. He found that Potato virus Y (PVY) could be
transmitted by the aphid, Myzus persicae, while another, Potato virus X (PVX)
could not. Also, PVY could not be mechanically transmitted to Datura stramonium,
while PVX could. Indicator plants remain a widely used diagnostic tool today.
4.2.3 Nucleic Acid-Based Detection
Since the advent of PCR (Mullis et al. 1986) nucleic acids have become a staple of
molecular diagnostics. They represent a signal for direct detection that is highly
specific and highly adaptable.
4.2.3.1 PCR
PCR has been widely used for the amplification of specific target nucleic acid
molecules, a key step in nucleic acid detection assays, present in low quantity in
various sources (Craw and Balachandran 2012; Zanoli and Spoto 2013).
Kuzdralinski et al. (2017) discussed on the basic steps involved in designing and
developing a specific PCR assay such as selection of DNA extraction and amplifica-
tion methods, identification of appropriate target gene, in silico analysis, and opti-
mization of PCR conditions by validation using environmental samples and
appropriate number of strains.
PCR is one of the most cost-effective nucleic acid-based diagnostic tools. A
significant drop in the cost of sequencing offers an advantage to obtain nucleotide
sequences for a large number of PCR amplicons and utilize them in accurately
identifying different closely related pathogen species or strains (Henson and French
1993). Another PCR variant, nested PCR (Kawada et al. 2004) reported to be more
sensitive with reduced impact of PCR inhibitors and is obviously a desirable assay
when the pathogen is in small amounts.
Despite a wide adoption of PCR in diverse fields, the requirement of a
thermocycler for PCR limits its application where the resource is limited (Zhao
et al. 2015; Zeng et al. 2019). Since 1990s, many novel DNA amplification
techniques that can be conducted at a constant temperature, called isothermal
amplification, have been developed to amplify nucleic acids without the need of
thermocycler (Zanoli and Spoto 2013; Zhao et al. 2015; Qi et al. 2018). These
include, for example, rolling circle amplification (RCA) (Walter and Strunk 1994),
helicase-dependent amplification (HDA) (Vincent et al. 2004), loop-mediated ampli-

fication (LAMP) (Notomi et al. 2000), nucleic acid sequence-based amplification
(NASBA) (Compton 1991), recombinase polymerase amplification (RPA)
(Piepenburg et al. 2006), and multiple displacement amplification (MDA) (Dean
et al. 2002; Zanoli and Spoto 2013).
Development of these isothermal DNA amplification techniques has opened the
door to the development of simple, fast diagnostic methods in a form of point-of-care
testing (POCT). Some of these are available as commercial kits in portable devices
(Chang et al. 2012; Craw and Balachandran 2012; Zanoli and Spoto 2013; Zhao
et al. 2015; Zeng et al. 2019). Zhao et al. (2015), divided the various types of
isothermal amplification techniques into three groups based on the kinetics of the
reaction: exponential amplification, linear amplification, and cascade amplification.
Among these groups, exponential isothermal amplification techniques are known to
provide higher amplification and detection sensitivity compared to other isothermal
techniques (Zhao et al. 2015; Qi et al. 2018). In this section, we focused on the
exponential isothermal amplification and discussed their application as a diagnostic
tool for the detection of target nucleic acids.
4.2.3.2 Nucleic Acid Sequence-Based Amplification (NASBA)

NASBA was developed in 1991 as a method that can specifically amplify single-
stranded RNA or DNA molecules (Compton 1991). NASBA is similar to a self-
sustained sequence replication (3SR), a transcription-based amplification system
(TAS) that mimics retrovirus replication by incorporating reverse transcriptase,
RNase H, and DNA-dependent RNA polymerase (Kwoh et al. 1989; Guatelli et al.
1990; Compton 1991; Blais et al. 1997; Deiman et al. 2002; Zhao et al. 2015).
NASBA is mediated by first, a conversion of single-stranded RNA into double-
stranded heteroduplex composed of the original RNA molecule and its complemen-
tary DNA (cDNA) by reverse transcription with a primer that has a promoter
sequence (e.g., T7) at its 50 portion. Subsequently, the RNA strand from the RNA:
cDNA heteroduplex is removed by RNase H leaving a single-stranded cDNA with a
promoter sequence at its 50 end. The single-stranded cDNA is then converted into
double-strand DNA (dsDNA) via the extension of a second primer that was annealed
to the single-stranded cDNA by reverse transcriptase. The promoter sequence
present in the dsDNA product will function to initiate the synthesis of negative-
sense RNA molecules (complementary to the original RNA molecules) by the
DNA-dependent RNA polymerase (e.g., T7 DNA polymerase). The negative-
sense RNA products will then serve as templates for another round of reverse
transcription resulting in the generation of more RNA:DNA heteroduplexes leading
to a cyclic reaction of the aforementioned whole process enabling an exponential
amplification of RNA molecules complementary to the initial target RNA (Compton
1991; Zanoli and Spoto 2013; Zhao et al. 2015). Although NASBA can be
conducted at 41 C where a billion copies of the target molecule can be synthesized
in about 2 h (Compton 1991; Deiman et al. 2002; Zanoli and Spoto 2013), it is
recommended to conduct initial denaturation to facilitate RNA-primer annealing for
reverse transcription (Blais et al. 1997; Simpkins et al. 2000; Abd El Galil et al.
68 M. Kunta et al.
2005; Zhao et al. 2015). In case that dsDNA is a starting material for NASBA, the
denaturation step should be incorporated for primer annealing to the target DNA
molecule followed by extension by reverse transcriptase (Compton 1991). The
detection of NASBA single-stranded RNA products can be achieved in several
ways including real-time detection using molecular beacon probe (Deiman et al.
2002; Polstra et al. 2002; Abd El Galil et al. 2005; Zanoli and Spoto 2013; Zhao et al.
2015), electrochemiluminescence (Simpkins et al. 2000; Deiman et al. 2002; Zanoli
and Spoto 2013; Zhao et al. 2015), and colorimetric analysis (Gill et al. 2006; Zhao
et al. 2015). Since its first report in 1991, NASBA was quickly adopted for pathogen
detection and diagnostics, especially for the detection of RNA viruses including
hepatitis C virus and human immunodeficiency virus (HIV) (Van Der Vliet et al.
1993; Vandamme et al. 1995; Hollingsworth et al. 1996; Craw and Balachandran
2012). In addition, the integration of NASBA into a lab-on-a-chip system after its
miniaturization without compromising the detection sensitivity has been reported for
novel pathogen detection (Gulliksen et al. 2004, 2005; Dimov et al. 2008; Craw and
Balachandran 2012).
4.2.3.3 Strand Displacement Amplification (SDA)

While NASBA exploited the retrovirus replication mechanism, the SDA technique
adopted the DNA replication process that takes place at a constant temperature
(Walker et al. 1992a, b; Zhao et al. 2015; Zeng et al. 2019). Since the development
of SDA technique in the early 1990s (Walker et al. 1992a, b), various modifications
have been added to the original SDA to improve its efficiency and the specificity of
DNA nick by endonucleases (Walker et al. 1992a; Mehrpouyan et al. 1997; Fang
et al. 2014; Zhang et al. 2016a, b; Wu et al. 2018; Zeng et al. 2019). SDA requires
two sets of primers, one set designed for both DNA nick and primer extension and
the other for primer extension which targets outside of the first primer-binding sites.
After heat denaturation of the template DNA, the first primer set that has an
endonuclease-recognition site at its 50 portion for DNA nick binds to the target
DNA and primes the synthesis of a new strand, producing double-stranded DNAs.
Then, the synthesized dsDNAs that have endonuclease recognition sites are
subjected to the cycles of DNA nick followed by DNA extension accompanied by
strand displacement of downstream DNA. The second primer set flanking the
binding sites of the first primer set produces new DNA strands of which dsDNA
product will then serve as a target for the first primer set to anneal, which will initiate
another round of the cycle of DNA nick and amplification of new DNA strands
(Walker et al. 1992a, b). The cycles of nicking DNA strand, primer extension, and
strand displacement enable the exponential amplification of single-stranded DNA
molecules complementary to the original target (Zhao et al. 2015). It has been shown
that SDA can be used for the detection of microRNAs for the purpose of disease
diagnosis (Shi et al. 2014).
4.2.3.4 Rolling Circle Amplification (RCA)

RCA technique utilizes DNA polymerases (e.g., Phi 29 DNA polymerase) that are
capable of continuous amplification and strand displacement of circular DNA
templates, resulting in the production of long single-stranded DNA molecules with

tandem repeats of the circular template (Nilsson et al. 1994; Walter and Strunk 1994;
Banér et al. 1998; Zanoli and Spoto 2013; Ali et al. 2014; Zhao et al. 2015). While
the linear amplification of nucleic acid by RCA is achieved by using a single primer
(Fire and Xu 1995; Liu et al. 1996; Zanoli and Spoto 2013), exponential RCA
method is done by including the second primer in the reaction which will hybridize
at multiple locations to the newly synthesized single-stranded DNA product with
tandem repeats of the circular target, initiating hyper-branched rolling circle DNA
amplification (Mothershed and Whitney 2006; Zanoli and Spoto 2013; Zhao et al.
2015). Since RCA requires small single-stranded DNA molecules as a template, long
dsDNA molecules, routinely obtained from biological samples, need to be converted
into small circular single-stranded DNAs for RCA either by padlock probes or by
primer-generation RCA (Nilsson et al. 1994; Banér et al. 1998; Murakami et al.
2009; Zanoli and Spoto 2013; Zhao et al. 2015). The padlock probe is composed of
two sequences, each of which is complementary to one-half of a single target region,
separated by a linker sequence (Nilsson et al. 1994; Banér et al. 1998). The
hybridization between the padlock probe and the target sequence followed by
DNA ligation circularizes the padlock probe forming a single-stranded circular
DNA which will be used as a template for RCA (Lizardi et al. 1998; Zanoli and
Spoto 2013; Zhao et al. 2015). The RCA mediated by the padlock probe is highly
specific to the target sequence, which is suited for SNP analysis (Lizardi et al. 1998;
Zanoli and Spoto 2013). Unlike padlock probe-mediated RCA, primer-generation
RCA only requires a single-stranded circularized probe that has a nucleotide
sequence complementary to the target and an endonuclease recognition site for a
DNA nick (Murakami et al. 2009). Once the circularized probe with a nick site
hybridizes to the single-stranded target, the linear RCA takes place producing a long
single-stranded DNA with tandem repeats of a sequence complementary to the
circular probe (Murakami et al. 2009). The single-stranded DNA product of linear
RCA will serve as a template molecule for the hybridization of multiple circular
probes and nicking reaction, resulting in the generation of primers for further RCA
(Murakami et al. 2009; Zanoli and Spoto 2013; Zhao et al. 2015). Primer-generation
RCA has been successfully used for a diagnostic purpose for the detection of
bacterial pathogens (Murakami et al. 2009).
4.2.3.5 Multiple Displacement Amplification (MDA)

Unlike SDA and other isothermal amplification methods that amplify specific target
nucleic acids, MDA and other whole genome amplification methods use random
primers enabling efficient amplification of template DNA for next-generation
sequencing (Dean et al. 2002; Zhao et al. 2015). Due to the nature of short random
primers (e.g., random hexamer), new DNA strand synthesis and strand displacement
are initiated at multiple locations on the template DNA which is mediated by Phi
29 DNA polymerase that has a strong strand displacement activity (Dean et al. 2002;
Zhao et al. 2015). It has shown that MDA can synthesize ~10 kb-long DNA
fragments in the amount of 20~30 μg from a very limited amount of starting
materials (Zhao et al. 2015). Although MDA can be directly used for various
70 M. Kunta et al.
types of biological samples including lysed cells without purification step, since the
amplification of DNA by MDA is nonspecific, any contaminated DNA in the sample
will be amplified, causing that only small fraction of the amplified MDA products
were derived from specific target molecules (Raghunathan et al. 2005; Zanoli and
Spoto 2013). This kind of amplification bias of MDA can be reduced by reducing the
reaction volume to the nanoliter level in microfluidic devices (Zhang and Xing 2010;
Zanoli and Spoto 2013).
4.2.3.6 Loop-Mediated Isothermal Amplification (LAMP)

LAMP, developed by Notomi et al. (2000), uses DNA polymerases with strand
displacement activity and two to three different primer sets (outer and inner primer
sets and/or loop primers), each of which targets specific nucleotide sequence present
on the sample in a single reaction, providing greatly improved specificity compared
to other isothermal amplification methods (Notomi et al. 2000; Zanoli and Spoto
2013; Zhao et al. 2015). The two inner primers, forward inner primer (FIP) and
backward inner primer (BIP), are comprised of two different sequences complemen-
tary to sense and antisense strands of target DNA segments, of which LAMP
products will form a loop structure at its each end that will provide annealing sites
for both inner primers during LAMP reaction (Notomi et al. 2000). The outer
primers, F3 (Forward) and B3 (Backward) flanking the two inner primer-binding
sites, synthesize a new DNA strand by strand displacement activity of the DNA
polymerase following the first LAMP reaction initiated by two inner primers
(Notomi et al. 2000). The new strands synthesized by strand displacement from
outer primers are then served as templates for the LAMP reaction with two inner
primers which will produce new single-stranded DNA molecules with loop
structures that subsequently enters the cycling LAMP reaction mediated by two
inner primers, resulting in geometrically and continuously branched out LAMP
products from each single-stranded LAMP product, which can accumulate 109
copies of target molecule within an hour (Notomi et al. 2000; Zanoli and Spoto
2013; Zhao et al. 2015). The final LAMP products can be detected in real-time and
by end-point assays (Xie et al. 2014; Yi et al. 2014; Zhang et al. 2014a, b). LAMP
has been widely adopted for the diagnostic purpose for the detection of pathogens as
well as for SNP analysis in various fields either with or without reverse transcription
step depending on the biochemical status of target molecules (Thi et al. 2004;
Ohtsuka et al. 2005; Misawa et al. 2007; Curtis et al. 2008; Mori and Notomi
2009; Zanoli and Spoto 2013; Keremane et al. 2015; Choi et al. 2018).
4.2.3.7 Helicase-Dependent Amplification (HDA)

HDA exploited DNA helicase-mediated DNA replication process together with
other single-strand-binding proteins and DNA polymerases in the reaction, which
can amplify long target DNA molecules (Vincent et al. 2004; Zanoli and Spoto
2013; Zhao et al. 2015). The original HDA, developed with E. coli UvrD helicase,
was replaced with thermostable UvrD helicase derived from thermophilic bacteria to
improve the specificity and efficiency of HDA and to amplify longer DNA targets
(Mechanic et al. 2000; Vincent et al. 2004; An et al. 2005; Matson and Robertson
2006; Zanoli and Spoto 2013). The addition of helicase in the target DNA fraction
mediates unwinding of dsDNA that generates two single-stranded DNA molecules
which are accessible for two sequence-specific primers for new strand synthesis by
DNA polymerase resulting in dsDNA formation. The newly synthesized dsDNA
serves as substrates for DNA helicases in the following HDA reactions, resulting in
an exponential amplification of the target DNA molecules (Vincent et al. 2004;
Zanoli and Spoto 2013). HDA has been successfully used for the detection of
bacterial and viral pathogens as well as SNP detection (An et al. 2005; Motré et al.
2008; Li et al. 2011a, b; Zanoli and Spoto 2013; Chen et al. 2015; Zhao et al. 2015).
4.2.3.8 Recombinase Polymerase Amplification (RPA)

RPA uses recombinase that interacts with primers forming a nucleoprotein complex
which will be directed to the target sequences on the dsDNA molecules for primer-
template DNA hybridization leading to a new strand synthesis accompanied by a
strand displacement (Piepenburg et al. 2006; Zanoli and Spoto 2013; Zhao et al.
2015). In RPA, the nucleoprotein complex formed by primer and recombinase scans
the dsDNA to locate the primer target sequence, then displaces the non-template
strand which will be stabilized by single-strand-binding proteins while the primer
extension takes place by DNA polymerase (Piepenburg et al. 2006; Zanoli and Spoto
2013). By incorporating two primers (forward and reverse primers), the primer
extension events of each primer will generate a complete target amplicon which
will be used as a template for continuous RPA enabling an exponential amplification
of the target (Piepenburg et al. 2006; Zanoli and Spoto 2013; Zhao et al. 2015).
TwistDx (Cambridge, UK) commercialized RPA kits for the detection of various
pathogens (Shen et al. 2011; Crannell et al. 2014; Rohrman and Richards-Kortum
2015; Zhao et al. 2015).
4.2.4 Real-Time Polymerase Chain Reaction (RT-PCR)
The discovery of a thermostable DNA polymerase, Taq DNA polymerase I

originating from a thermophilic bacteria, Thermus aquaticus, followed by its adop-
tion in PCR made the PCR a major tool for the amplification of target nucleic acid
molecules from various source materials (Chien et al. 1976; Saiki et al. 1988; Kralik
and Ricchi 2017). Shortly after the utilization of a thermostable DNA polymerase for
PCR, the concept of “real-time” monitoring of DNA amplification was probed using
either a hydrolysis probe paired with 50 nuclease activity of Taq DNA polymerase or
an intercalating DNA-binding dye (Holland et al. 1991; Higuchi et al. 1992; Navarro
et al. 2015; Kralik and Ricchi 2017). The commercialization of real-time PCR
instruments from various vendors, which has a dual function of thermocycling and
fluorescence detection after each PCR cycle, made the real-time PCR technique
quickly widespread and then resulted in the development of various real-time PCR
protocols for diagnostic purposes, quantification of gene expression, and mutation
detection (Navarro et al. 2015; Kralik and Ricchi 2017). The real-time PCR
techniques can be categorized into two major groups based on the detection
72 M. Kunta et al.
chemistry of amplicons (Navarro et al. 2015). The first group utilizes intercalating
fluorescent dyes and the second group uses fluorophore-labeled oligonucleotides
(Navarro et al. 2015). Unlike the intercalating dye-based methods, the latter group
adopted various types or structures of fluorophore-labeled oligos for the detection of
amplicons with improved specificity (Navarro et al. 2015).
4.2.4.1 Real-Time PCR with Intercalating Fluorescent Dyes

The applicability of DNA-binding dyes for real-time detection of the amplicon was
first proved by Higuchi et al. (1992) using ethidium bromide. Among those
DNA-binding dyes used for real-time PCR (e.g., SYBR Green I, SYTO9, BEBO,
etc.), SYBR Green I is the most widely used fluorescent dye, which is often included
in a commercial master mix for real-time PCR assays (Ririe et al. 1997; Wittwer
et al. 1997). These dyes emit fluorescence when they bind to the minor groove of
double-stranded DNA molecules, which is then measured during the course of the
PCR. As the DNA-binding dyes preferentially bind to double-stranded DNA
molecules, both specific and nonspecific amplicons as well as, if any, primer-dimers
can be detected in the real-time PCR assay. To verify the specificity of the real-time
PCR assay with DNA-binding dyes, a melt-curve analysis must be conducted at the
end of the PCR (Navarro et al. 2015). Since the melting temperature of PCR products
in the reaction is determined by their length and nucleotide composition, the
specificity of the real-time PCR assay can be easily evaluated by melt-curve analysis
(Ririe et al. 1997). Although SYBR Green I was proven to work for real-time PCR, it
also has some drawbacks such as dye instability, PCR inhibition at high dye
concentration and dye-redistribution, which made SYBR Green I unsuitable for
high-resolution melt curve analysis (HRM) (Wittwer et al. 1997; Nath et al. 2000;
Monis et al. 2005; Varga and James 2006; Mao et al. 2007). EvaGreen developed by
Biotium Inc. (Hayward, CA, USA) has some advantage over SYBR Green I in terms
of dye stability and less PCR inhibitory effect compared to SYBR Green I which
made EvaGreen suitable for real-time PCR as well as HRM (Ihrig et al. 2006; Wang
et al. 2006; Mao et al. 2007). Despite the drawbacks of SYBR Green I, various real-
time PCR assays using SYBR Green I as well as EvaGreen were successfully used
for the detection of pathogen and genetic variability (e.g., SNP) (Buh Gašparič et al.
2010; Li et al. 2010; Carrasco et al. 2013; He et al. 2014; Navarro et al. 2015).
4.2.4.2 Real-Time PCR with Fluorophore-Labeled Oligonucleotides

Fluorescent molecules, fluorophores, absorb energy from the light of a certain
wavelength and emit the energy (donor fluorophore) as a form of light of a longer
wavelength that can excite or be quenched by another fluorophore (acceptor
fluorophore) nearby via fluorescence resonance energy transfer (FRET) (Cardullo
et al. 1988; Mergny et al. 1994; Howell 2006). Real-time PCR adopts these two
different FRET mechanisms for the detection of amplicons in the reaction mixture:
(i) FRET-quenching by acceptor (e.g., TaqMan, Scorpion, Molecular Beacon, etc.)
or (ii) FRET leading to fluorescence emission from acceptor (e.g., Hybprobes,
Angler®, etc.) (Mergny et al. 1994; Marras 2006; Navarro et al. 2015).
I. FRET-quenching Oligos
(i) Hydrolysis Probe (TaqMan Probe)
Hydrolysis probes are modified, non-extendible oligonucleotides which are

designed to hybridize to a specific sequence within the real-time PCR amplicon
(Holland et al. 1991; Gibson et al. 1996; Heid et al. 1996). The hydrolysis probe has
donor and acceptor fluorophores at the 50 and 30 ends, respectively. The fluorescence
of the donor fluorophore in the probe is quenched by the acceptor fluorophore
(quencher) due to the physical proximity of both fluorophores (Holland et al.
1991; Gibson et al. 1996; Navarro et al. 2015). The binding of the hydrolysis
probe to the target region during real-time PCR leads to the release of donor
fluorophore from 50 end of the probe due to the 50 exonuclease activity of Taq
DNA polymerase, which results in the increase of the fluorescence that will be
monitored after each PCR cycle (Gibson et al. 1996; Heid et al. 1996). Real-time
PCR using hydrolysis probes can be multiplexed by using fluorophores with differ-
ent excitation and emission wavelengths and has been routinely applied in diverse
applications such as pathogen detection, quantification of gene expression, and SNP
detection (Navarro et al. 2015).
(ii) Scorpion Probe
Scorpion probe has a stem-loop (hairpin) structure that functions as a PCR primer
as well as a probe (Whitcombe et al. 1999). The scorpion probe is composed of three
major functional units: (i) fluorophores (a donor fluorophore at the 50 end and an
internal quencher), (ii) a loop working as a probe, which is flanked with a
fluorophore and a quencher as well as complementary sequences leading to the
formation of a stem structure followed by (iii) an oligonucleotide sequence at its 30
region which will act as a PCR primer (Whitcombe et al. 1999). Like the hydrolysis
probe, the physical proximity between the fluorophore and the quencher results in
the suppression of the fluorescence in the scorpion probe. During real-time PCR, the
primer sequence present at the 30 region of the scorpion probe initiates a DNA
synthesis complementary to the target DNA, of which product will have a copy of
the nucleotide sequence of the scorpion probe at the 50 end. The denaturation step of
the following PCR cycle will denature the scorpion probe sequence and allows the
probe sequence residing on the loop portion to bind to the target sequence of the
same newly synthesized product resulting in the increase of fluorescence due to a
lack of quenching mechanism (Whitcombe et al. 1999; Navarro et al. 2015). Due to
the structural feature (a hairpin) of the scorpion probe, nonspecific amplification and
the formation of primer-dimers can be minimized during PCR cycles (Nazarenko
et al. 2002; Navarro et al. 2015). Scorpion probes can be multiplexed and are used
for the detection of pathogens and genetic variability (e.g., SNPs, etc.) (Solinas et al.
2001; Naserpour Farivar et al. 2014; Zhang et al. 2014a, b; Navarro et al. 2015).
74 M. Kunta et al.
(iii) Molecular Beacon Probe
Molecular beacon probe has the feature of both hydrolysis probe and scorpion
probe in terms of the structure and mode of action. It has a hairpin structure where a
fluorophore and a quencher are located at the 50 and 30 end of the probe, respectively,
and like a hydrolysis probe, it contains a probe sequence on the loop (Tyagi and
Kramer 1996). The native hairpin structure of the molecular beacon probe, placing a
fluorophore and a quencher in close proximity, suppresses the fluorescence from the
molecular beacon probe (Tyagi and Kramer 1996). The hybridization of the probe to
the target DNA region takes place at the annealing step of PCR resulting in the
increase of fluorescence (Tyagi and Kramer 1996). The molecular beacon probe has
higher specificity due to its structural specificity than other hybridization probes and
can discriminate target sequences with single nucleotide variation (Bonnet et al.
1999; Navarro et al. 2015).
II. FRET-mediated Fluorescence Probe
(i) FRET Probe (Hybprobe)
FRET probe is consisted of two oligonucleotides, which are specifically designed

to bind two adjacent target sequences in the amplicon (Heller and Morrison 1985;
Navarro et al. 2015). The first oligo has a donor fluorophore at its 30 end, and the
second oligo carries a second fluorophore at its 50 end and a phosphate group at its 30
end preventing the primer extension (Cardullo et al. 1988; Morrison et al. 1989;
Navarro et al. 2015). These two oligos hybridize to the adjacent two target sequences
on the amplicon at the annealing step during PCR, placing the two fluorophores next
to each other. The second fluorophore emits the fluorescence by the FRET mecha-
nism from the first fluorophore (Bernard and Wittwer 2000; Navarro et al. 2015).
Real-time PCR with FRET probe can be multiplexed and was used for the detection
of pathogen and genotyping (Liew et al. 2006; Lim et al. 2008; Navarro et al. 2015).
(ii) Angler® and ResonSense® Probes
Angler®probe is composed of two nucleotide sequences, a reverse primer and a

probe sequence identical to the target region adjacent to the reverse primer-binding
site, which were connected by a linker (Lee et al. 2002; Navarro et al. 2015). The
real-time PCR with Angler®probe uses SYBR Gold as a donor fluorophore. The 50
end of Angler®probe has a fluorophore which emits its fluorescence when the
reverse primer portion of Angler®probe initiates a synthesis of a complementary
strand of the target DNA region. During the denaturation step of the following PCR
cycle, the probe sequence of Angler®probe binds to the complementary sequence of
the same strand that leads to the formation of double-stranded DNA to which the
intercalating fluorescent dye, SYBR Gold included in the reaction mix, will bind and
act as a donor fluorophore for the acceptor fluorophore residing at the 50 end of
Angler®probe which will emit fluorescence via FRET derived from SYBR Gold
(Lee et al. 2002). The real-time PCR with Angler®probe together with an
intercalating dye can distinguish nonspecific amplicon (SYBR Gold) and specific
amplicon (Angler®probe) without melt-curve analysis (Lee et al. 2002; Naserpour
Farivar et al. 2014).
ResonSense® probe functions as a primer that has an acceptor fluorophore at its
50 end, but unlike Angler® probe, ResonSense® probe itself acts as a primer and a
probe. The hybridization of ResonSense® probe to the target sequence will form a
double-strand DNA where SYBR Gold will bind and act as a donor fluorophore for
FRET leading to the emission of fluorescence from ResonSense® probe. Both
Angler® and ResonSense® probes were successfully used for the detection of
pathogen, mutation, and gene expression (Lee et al. 2002; Punia et al. 2004;
Sanchez et al. 2006; Naserpour Farivar et al. 2014; Navarro et al. 2015).
In addition to the real-time PCR methods described above, there are other
amplicon detection methods available for real-time PCR (Navarro et al. 2015).
The review paper published by Navarro et al. (2015) and the papers cited in the
review described details of these methods, some of which methods share a similar
molecular structure or add a molecular ligand for improved specificity (e.g., hairpin
structure in Scorpion and molecular beacon probes; MGB (minor groove-binding
ligand)) for the amplicon detection or adopt a new approach shown in Cyclicon,
Amplifluor®, Yin-Yang and Snake assays (Tyagi and Kramer 1996; Whitcombe
et al. 1999; Kandimalla and Agrawal 2000; Kutyavin et al. 2000; Li et al. 2002;
Nazarenko et al. 2002; Lukhtanov et al. 2007; Kutyavin 2010; Navarro et al. 2015).
4.3 Methods Under Active Development
4.3.1 Programmable Nucleic Acid-Binding Proteins
Advances in molecular biology and interest in the fundamental organization of

eukaryotic chromosomes led to the identification of nucleic acid-binding proteins
that bind to specific nucleotide sequences (Miller et al. 1985). Biochemical charac-
terization of these proteins led to the conclusion that alterations in amino acid
sequence could be used to alter their affinity for unique nucleotide sequences
(Berg 1988; Choo and Klug 1994a, b) and subsequent targeting of genetic
differences (Choo et al. 1994). Further development led to the use of zinc finger
(ZnF) proteins in diagnostics while exploration, in other fields, led to the discovery
of transcription activator-like effector (TALE) proteins and clustered regularly
interspaced palindromic repeat (CRISPR)-associated (CAS) proteins. These proteins
have all been adapted for use in diagnostics and, in the case of CRISPR-Cas in
particular, represent active areas of research. The diagnostic approaches proposed for
each of these protein classes are diverse and, below, we present a comprehensive
summary of each method organized by protein class (Table 4.1). Each approach
shows substantial promise, but their use, to date, has been limited.
76
Table 4.1 Characteristics of diagnostics systems based on programmable nucleic acid binding proteins
Signal
Type System name Effector Target amplification Readout Sensitivity References
ZFP SEER Fused ZFP-GFP DNA – Fluorescence 4 μM Stains et al. (2005)
SEER-LAC Fused DNA – Colorimetric 5 nM Ooi et al. (2006) and Kim
ZFP-β-lactamase et al. (2011)
– ZFP-GST/anti-GST DNA PCR Chemiluminescence/ 10 copies Osawa et al. (2008, 2009)
antibody ELISA
– ZFP/ZFP-luciferase DNA PCR Bioluminescence 10 copies; Abe et al. (2012), Shi et al.
62 pM (2017), and Takano et al.
(2017)
– ZFP/ZFP-biotin DNA – Chemiluminescence 0.5 nM Kim and Kim (2016)
– ZFP/ZFP-glucose DNA PCR Electrochemistry 10 copies Lee et al. (2017)
dehydrogenase
TALE – TALE/ DNA PCR Chemiluminescence 1.66 mM Honarmand et al. (2014)
TALE-β-lactamasee
CRISPR NASBACC Cas9 RNA NASBA Colorimetric 1 fM Pardee et al. (2016)
PC reporter dCas9-luciferase DNA PCR Bioluminescence 1 copy/ Zhang et al. (2017)
500 μL
– dCas9 DNA – Fluorescence 1 CFU/ Guk et al. (2017)
mL
ctPCR Cas9 DNA PCR Electrophoresis/ 5 ng of Wang et al. (2018)
Fluorescence amplicon
CAS-EXPAR Cas9 DNA/ EXPAR Fluorescence aM Huang et al. (2018)
RNA
RCH dCas9 microRNA RCA Colorimetric 35 aM Qiu et al. (2018)
CARP Cas9 DNA PCR Electrophoresis/ 2 pg of Zhang et al. (2018)
Fluorescence amplicon
SHERLOCK Cas13a DNA/ RPA Fluorescence 2 aM Gootenberg et al. (2017)
RNA
M. Kunta et al.
4
SHERLOCK CcaCas13b, DNA/ RPA Fluorescence; 8 zM Gootenberg et al. (2018)

V2 PsmCas13b, RNA Colorimetric
LwaCas13a
DETECTR Cas12a DNA RPA Fluorescence aM Chen et al. (2018)
HOLMES Cas12a DNA/ PCR/RT-PCR Fluorescence 1–10 aM Li et al. (2018)
RNA
HOLMES V2 Cas12b DNA/ LAMP; Fluorescence 10 aM Li et al. (2019)
RNA RT-LAMP;
Asymmetric PCR
Cas14- Cas14 ssDNA RPA Fluorescence aM Aquino-Jarquin (2019)
DETECTR
Modern Tools for Detection and Diagnosis of Plant Pathogens
77
78 M. Kunta et al.
4.3.1.1 Zinc Finger Proteins

Comprising a highly diverse protein family, zinc finger (ZnF) proteins share a motif
incorporating at least one zinc ion, which stabilizes their secondary structure. The
domains formed typically involve molecular interactions such as binding DNA,
RNA, proteins, or small molecules (Laity et al. 2001). Indeed, this specificity
in binding particular DNA sequences first propelled these proteins to prominence
in biotechnology. ZnFs were the first proteins engineered for gene editing, resulting
in a diverse set of proteins capable of targeting desired genomic regions (Urnov et al.
2010; Perez-Pinera et al. 2012).
Stains et al. (2005) first took advantage of the sequence-specific binding of ZnFs
for diagnostics. Building upon the availability of ZnFs to target desired sequences of
DNA, they developed the sequence-enabled reassembly of proteins (SEER)
approach. In order to prepare a fluorescent reporter for a desired DNA sequence,
they split the green fluorescent protein (GFP), thereby removing its chromophore
function, and fused each half to one of two ZnFs designed to specifically bind to
unique DNA sequences. In the presence of the target DNA sequence, each ZnF binds
to its respective nucleic acid sequence, the GFP halves are brought into proximity
allowing reassembly, which catalyzes the chromophore formation producing a
detectable fluorescent signal.
Ooi et al. (2006) subsequently improved the sensitivity of the SEER system by
assessing the impact of distance (in nucleotides) between ZnF-binding sites. Simi-
larly, they improved specificity by altering the length of the amino acid linker
between the ZnFs and the signal domains. Finally, they converted the system to a
colorimetric reporter by exchanging GFP for β-lactamase.
Subsequent work expanded on these methods by introducing new signaling
systems, improving sensitivity, and removing the need for DNA amplification
(Table 4.1).
4.3.1.2 Transcription Activator-Like Effector Proteins

Transcription activator-like effector (TALE) proteins are a class of DNA-binding
proteins secreted by Xanthamonas sp. bacteria to manipulate host plant gene expres-
sion to aid colonization (Boch and Bonas 2010; Bogdanove et al. 2010). Unlike
ZnFs, which target three nucleotide base arrays for binding, TALEs target single
nucleotides providing a much more modular system (Sanjana et al. 2012; Sun and
Zhao 2013). As a result, TALEs can theoretically target any sequence and show
reduced off-target DNA-binding capabilities (Zischewski et al. 2017; Nerys-Junior
et al. 2018).
Similar to ZnFs, TALE domains can be fused with other protein domains to
confer novel function (Li et al. 2011a, b). This approach was taken by Honarmand
et al. (2014) in the development of the first TALE-based detection system. By
constructing two TALE arrays to bind uniquely to the target DNA, one could be
immobilized on a nitrocellulose substrate, while the second was fused with a reporter
protein. Upon addition of the test DNA, the nitrocellulose-bound TALE captures
those DNA strands that present the appropriate sequence. If the target DNA carries
the second TALE recognition sequence, the TALE-reporter construct binds. The
presence of both TALE recognition sequences immobilizes the TALE-reporter

construct with the target DNA acting as a linker. Immobilization allows visualization
and detection.
TALE approaches differ from ZnFs and CRISPR systems (see below) in several
ways that benefit diagnosticians. TALE proteins are more stable than ZnF proteins,
smaller than Cas proteins, do not require neighboring protospacer adjacent motif
(PAM) sites for sequence recognition, and can target RNA-DNA hybrids (Yin et al.
2012; Pattanayak et al. 2013). While these advantages of TALE-based diagnostic
systems warrant further attention, no other TALE-based diagnostic systems have
been developed. These useful proteins apparently have lost the attention of
researchers drawn to the newer CRISPR systems (Batista and Pacheco 2018).
4.3.1.3 Clustered Regularly Interspaced Short Palindromic

Repeat-Associated Proteins
Clustered regularly interspaced short palindromic repeats (CRISPR) are genomic
elements that retain a genetic memory with Lamarckian-style inheritance for many
lineages of bacteria and archea. Together with CRISPR-associated (Cas) proteins,
this genetic system forms an adaptive immune system that acquires sequences from
foreign genomes and recalls this information for sequence-specific recognition and
cleavage by Cas endonucleases (Mojica et al. 2005; Barrangou et al. 2007). With the
development of engineered Cas9 endonucleases for genome editing, these systems
have experienced intense research interest (Barrangou and Doudna 2016). CRISPR-
associated proteins (Cas) and single-guide RNAs (sgRNA) have been adapted to
function in sequence-specific genomic and epigenetic editing (Wang et al. 2016;
Kungulovski and Jeltsch 2016), transcriptional regulation (Didovyk et al. 2016),
loss-of-function screening (Shalem et al. 2015; Wade 2015), characterization of
enhancer and regulatory sequences (Rajagopal et al. 2016; Lopes et al. 2016),
visualization of DNA (Ma et al. 2016), creation of gene drives (Champer et al.
2016), antimicrobial and antiviral development (Fagen et al. 2017), suppression of
the uptake of antibiotic resistance genes (Garneau et al. 2010), and in data storage
(Shipman et al. 2016).
The excitement over CRISPR-Cas systems likely stems from the ease of design-
ing and producing sgRNAs to target-specific sequences. This excitement has led to
the exploration of CRISPR systems across taxa which has revealed substantial
diversity (Luo et al. 2016; Koonin et al. 2017) only beginning to be exploited to
advance diagnostics. To date, four Cas endonucleases have been utilized for
diagnostics: Cas9, Cas13 (formerly known as C2c2), Cas12 (formerly known as
Cpf1), and Cas14.
I. Cas9-based Systems
Pardee et al. (2016) pioneered the use of RNA-guided endonucleases for

diagnostics. Using the nucleic acid sequence-based amplification method
(NASBA; Compton 1991) to initially amplify the target RNA, they employed
CRISPR-Cas9 as a sequence-specific nuclease to identify strain-specific sequences
80 M. Kunta et al.
and toehold switches to control signal expression in a cell-free, paper-based system

to detect the RNA genome of Zika virus and distinguish between strains of the virus.
Specifically, they reverse transcribed the RNA target to DNA using a sequence-
specific reverse primer to produce an RNA/DNA duplex. They degraded the RNA
strand allowing a forward primer containing a T7 promoter to bind and initiate
synthesis of second-strand DNA. The T7 promoter within the newly created double-
stranded DNA allowed T7 RNA polymerase to amplify the RNA template (Deiman
et al. 2002; Zhao et al. 2015). Due to NASBA primer design, the RNA product
contained the trigger sequence of a toehold switch.
Toehold switches are synthetic riboregulator RNAs that contain hairpin structures
that block a ribosome-binding site until the switch is bound to a complementary
trigger RNA. In this system, complementary binding of the trigger RNA to the
toehold switch drives conformational changes that reveal a ribosomal-binding site
and initiate translation of a reporter gene. Pardee et al. utilized CRISPR-Cas9 to
discriminate between strains of Zika virus, at the single nucleotide level, by
identifying a target region in one strain containing a PAM sequence required for
Cas9 to initiate cleavage. In the presence of the appropriate PAM, Cas9 destroys the
trigger sequence which can no longer cause conformational changes in the toehold
switch so reporter gene expression remains suppressed. In the absence of the
appropriate PAM, the full trigger sequence is produced and results in reporter gene
expression. Careful choice of target region and design of NASBA reverse primers
allowed Pardee et al. to control signal production and distinguish strains of Zika
virus.
Others have used CRISPR-Cas9 to recognize and generate the amplified signal.
Huang et al. (2018) used a sequence-specific sgRNA along with an antisense
PAM-presenting oligonucleotide (PAMmer) to nick single-stranded target DNA
and generate the fragment for amplification and detection. The fragment, paired
with an oligonucleotide designed for exponential amplification reaction (EXPAR;
Van Ness et al. 2003), primes DNA polymerase to synthesize the remaining EXPAR
template (designed as a copy of the target fragment). Nicking allows this fragment to
be displaced by DNA polymerase and bind with a new EXPAR template initiating
isothermal amplification (Zhang et al. 2016a, b). SYBR Green is used to detect this
amplified double-stranded DNA product.
Zhang et al. (2018) similarly developed a Cas9/sgRNAs-associated reverse PCR
(CARP) diagnostic tool using two sgRNAs to cleave genomic DNA into a target
fragment. However, they employed T4 DNA ligase to concatenate or circularize the
resulting fragment. Adding reverse primers designed to initiate exponential amplifi-
cation of the target fragment in its concatenated or circularized form rather than its
original orientation allows the production of double-stranded DNA capable of being
visualized on agarose gels or with SYBR Green.
Wang et al. (2018) developed CRISPR-typing PCR (ctPCR) to detect and identify
human papillomavirus (HPV) subtypes. The first step relies on traditional PCR with
universal primers to amplify a target region associated with the virus. Amplicons
with sequences matching a pair of specific sgRNAs are cleaved by Cas9, incubated
with Taq polymerase to induce A-tailing, and ligated to a T adaptor. A second round
of PCR using primers matching the T adaptor produced specific fragments for
visualization.
Other approaches take advantage of dead Cas9 (dCas9), a mutated form of the
Cas9 protein that lacks the characteristic endonuclease activity of the native version.
Zhang et al. (2017) utilized dCas9 as a protein reassembly reporter system similar to
those used with Znfs and TALE. Designing sgRNAs to target neighboring
sequences, they reassembled split halves of the bioluminescent enzyme, luciferase,
bound to dCas9 proteins in a system they called PC reporter (Zhang et al. 2014a, b).
A similar approach was later used by Qiu et al. (2018) to detect microRNAs
amplified by rolling circle amplification and signaled by reassembly of split halves
of horseradish peroxidase bound to dCas9 proteins. A different approach using
dCas9 is based on fluorescent in situ hybridization (FISH; Guk et al. 2017). Using
magnetic bead-labeled dCas9 and a sequence-specific sgRNA, Guk et al. captured
and physically separated target DNA for visualization using SYBR Green without
the need for amplification.
II. Cas13-based Systems
Unlike the well-known Cas9 RNA-guided deoxyribonuclease (DNase), Cas13a

(previously called C2c2; Shmakov et al. 2015) is an RNA-guided ribonuclease
(Abudayyeh et al. 2016). In addition to RNA-guided cleavage, binding with the
target RNA also activates a nonspecific ribonuclease activity (Abudayyeh et al.
2016). This “collateral” cleavage was used by Gootenberg et al. (2017) to develop
the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) diagnostic
system. This system accepts test material in the form of double-stranded DNA or
RNA for amplification by recombinase polymerase amplification (RPA) or reverse
transcriptase-RPA (RT-RPA), respectively (Piepenburg et al. 2006). The DNA
product is then transcribed to RNA using T7 RNA polymerase and exposed to
Cas13a. If the test sample contains the target site, Cas13a binds and initiates
nonspecific “collateral” cleavage of single-stranded RNAs. By providing reporter
RNAs labeled with a fluorophore at one end and a quencher at the other, the
nonspecific “collateral” ribonuclease activity of Cas13a releases the fluorophore
from its quencher only in the presence of the target RNA.
Two updates to the SHERLOCK method have been published. One, heating
unextracted diagnostic samples to obliterate nucleases (HUDSON), focused on
preparing samples for analysis to facilitate SHERLOCK deployment in the field
(Myhrvold et al. 2018). The second extended the functionality and specificity of
SHERLOCK. Aiming to extend their diagnostic method to simultaneously detect
multiple targets, Gootenberg et al. (2018) characterized the cleavage preferences of
Cas13a and Cas13b orthologs. These orthologs, paired with Cas12a (see below;
Chen et al. 2018), allowed Gootenberg et al. to detect four targets in a single reaction.
By altering the RPA primer concentrations and reaction volumes, they achieved the
detection of as little as 8 zM target concentrations. By incorporating Csm6, a
CRISPR type III nuclease that is activated by Cas13 collateral cleavage products,
and a unique reporter molecule, they were able to amplify the signal strength for
82 M. Kunta et al.
reporting. Finally, they developed a lateral flow readout system by replacing the
fluorophore quencher with biotin. Samples added to the test strip first acquire a
nanoparticle-bound anti-fluorophore antibody, then a streptavidin band captures the
biotin bound reporters and only those cleaved products accumulate at the antibody
capture line. Combined with HUDSON, this method offers a sensitive, multiplexed,
field-deployable diagnostic system.
III. Cas12-based Systems
While Cas13 systems can be converted to work with DNA target molecules
through reverse transcription, Cas12 is a DNase with collateral activity that does
not require an additional step for DNA targets. As such, SHERLOCK-like diagnos-
tic systems have emerged since the characterization of Cas12 proteins. One-hour
low-cost multipurpose highly efficient system (HOLMES; Li et al. 2018) and DNA
endonuclease-targeted CRISPR trans reporter (DETECTR; Chen et al. 2018) work
on a similar framework to SHERLOCK. With target amplification, by PCR or RPA
(HOLMES and DETECTR, respectively), each uses collateral cleavage of single-
stranded DNA reporters capped with fluorophores and quenchers.
These approaches were expanded upon by Wang et al. 2019 and Li et al. 2019 to
incorporate amplification and signal production in a single tube to improve ease of
use and avoid cross-contamination. The Cas12a-based visual detection
(Cas12aVDet) utilizes RPA, a portable heater, and a blue light for visualization
(Wang et al. 2019). HOLMES version 2 (Li et al. 2019) used loop-mediated
isothermal amplification (LAMP; Notomi et al. 2000) for single tube testing and
expanded the method for single nucleotide polymorphism (SNP) and RNA detection
as well as quantitating DNA methylation.
IV. Cas14-based Systems
Through the use of metagenomic sequencing of uncultivated archea, Harrington

et al. (2018) discovered a new Cas endonuclease, Cas14. Substantially smaller than
other Cas endonucleases, this protein is an RNA-guided DNA endonuclease that,
unlike other DNA-targeting endonucleases, selectively targets single-stranded DNA.
Like Cas13 and Cas12, nonspecific collateral cleavage is activated in Cas14 by
binding with the target DNA. However, unlike other Cas endonucleases, Cas14 does
not require a PAM site.
Harrington et al. (2018) proposed that Cas14a could be used to modify the
DETECTR platform. As proof of principle, they used Cas14-DETECTR to detect
an SNP in the human HERC2 gene which is responsible for eye color. This
diagnostic system will be useful to detect single nucleotide polymorphisms (SNPs)
and single-stranded DNA viruses.
4.3.2 Optical Biosensors
Optical biosensor technologies for plant disease detection focus on identifying

changes in the host plant properties due to biotic or abiotic stresses, by determining
unique spectral profiles which correlate with these conditions (Martinelli et al.
2015). Generally speaking, plant diseases will modify the physiological state of
plant leaves, and any modification to a leaf constituent will correspondingly modify
the spectral properties of the leaves by affecting its light scattering and absorption
characteristics (Mahlein 2016). Optical technologies therefore rely on light
interrogation of a diseased plant to measure and then translate the resulting reflected
light into optical spectra, which can be analyzed for unique spectral biomarkers
associated with a particular disease state.
In this way, optical techniques for disease detection can be classified as indirect,
in comparison to direct molecular or serological identifiers of pathogens such as
PCR and ELISA based approaches (Fang and Ramasamy 2015; Martinelli et al.
2015). Other indirect technologies exist, such as chemical analyzers that detect the
release of new gaseous plant volatiles (Schmelz et al. 2003; McCartney et al. 2016)
or nuclear magnetic resonance spectroscopy-based detection of changes to plant
metabolite profiles (López-Gresa et al. 2010; Freitas et al. 2015). By correlating host
response to pathogen presence, indirect approaches are not necessarily reliant on the
level of pathogen in a collected sample to determine whether the plant is diseased.
Therefore, they provide the capability to overcome sampling issues associated with
direct approaches due to uneven, low-level pathogen distribution in the host and can
provide a more efficient means for determining disease state, especially in the early
stages of development.
Optical technologies offer some additional advantages for plant disease detection.
They can noninvasively interrogate plants (Behmann et al. 2015), which in many
cases completely alleviates any requirement for sample collection. Further, spectral
imaging-based solutions including hyperspectral remote sensing-based approaches
provide a rapid assessment of large-area vegetation or crops, improving the odds for
identifying disease in a timely manner. However, the sensitivity of spectral-imaging
systems varies based on the desired spatial resolution. Aerial or drone-mounted
hyperspectral systems provide very large area coverage, but have limited spatial
resolution often resulting in entire plant or canopy averaging, limiting the sensitivity
for detection of small-scale, leaf-level physiological changes.
Optical techniques utilize several spectroscopy approaches to identify the spectral
biomarkers associated with plant disease. Primarily, these include visible and
near-infrared (VIS-NIR) spectroscopy, fluorescence spectroscopy, and Raman spec-
troscopy techniques. VIS-NIR spectroscopy technologies focus on measuring the
reflection of light from a leaf, which is affected by absorption due to leaf pigments
and scattering due to heterogeneous cellular and intercellular structures. The visible
spectrum (~ 400–700 nm) is dominated by pigment absorption, and in most plant
leaves chlorophyll is the main absorber, including chlorophyll a and b, which absorb
significantly in the blue and red spectral regions (Peñuelas and Filella 1998). There
84 M. Kunta et al.
are other pigments that also contribute to the visible absorption and include xantho-
phyll, carotene, and anthocyanin (Christensen 2004).
Fluorescence detection is also used to measure the photosynthetic activity of
chlorophyll, both through natural light stimulus (Berdugo et al. 2014) and laser-
induced approaches (Belasque et al. 2008). Since chlorophyll function presents as an
easily accessible optical biomarker to analyze for a disease state, it is utilized
extensively in optical plant disease detection (Mahlein 2016). However, chlorophyll
function is also affected by other plant stressors such as nutrient deficiency, and
often needs to be measured in conjunction with other biomarkers to distinguish
between other plant stress states (Lowe et al. 2017; Zarco-Tejada et al. 2018).
In the near-infrared spectrum, the absorption of pigments is not significant, and
leads to relatively flat reflectance from ~ 700 to 1200 nm. The near-infrared is mainly
dominated by scattering due to inhomogeneous refractive index distribution arising
from cellular and intercellular structures such as the cellulose to air interfaces
(Mahlein 2016; Kuska et al. 2015). Near-infrared spectral signatures can therefore
be correlated with certain structural changes associated with disease progression.
Raman spectroscopy, on the other hand, can utilize infrared plant interrogation in a
different manner. Unlike VIS-NIR, Raman spectroscopy focuses on utilizing near-
infrared laser systems to probe for chemically specific information, and therefore can
determine localized changes in leaf chemical composition (e.g., carbohydrates,
proteins, lipids) and associate those changes with the disease (Pérez et al. 2016).
By determining unique spectral biomarkers for disease states, optical techniques
have been shown to be sensitive to many disease conditions (e.g., fungal, viral,
bacterial) across various plant species. Bravo et al. (2003) investigated
Pucciniastriiformis (yellow rust) infection in wheat plants using a ground-mounted
spectral imaging system. Rumpf et al. (2010) analyzed hyperspectral data collected
on sugar beet experiencing Cercospora leaf spot, leaf rust and powdery mildew, and
leveraged machine learning analytics to perform early disease differentiation. More
recent demonstrations elsewhere have also harnessed recent developments in
machine learning and other variants of artificial intelligence (AI) analytics to
improve accuracy for disease detection. Some examples include Huanglongbing
detection in citrus trees (Li et al. 2013), Tobacco mosaic virus in tobacco plants (Zhu
et al. 2017), Xylella fastidiosa in olive trees (Zarco-Tejada et al. 2018),
Phytophthora infestans in potato plants (Franceschini et al. 2019); reviews presented
elsewhere provide comprehensive lists of other demonstrations and applications for
plant disease detection (e.g., Mahlein 2016; Lowe et al. 2017; Sankaran et al. 2010;
Sylvain and Cecile 2018).
Cellular Analysis and Notification of Antigen Risks and Yields (CANARY®) is a
biosensor that utilizes genetically engineered B lymphocytes expressing biolumi-
nescent protein and pathogen-specific antibodies on the membrane surface for
specific and sensitive pathogen detection (Rider et al. 2003). CANARY® possesses
an optimal combination of the sensitivity of PCR and speed of lateral flow devices
and is under development and evaluation for several pathogens including Ralstonia
solanacearum, Phytophthora spp., and potyviruses (Nargi 2006).
To summarize, the development of optical biomarker-based technologies for

plant disease detection has progressed steadily over the last couple of decades.
Recent advances in the mobilization of optical technologies coupled with emerging
AI analytics are rapidly improving the availability and effectiveness of such
solutions. Optical technologies offer some key advantages including indirect detec-
tion, noninvasive interrogation with no sample preparation, and rapid, large-area
assessment for plant disease. In certain cases where sensitivity is an issue, the high-
throughput nature of optical detection can be used to guide other detection
technologies for follow-up or confirmation on disease state. Ultimately, large-scale
detection methods are needed to identify and monitor for diseases in order to prevent
epidemics, and optical technologies are showing some promise toward providing
these capabilities.
Traditional methods of fungal identification by visual observation for disease
symptoms, incubation methods, fungal isolations, colony and spore morphology,
and microscopy are tedious, time-consuming, and may lead to wrong identifications.
Fungal diagnosis using PCR-based methods has evolved rapidly and became the
gold standard. Several diagnostic markers such as ribosomal genes, particularly the
internal transcribed spacer (ITS) region, have been successfully used in fungal
identification up to the species level (White et al. 1990). Analysis of more than
one DNA locus is beneficial in phylogenetic interpretation and species identification
of new isolates especially in certain genera such as Mycosphaerella, Colletotrichum,
and Fusarium. Next-generation sequencing (NGS) is a versatile tool being used not
only for diagnostics but also to learn the whole genome or transcriptome of the
fungal isolates. Another technology that gained popularity for rapid and reliable
diagnostics is matrix-assisted laser desorption ionization time-of-flight mass spec-
trometry (MALDI-TOF MS) fingerprinting. A combination of techniques,
multilocus PCR, and electrospray ionization –mass spectrometry (ESI-MS) can
rapidly detect multiple fungal pathogens and provide genetic information in a single
assay.
Microscopy, serological techniques such as ELISA including plant-trapped anti-
gen ELISA, and double antibody sandwich ELISA and PCR-based technologies,
and isothermal amplification such as LAMP, rolling circle amplification (RCA), and
nucleic acid sequence-based amplification (NASBA) are routinely employed in the
detection of phytopathogens. Recent diagnostic assays are all based on the nucleo-
tide sequences of pathogen RNA or DNA, but even these can be combined with
earlier assays such as immuno-capture PCR (Mulholland 2009). Modern diagnostic
techniques such as CANARY® can accurately determine the minute amounts of the
pathogen in the infected plant tissue very rapidly and is easy to use. Prokaryotic
CRISPR-Cas immune systems have contributed to the advancement of the biotech-
nology field with an ability to precisely edit the genomes, gene disruption, and gene
repression (Hsu et al. 2014). Several diagnostic methods based on CRISPR/Cas9 to
detect nucleic acid of the pathogen were recently developed.
Despite the advances in molecular diagnosis, biological indicators still have their
place. They are still required for newly discovered viruses to complete Koch’s
postulates, and they are also essential for indexing diseases for which no pathogen
86 M. Kunta et al.
has been identified. Even for viruses which are well characterized, the plant is still
the only indicator for symptoms.
4.4 Conclusions
Critical to all efforts to fight plant disease, detection, and diagnosis of pathogens is
the first step toward any treatment, suppression, or containment of the pathogen.
While the variety of pathogens is innumerable, so too is the ingenuity of
diagnosticians. Here we have provided an overview of and an entrance into the
various methods that are being used or being developed to detect and diagnose plant
pathogens. The variety of well-established methods should allow for the develop-
ment of a diagnostic strategy for any known plant pathogen. The methods under
development offer exciting promises for quicker, cheaper, and easier deployment of
more sensitive and more accurate diagnostic methods. Much work is needed yet for
these methods, but we encourage their exploration and implementation where
appropriate.
References
Abd El Galil KH, El Sokkary MA, Kheira SM, Salazar AM, Yates MV, Chen W, Mulchandani A
(2005) Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A
virus. Appl Environ Microbiol 71:7113–7116
Abe K, Kumagai T, Takahashi C, Kezuka A, Murakami Y, Osawa Y, Motoki H, Matsuo T,
Horiuchi M, Sode K, Igimi S, Ikebukuro K (2012) Detection of pathogenic bacteria by using
zinc finger protein fused with firefly luciferase. Anal Chem 84:8028–8032
Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DBT, Shmakov S,
Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV,
Zhang F (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting
CRISPR effector. Science 353:aaf5573
Ali MM, Li F, Zhang Z, Zhang K, Kang D-K, Ankrum JA, Le XC, Zhao W (2014) Rolling circle
amplification: a versatile tool for chemical biology, materials science and medicine. Chem Soc
Rev 43:3324–3341
Alvarez A (2004) Integrated approaches for detection of plant pathogenicbacteria and diagnosis of
bacterial diseases. Annu Rev Phytopathol 42:339–366
An L, Tang W, Ranalli TA, Kim HJ, Wytiaz J, Kong H (2005) Characterization of a thermostable
UvrD helicase and its participation in helicase-dependent amplification. J Biol Chem
280:28952–28958
Aquino-Jarquin G (2019) CRISPR-Cas14 is now part of the artillery for gene editing and molecular
diagnostic. Nanomedicine: NBM 18:428–431. https://doi.org/10.1016/j.nano.2019.03.006
Banér J, Nilsson M, Mendel-Hartvig M, Landegren U (1998) Signal amplification of padlock
probes by rolling circle replication. Nucleic Acids Res 26:5073–5078
Barrangou R, Doudna JA (2016) Applications of CRISPR technologies in research and beyond. Nat
Biotechnol 34:933–941
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DS, Horvath P
(2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science
325:1709–1712
Batista AC, Pacheco LGC (2018) Detecting pathogens with Zinc-finger, TALE, and CRISPR-based
programmable nucleic acid binding proteins. J Microbiol Methods 152:98–104
Bawden FC, Pirie NW, Bernal JD, Fankuchen I (1936) Liquid crystalline substances from virus-
infected plants. Nature 138:1051–1052
Beale HP (1928) Immunologic reactions with tobacco mosaic virus. Proc Soc Exp Biol Med 25:602
Behmann J, Mahlein AK, Rumpf T, Römer C, Plümer L (2015) A review of advanced machine
learning methods for the detection of biotic stress in precision crop protection. Precis Agric
16:239–260
Belasque J Jr, Gasparoto MCG, Marcassa LG (2008) Detection of mechanical and disease stresses
in citrus plants by fluorescence spectroscopy. Appl Optics 47(11):1922–1926
Berdugo C, Zito R, Paulus S, Mahlein A-K (2014) Fusion of sensor data for the detection and
differentiation of plant diseases in cucumber. Plant Pathol 63:1344–1356
Berg JM (1988) Proposed structure for the zinc binding domains from transcription factor IIIA and
related proteins. Proc Natl Acad Sci U S A 85:99–102
Bernal JD, Fankuchen I (1937) Structure types of protein “crystals” from virus-infected plants.
Nature 139:923–924
Bernard PS, Wittwer CT (2000) Homogeneous amplification and variant detection by fluorescent
hybridization probes. Clin Chem 46:147–148
Blais BW, Turner G, Sooknanan R, Malek LT (1997) A nucleic acid sequence-based amplification
system for detection of Listeria monocytogeneshlyA sequences. Appl Environ Microbiol
63:310–313
Boch J, Bonas U (2010) Xanthomonas AvrBs3 family-type III effectors: discovery and function.
Bogdanove AJ, Schornack S, Lahaye T (2010) TAL effectors: finding plant genes for disease and
defense. Curr Opin Plant Biol 13:394–401
Bonnet G, Tyagi S, Libchaber A, Kramer FR (1999) Thermodynamic basis of the enhanced
specificity of structured DNA probes. Proc Natl Acad Sci U S A 96:6171–6176
Bravo C, Moshou D, West J, McCartney A, Ramon H (2003) Early disease detection in wheat fields
using spectral reflectance. Biosyst Eng 84:137–145
Buh Gašparič M, Tengs T, La Paz JL, Holst-Jensen A, Pla M, Esteve T, Žel J, Gruden K (2010)
Comparison of nine different real-time PCR chemistries for qualitative and quantitative
applications in GMO detection. Anal Bioanal Chem 396:2023–2029
Cardullo RA, Agrawal S, Flores C, Zamecnik PC, Wolf DE (1988) Detection of nucleic acid
hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A
85:8790–8794
Carrasco N, Roozenburg I, Voorbergen-Laarman M, Itoh N, Engelsma MY (2013) Development of
a real-time PCR for detection of the oyster pathogen Nocardiacrassostreae based on its homo-
geneous 16S-23S rRNA intergenic spacer region. J Invertebr Pathol 114:120–127
Champer J, Buchman A, Akbari OS (2016) Cheating evolution: engineering gene drives to
manipulate the fate of wild populations. Nat Rev Genet 17:146–159
Chang CC, Chen CC, Wei SC, Lu HH, Liang YH, Lin CW (2012) Diagnostic devices for
isothermal nucleic acid amplification. Sensors (Switzerland) 12:8319–8337
Chen F, Zhao Y, Fan C, Zhao Y (2015) Mismatch extension of DNA polymerases and high-
accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved
isothermal amplification. Anal Chem 87:8718–8723
Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (2018) CRISPR-
Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science
360:436–439
Chien A, Edgar DB, Trela JM (1976) Deoxyribonucleic acid polymerase from the extreme
thermophile Thermus aquaticus. J Bacteriol 127:1550–1557
Choi CW, Hyun JW, Hwang RY, Powell CA (2018) Loop-mediated isothermal amplification assay
for detection of CandidatusLiberibacterasiaticus, a causal agent of citrus Huanglongbing. Plant
Pathol J 34:499–505
88 M. Kunta et al.
Choo Y, Klug A (1994a) Toward a code for the interactions of zinc fingers with DNA: selection of
randomized fingers displayed on phage. Proc Natl Acad Sci U S A 91:11163–11167
Choo Y, Klug A (1994b) Selection of DNA binding sites for zinc fingers using rationally
randomized DNA reveals coded interactions. Proc Natl Acad Sci U S A 91:11168–11172
Choo Y, Sánchez-Garciá I, Klug A (1994) In vivo repression by a site-specific DNA-binding
protein designed against an oncogenic sequence. Nature 372:642–645
Christensen LK (2004) NPK deficiencies discrimination by use of spectral and spatial response.
Trabajo de gradopresentadocomorequisito para optar al Título de Doctor en Filosofía.
Dinamarca: THE Royal Veterinary and Agricultural University, Department of agricultural
Sciences AgroTechonology
Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme-linked immuno-
sorbent assay for the detection of plant viruses. J Gen Virol 34:475–483
Compton J (1991) Nucleic acid sequence-based amplification. Nature 350:91–92
Crannell ZA, Rohrman B, Richards-Kortum R (2014) Quantification of HIV-1 DNA using real-
time recombinase polymerase amplification. Anal Chem 86:5615–5619
Craw P, Balachandran W (2012) Isothermal nucleic acid amplification technologies for point-of-
care diagnostics: a critical review. Lab Chip 12:2469–2486
Curtis KA, Rudolph DL, Owen SM (2008) Rapid detection of HIV-1 by reverse-transcription, loop-
mediated isothermal amplification ( RT-LAMP ). J Virol Methods 151:264–270
de Bary A (1861) Die gegenwärtig herrschende Kartoffelkrankheit, ihreUrsache und ihre
Verhütung. Eine pfl anzenphysiologische Untersuchungin allgemein verständlicher Form
dargestellt. A. Förster‘sche Buchhandlung (Arthur Felix), Leipzig
Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P, Sun Z, Zong Q, Du Y, Du J et al
(2002) Comprehensive human genome amplification using multiple displacement amplification.
Proc Natl Acad Sci U S A 99:5261–5266
Deiman B, van Aarle P, Sillekens P (2002) Characteristics and applications of nucleic acid
sequence-based amplification (NASBA). Mol Biotechnol 20:163–179
Derrick KS (1973) Quantitative assay for plant viruses using serologically specific electron
microscopy. Virology 56:652–653
Didovyk A, Borek B, Tsimring L, Hasty J (2016) Transcriptional regulation with CRISPR-Cas9:
principles, advances, and applications. Curr Opin Biotechnol 40:177–184
Dimov IK, Garcia-Cordero JL, O’Grady J, Poulsen CR, Viguier C, Kent L, Daly P, Lincoln B,
Maher M, O’Kennedy R et al (2008) Integrated microfluidic tmRNA purification and real-time
NASBA device for molecular diagnostics. Lab Chip 8:2071–2078
Edwardson JR (1974) Some Properties of the Potato Virus Y Group. Institute of Food and
Agricultural Sciences, University of Florida, Gainesville
Fagen JR, Collias D, Singh AK, Beisel CL (2017) Advancing the design and delivery of CRISPR
antimicrobials. Curr Opin Biomed Eng 4:57–64
Fang Y, Ramasamy RP (2015) Current and prospective methods for plant disease detection.
Biosensors 5:537–561
Fang Z, Wu W, Lu X, Zeng L (2014) Lateral flow biosensor for DNA extraction-free detection of
salmonella based on aptamer mediated strand displacement amplification. Biosens Bioelectron
56:192–197
Fire A, Xu S-Q (1995) Rolling replication of short DNA circles. Proc Natl Acad Sci 92:4641–4645
Franceschini MHD, Bartholomeus H, Van Apeldoorn DF, Suomalainen J, Kooistra L (2019)
Feasibility of unmanned aerial vehicle optical imagery for early detection and severity assess-
ment of late blight in potato. Remote Sens (Basel) 11:224
Freitas DDS, Carlos EF, Gil MCSDS, Vieira LGE, Alcantara GB (2015) NMR-based metabolomic
analysis of Huanglongbing-asymptomatic and-symptomatic citrus trees. J Agric Food Chem 63
(34):7582–7588
Garneau JE, Dupuis M-E, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P,
Magadán AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacterio-
phage and plasmid DNA. Nature 468:67–71
Gibson UEM, Heid CA, Williams PM (1996) A novel method for real time quantitative RT-PCR.
Genome Res 6:995–1001
Gill P, Ramezani R, Amiri MVP, Ghaemi A, Hashempour T, Eshraghi N, Ghalami M, Tehrani HA
(2006) Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for
molecular detection of M. tuberculosis. Biochem Biophys Res Commun 347:1151–1157
Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N,
Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung
DT, Sabeti PC, Collins JJ, Zhang F (2017) Nucleic acid detection with CRISPR13a/C2c2.
Science 356:438–442
Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F (2018) Multiplexed and
portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360:439–444
Gratia A (1933) Pluralité antigenique et identification serologique des virus de plantes. Compt Rend
Soc Biol 114:923
Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richman DD, Gingeras TR (1990)
Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after
retroviral replication. Proc Natl Acad Sci U S A 87:1874–1878
Guk K, Keem JO, Hwang SG, Kim H, Kang T, Lim E-K, Jung J (2017) A facile, rapid and sensitive
detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/
sgRNA complex. Biosens Bioelectron 95:67–71
Gulliksen A, Solli L, Karlsen F, Rogne H, Hovig E, Nordstrøm T, Sirevåg R (2004) Real-time
nucleic acid sequence-based amplification in nanoliter volumes. Anal Chem 76:9–14
Gulliksen A, Solli LA, Drese KS, Sörensen O, Karlsen F, Rogne H, Hovig E, Sirevåg R (2005)
Parallel nanoliter detection of cancer markers using polymer microchips. Lab Chip 5:416–420
Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, Cofsky JC, Kyrpides NC,
Banfield JF, Doudna JA (2018) Programmed DNA destruction by miniature CRISPR-Cas14
enzymes. Science 362:839–842
He P, Chen Z, Luo J, Wang H, Yan Y, Chen L, Gao W (2014) Multiplex real-time PCR assay for
detection of pathogenic Vibrio parahaemolyticus strains. Mol Cell Probes 28:246–250
Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res
6:986–994
Heller MJ, Morrison LE (1985) Chemiluminescent and fluorescent probes for DNA hybridization
systems. In: Kingsbury D, Falkow S (eds) Rapid detection and identification of infectious
agents. Academic, New York, pp 245–256
Henson JM, French R (1993) The polymerase chain reaction and plant disease diagnosis. Annu Rev
Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of
specific DNA sequences. Nat Biotechnol 10:413–417
Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain
reaction product by utilizing the 50 ! 30 exonuclease activity of Thermus aquaticus DNA
polymerase. Proc Natl Acad Sci U S A 88:7276–7280
Hollingsworth RC, Sillekens P, Van Deursen P, Neal KR, Irving WL, James V, Jones DA,
Hollingsworth R, Irving W, Neal K et al (1996) Serum HCV RNA levels assessed by quantita-
tive NASBA®: stability of viral load over time, and lack of correlation with liver disease. J
Hepatol 25:301–306
Holmes FO (1929) Local lesions in tobacco mosaic. Bot Gaz 87:39–55
Honarmand A, Mayal R, George I, Oberding L, Dastidar H, Fegan J, Chaudhuri S, Dole J, Feng S,
Hoang D, Moges R, Osgood J, Remondini T, van der Meulen WK, Wang S, Wintersinger C,
Zucoloto AZ, Chatfield-Reed K, Arcellana-Panlilio M, Nygren A (2014) A multiplexed tran-
scription activator-like effector system for detecting specific DNA sequences. ACS Synth Biol
3:953–955
Hooke R (1665) Micrographia. Royal Society, London
Howell WM (2006) Detection of DNA hybridization using induced fluorescence resonance energy
transfer. In: Didenko VV (ed) Fluorescent energy transfer nucleic acid probes: designs and
protocols. Humana Press, Totowa, pp 33–41
90 M. Kunta et al.
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome
engineering. Cell 157:1262–1278
Huang M, Zhou X, Wang H, Xing D (2018) Clustered regularly interspaced short palindromic
repeats/Cas9 triggered isothermal amplification for site-specific nucleic acid detection.
90:2193–2200
Ihrig J, Lill R, Mühlenho U (2006) Application of the DNA-specific dye EvaGreen for the routine
quantification of DNA in microplates. Anal Biochem 359:265–267
Iwanowski D (1892) Über die Mozaikkrankheit der Tabakspflanze. Bull Acad Imp Sci St
Petersberg NS III 35:65–70. (cited by Mathews 1970)
Kandimalla ER, Agrawal S (2000) ‘Cyclicons’ as hybridization-based fluorescent primer-probes:
synthesis, properties and application in real-time PCR. Bioorg Med Chem 8:1911–1916
Kausche GA, Pfankuch E, Ruska A (1939) Die Sichtbormachung von pflanz-lichem Virus
imÜbermikroskop. Naturwissenschaften 29:573–574
Kawada J, Kimura H, Ito Y, Hoshino Y, Tanaka-Kitajima N, Ando Y, Futamura M, Morishima T
(2004) Comparison of real-time and nested PCR assays for detection of herpes simplex virus
DNA. Microbiol Immunol 48:411–415
Keremane ML, Ramadugu C, Rodriguez E, Kubota R, Shibata S, Hall DG, Roose ML, Jenkins D,
Lee RF (2015) A rapid field detection system for citrus huanglongbing associated
“CandidatusLiberibacterasiaticus” from the psyllid vector, DiaphorinacitriKuwayama and its
implications in disease management. Crop Prot 68:41–48
Kim MS, Kim J (2016) Multiplexed detection of pathogen-specific DNA using engineered zinc
finger proteins without target amplification. Anal Methods 8:6696–6700
Kim M, Stybayeva G, Lee JY, Revzin A, Segal DJ (2011) A zinc finger protein array for the visual
detection of specific DNA sequences for diagnostic applications. Nucleic Acids Res 39:e29
Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined
specificity. Nature (London) 256:95–497
Koonin EV, Madarova KS, Zhang F (2017) Diversity, classification and evolution of CRISPR-Cas
systems. Curr Opin Microbiol 37:67–78
Kralik P, Ricchi M (2017) A basic guide to real time PCR in microbial diagnostics: definitions,
parameters, and everything. Front Microbiol 8:108
Kungulovski G, Jeltsch A (2016) Epigenome editing: state of the art, concepts, and perspectives.
Trends Genet 32:101–113
Kuska M, Wahabzada M, Leucker M, Dehne HW, Kersting K, Oerke EC, Mahlein AK et al (2015)
Hyperspectral phenotyping on the microscopic scale: towards automated characterization of
plant-pathogen interactions. Plant Methods 11:28
Kutyavin IV (2010) New approach to real-time nucleic acids detection: folding polymerase chain
reaction amplicons into a secondary structure to improve cleavage of Förster resonance energy
transfer probes in 50 -nuclease assays. Nucleic Acids Res 38:e29
Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger
DK, Lokhov SG, Gall AA et al (2000) 3’-Minor groove binder-DNA probes increase sequence
specificity at PCR extension temperatures. Nucleic Acids Res 28:655–661
Kuzdralinski A, Kot A, Szczerba H, Nowak M, Muszynska M (2017) A review of conventional
PCR assays for the detection of selected phytopathogens of wheat. J Mol Microbiol Biotechnol
27:175–189
Kwoh DY, Davis GR, Whitfield KM, Chappelle HL, DiMichelle LJ, Gingeras TR (1989)
Transcription-based amplification system and detection of amplified human immunodeficiency
virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad Sci U S A
86:1173–1177
Laity JH, Lee BM, Wright PE (2001) Zinc finger proteins: new insights into structural and
functional diversity. Curr Opin Struct Biol 11:39–46
Lee MA, Siddle AL, Page RH (2002) ResonSense®: simple linear fluorescent probes for quantita-
tive homogeneous rapid polymerase chain reaction. Anal Chim Acta 457:61–70
Lee J, Yoshida W, Abe K, Nakabayashi K, Wakeda H, Hata K, Marquette CA, Blum LJ, Sode K,
Ikebukuro K (2017) Development of an electrochemical detection system for measuring DNA
methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc
finger protein. Biosens Bioelectron 93:118–123
Li Q, Luan G, Guo Q, Liang J (2002) A new class of homogeneous nucleic acid probes based on
specific displacement hybridization. Nucleic Acids Res 30:e5
Li YD, Chu ZZ, Liu XG, Jing HC, Liu YG, Hao DY (2010) A cost-effective high-resolution
melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in
plants. J Integr Plant Biol 52:1036–1042
Li T, Huang S, Jian WZ, Wright D, Spalding MH, Weeks DP, Yang B (2011a) TAL nucleases
(TALNs): hybrid proteins composed of TAL effectors and Tokl DNA-cleavage domain. Nucleic
Acids Res 39:359–372
Li Y, Jortani SA, Ramey-Hartung B, Hudson E, Lemieux B, Kong H (2011b) Genotyping three
SNPs affecting warfarin drug response by isothermal real-time HDA assays. Clin Chim Acta
412:79–85
Li H, Lee WS, Wang K (2013) Airborne hyperspectral imaging based citrus greening disease
detection using different dimension reduction methods. In: 2013 Kansas City, Missouri, July
21-July 24, 2013. American Society of Agricultural and Biological Engineers, p 1
Li S, Cheng Q-X, Wang J-M, Li X-Y, Zhang Z-L, Gao S, Cao R-B, Zhao G-P, Wang J (2018)
CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov 4:20
Li L, Li S, Wu N, Wu J, Wang G, Zhao G, Wang J (2019) HOLMESv2: ACRISPR-Cas12b-assisted
platform for nucleic acid detection and DNA methylation quantitation. ACS Synth Biol
8:2228–2237
Liew M, Nelson L, Margraf R, Mitchell S, Erali M, Mao R, Lyon E, Wittwer C (2006) Genotyping
of human platelet antigens 1 to 6 and 15 by high-resolution amplicon melting and conventional
hybridization probes. J Mol Diagn 8:97–104
Lim SY, Kim BJ, Lee MK, Kim K (2008) Development of a real-time PCR-based method for rapid
differential identification of Mycobacterium species. Lett Appl Microbiol 46:101–106
Liu D, Daubendiek SL, Zillman MA, Ryan K, Kool ET (1996) Rolling circle DNA synthesis: small
circular oligonucleotides as efficient templates for DNA polymerases. J Am Chem Soc
118:1587–1594
Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC (1998) Mutation detection and
single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19:225–232
Lopes G, Korkmaz G, Agami R (2016) Applying CRISPR-Cas9 tools to identify and characterize
transcriptional enhancers. Nat Rev Mol Cell Biol 17(9):597–604
López-Gresa MP, Maltese F, Bellés JM, Conejero V, Kim HK, Choi YH, Verpoorte R (2010)
Metabolic response of tomato leaves upon different plant–pathogen interactions. Phytochem
Anal 21:89–94
Lowe A, Harrison N, French AP (2017) Hyperspectral image analysis techniques for the detection
and classification of the early onset of plant disease and stress. Plant Methods 13:80
Lukhtanov EA, Lokhov SG, Gorn VV, Podyminogin MA, Mahoney W (2007) Novel DNA probes
with low background and high hybridization-triggered fluorescence. Nucleic Acids Res 35:e30
Luo ML, Leenay RT, Beisel CL (2016) Current and future prospects for CRISPR-based tools in
bacteria. Biotechnol Bioeng 113:930–943
Ma H, Tu L-C, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T (2016) Multiplexed
labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat
Mahlein AK (2016) Plant disease detection by imaging sensors–parallels and specific demands for
precision agriculture and plant phenotyping. Plant Dis 100(2):241–251
Mao F, Leung WY, Xin X (2007) Characterization of EvaGreen and the implication of its
physicochemical properties for qPCR applications. BMC Biotechnol 7:76
Marras SAE (2006) Selection of fluorophore and quencher pairs for fluorescent nucleic acid
hybridization probes. In: VDidenko V (ed) Fluorescent energy transfer nucleic acid probes:
designs and protocols. Humana Press, Totowa, pp 3–16
92 M. Kunta et al.
Martinelli F, Scalenghe R, Davino S, Panno S, Scuderi G, Ruisi P, Davis CE et al (2015) Advanced

methods of plant disease detection. A review. Agron Sustain Dev 35:1–25
Mathews REF (1970) Plant virology. Academic, New York
Matson SW, Robertson AB (2006) The UvrD helicase and its modulation by the mismatch repair
protein MutL. Nucleic Acids Res 34:4089–4097
Mayer A (1886) Über die Mosaikktankheit des Tabaks. Landwirsch Versuchs Stationen
32:452–467. (cited by Mathews 1970)
McCartney MM, Spitulski SL, Pasamontes A, Peirano DJ, Schirle MJ, Cumeras R, Dike SC et al
(2016) Coupling a branch enclosure with differential mobility spectrometry to isolate and
measure plant volatiles in contained greenhouse settings. Talanta 146:148–154
Mechanic LE, Frankel BA, Matson SW (2000) Escherichia coli MutL loads DNA helicase II onto
DNA. J Biol Chem 275:38337–38346
Mehrpouyan M, Bishop JE, Ostrerova N, Van Cleve M, Lohman KL (1997) A rapid and sensitive
method for non-isotopic quantitation of HIV-1 RNA using thermophilic SDA and flow
cytometry. Mol Cell Probes 11:337–347
Mergny JL, Boutorine AS, Garestier T, Belloc F, Rougée M, Bulychev NV, Koshkin AA,
Bourson J, Lebedev AV, Valeur B et al (1994) Fluorescence energy transfer as a probe for
nucleic acid structures and sequences. Nucleic Acids Res 22:920–928
Miller J, McLachlan AD, Klug A (1985) Repetitive zinc-binding domains in the protein transcrip-
tion factor III A from Xenopus oocytes. EMBO J 4:1609–1614
Misawa Y, Yoshida A, Saito R, Yoshida H, Okuzumi K, Ito N, Okada M, Moriya K, Koike K
(2007) Application of loop-mediated isothermal amplification technique to rapid and direct
detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J Infect
Chemother 13:134–140
Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E (2005) Intervening sequences of regularly
spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60:174–182
Monis PT, Giglio S, Saint CP (2005) Comparison of SYTO9 and SYBR Green I for real-time
polymerase chain reaction and investigation of the effect of dye concentration on amplification
and DNA melting curve analysis. Anal Biochem 340:24–34
Mori Y, Notomi T (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and
cost-effective diagnostic method for infectious diseases. J Infect Chemother 15:62–69
Morrison LE, Halder TC, Stols LM (1989) Solution-phase detection of polynucleotides using
interacting fluorescent labels and competitive hybridization. Anal Biochem 183:231–244
Mothershed EA, Whitney AM (2006) Nucleic acid-based methods for the detection of bacterial
pathogens: present and future considerations for the clinical laboratory. Clin Chim Acta
363:206–220
Motré A, Li Y, Kong H (2008) Enhancing helicase-dependent amplification by fusing the helicase
with the DNA polymerase. Gene 420:17–22
Mulholland V (2009) Immunocapture-PCR for plant virus detection. Methods Mol Biol
508:183–192
Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H (1986) Specific enzymatic amplification of
DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51:263–273
Murakami T, Sumaoka J, Komiyama M (2009) Sensitive isothermal detection of nucleic-acid
sequence by primer generation-rolling circle amplification. Nucleic Acids Res. https://doi.org/
10.1093/nar/gkn1014
Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, Kellner MJ, Tan
AL, Paul LM, Parham LA, Garcia KF, Barnes KG, Chak B, Mondini A, Nogueira ML, Isern S,
Michael SF, Lorenzana I, Yozwiak NL, MacInnis BL, Bosch I, Gehrke L, Zhang F, Sabeti PC
(2018) Field-deployable viral diagnostics using CRISPR-Cas13. Science 360:444–448
Nagaraj AN (1965) Immunofluorescence studies on synthesis and distribution of tobacco mosaic
virus antigen in tobacco. Virology 25:133–142
Nargi F (2006) CANARY B-cell sensor for rapid identification of plant pathogens. (Abstr.).
Phytopathology 96:S83
Naserpour Farivar T, Johari P, Najafipour R, Farzam A, Nasirian N, HajManouchehri F,
JahaniHashemi H, Azimi A, Bahrami M (2014) The relationship between gastric cancer and
Helicobacter pylori in formaldehyde fixed paraffin embedded gastric tissues of gastric cancer
patients-scorpion real-time PCR assay findings. Pathol Oncol Res 20:113–117
Nath K, Sarosy JW, Hahn J, Di Como CJ (2000) Effects of ethidium bromide and SYBR® Green I
on different polymerase chain reaction systems. J Biochem Biophys Methods 42:15–29
Navarro E, Serrano-Heras G, Castaño MJ, Solera J (2015) Real-time PCR detection chemistry. Clin
Chim Acta 439:231–250
Nazarenko I, Lowe B, Darfler M, Ikonomi P, Schuster D, Rashtchian A (2002) Multiplex quantita-
tive PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res 30:
e37
Nerys-Junior A, Braga-Dias LP, Pezzuto P, Cotta-de-Almeida V, Tanuri A (2018) Comparison of
the editing patterns and ediging efficiencies of TALEN and CRISPR-Cas9 when targeting the
human CCR5 gene. Genet Mol Biol 41:167–179
Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary B, Landegren U (1994)
Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265
(5181):2085–2088
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-
mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63
O’Donnell IJ, Shukla DD, Gough KH (1982) Electro-blot radioimuunoassay of virus-infected plant
sap – a powerful new technique for detecting plant viruses. J Virol Methods 4:19–26
Ohtsuka K, Yanagawa K, Takatori K, Hara-Kudo Y (2005) Detection of Salmonella enterica in
naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characteri-
zation of Salmonella isolates. Appl Environ Microbiol 71:6730–6735
Ooi AT, Stains CI, Ghosh I, Segal DJ (2006) Sequence-enabled reassembly of β-lactamase (SEER-
LAC): a sensitive method for the detection of double-stranded DNA. Biochemistry
45:3620–3625
Osawa Y, Ikebukuro K, Motoki H, Matsuo T, Horiuchi M, Sode K (2008) The simple and rapid
detection of specific PCR products from bacterial genomes using Zn finger proteins. Nucleic
Acids Res 36:e68
Osawa Y, Ikebukuro K, Sode K (2009) Zn finger-based direct detection system for PCR products of
Salmonella spp. and the influenza A virus. Biotechnol Lett 31:725–733
Ouchterlony O (1962) Diffusion-in-gel methods for immunological analysis II. Prog Allergy
6:30–154
Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghai N,
Fan M, Daringer NM, Bosch I, Dudley DM, O’Connor DH, Gehrke L, Collins JJ (2016) Rapid,
low-cost detection of zika virus using programmable biomolecular components. Cell
165:1255–1266
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughouput profiling
of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat
Peñuelas J, Filella I (1998) Visible and near-infrared reflectance techniques for diagnosing plant
physiological status. Trends Plant Sci 3:151–156
Pérez MRV, Mendoza MGG, Elías MGR, González FJ, Contreras HRN, Servín CC et al (2016)
Raman spectroscopy an option for the early detection of citrus Huanglongbing. Appl Spectrosc
70:829–839
Perez-Pinera P, Ousterout DG, Gersbach CA (2012) Advances in targeted genome editing. Curr
Opin Chem Biol 16:268–277
Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination
proteins. PLoS Biol 4:1115–1121
Polstra AM, Goudsmit J, Cornelissen M (2002) Development of real-time NASBA assays with
molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes. BMC
Infect Dis 2:1–10
Punia P, Cane P, Teo C-G, Saunders N (2004) Quantitation of hepatitis B lamivudine resistant
mutants by real-time amplification refractory mutation system PCR. J Hepatol 40:986–992
94 M. Kunta et al.
Qi H, Yue S, Bi S, Ding C, Song W (2018) Isothermal exponential amplification techniques: from

basic principles to applications in electrochemical biosensors. Biosens Bioelectron
110:207–217
Qiu X-Y, Zhu L-Y, Zhu C-S, Ma J-X, Hou T, Wu X-M, Xie S-S, Min L, Tan D-A, Zhang D-Y, Zhu
L (2018) Highly effective and low-cost microRNA detection with CRISPR-Cas9. ACS Synth
Biol 7:807–813
Raghunathan A, Ferguson HR, Bornarth CJ, Song W, Driscoll M, Lasken RS (2005) Genomic
DNA amplification from a single bacterium. Appl Environ Microbiol 71:3342–3347
Rajagopal N, Srinivasan S, Kooshesh K, Guo Y, Edwards MD, Banerjee B, Syed T, Emons BJM,
Gifford DK, Sherwood RI (2016) High-throughput mapping of regulatory DNA. Nat Biotechnol
34:167–174
Rider P, Nargi H, Schwoebel M, Blanchard B, Young C, Hollis (2003) AB cell-based sensor for
rapid identification of pathogens. Science 301:213–215
Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting
curves during the polymerase chain reaction. Anal Biochem 245:154–160
Rohrman B, Richards-Kortum R (2015) Inhibition of recombinase polymerase amplification by
background DNA: a lateral flow-based method for enriching target DNA. Anal Chem
87:1963–1967
Rumpf T, Mahlein AK, Steiner U, Oerke EC, Dehne H, Plümer L (2010) Early detection and
classification of plant diseases with support vector machines based on hyperspectral reflectance.
Rybicki EP, von Wechmar MB (1982) Enzyme-assisted immune detection of plant virus proteins
electroblotted onto nitrocellulose paper. J Virol Methods 5:267–278
Saiki RK, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed
enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
Sanchez JA, Abramowitz JD, Salk JJ, Reis AH, Rice JE, Pierce KE, Wangh LJ (2006)
Two-temperature LATE-PCR endpoint genotyping. BMC Biotechnol 6:44
Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F (2012) A transcription activator-like
effector toolbox for genome editing. Nat Protoc 7:171–192
Schmelz EA, Engelberth J, Alborn HT, O’donnell P, Sammons M, Toshima H, Tumlinson JH
(2003) Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds
in plants. Proc Natl Acad Sci 100:10552–10557
Shalem O, Sanjana NE, Zhang F (2015) High-throughput functional genomics using CRISPR-
Cas9. Nat Rev Genet 16:299–311
Shen F, Davydova EK, Du W, Kreutz JE, Piepenburg O, Ismagilov RF (2011) Digital isothermal
quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase
amplification reactions on SlipChip. Anal Chem 83:3533–3540
Shi C, Liu Q, Ma C, Zhong W (2014) Exponential strand-displacement amplification for detection
of micrornas. Anal Chem 86:336–339
Shi C, Xu Q, Ge Y, Jiang L, Huang H (2017) Luciferase-Zinc-Finger system for the rapid detection
of pathogenic bacteria. J Agric Food Chem 65:6674–6681
Shipman SL, Nivala J, Macklis JD, Church GM (2016) Molecular recordings by directed CRISPR
spacer acquisition. Science 353:aaf1175
Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg S, Semenova E, Minakhin L,
Joung J, Konermann S, Severinova K, Zhang F, Koonin E (2015) Discover and functional
characterization of diverse class 2 CRISPR-Cas systems. Mol Cell 60:385–397
Simpkins SA, Chan AB, Hays J, Pöpping B, Cook N (2000) An RNA transcription-based amplifi-
cation technique (NASBA) for the detection of viable Salmonella enterica. Lett Appl Microbiol
30:75–79
Smith KM (1931) On the composite nature of certain potato virus diseases of the mosaic group as
revealed by the use of plant indicators and selective methods of transmission. Proc Roy Soc
Lond A B109:251–266
Solinas A, Brown LJ, McKeen C, Mellor JM, Nicol JTG, Thelwell N, Brown T (2001) Duplex
Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res 29:e96
Stains CI, Porter JR, Ooi AT, Segal DJ, Ghosh I (2005) DNA sequence-enabled reassembly of the
green fluorescent protein. J Am Chem Soc 127:10782–10783
Stanley WM (1935) Isolation of a crystalline protein possessing the properties of tobacco mosaic
virus. Science 81:644–645
Sun N, Zhao H (2013) Transcription activator-like effector nucleases (TALENs): a highly efficient
and versatile tool for genome editing. Biotechnol Bioeng 110:1811–1821
Sylvain T, Cecile LG (2018) Disease identification: a review of vibrational spectroscopy
applications. In: Comprehensive analytical chemistry, vol 80. Elsevier, pp 195–225
Takano E, Shimura N, Akiba T, Kitayama Y, Sunayama H, Abe K, Ikebukuro K, Takeuchi T
(2017) Pipette tip biosensors for bacterial double-stranded DNA using bioluminescence induced
by zinc finger luciferase. Michrochim Acta 184:1595–1601
Thi H, Thai C, Le MQ, Vuong CD, Parida M, Minekawa H, Notomi T, Hasebe F (2004)
Development and evaluation of a novel loop-mediated isothermal amplification method for
rapid detection of severe acute respiratory syndrome Coronavirus. J Clin Microbiol
42:1956–1961
Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat
Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered
zinc finger nucleases. Nat Rev Genet 11:636–646
Van Der Vliet GME, Schukkink RAF, Van Gemen B, Schepers P, Klatser PR (1993) Nucleic acid
sequence-based amplification (NASBA) for the identification of mycobacteria. J Gen Microbiol
139:2423–2429
van Lent JWM, Verduin BJM (1985) Specific gold-labelling of antibodies bound to plant viruses in
mixed suspensions. Neth J Plant Pathol 91:205–213
Van Ness J, Van Ness LK, Galas DJ (2003) Isothermal reactions for the amplification of
oligonucleotides. PNAS 100:4504–4509
Vandamme AM, Van Dooren S, Kok W, Goubau P, Fransen K, Kievits T, Schmit JC, De Clercq E,
Desmyter J (1995) Detection of HIV-1 RNA in plasma and serum samples using the NASBA
amplification system compared to RNA-PCR. J Virol Methods 52:121–132
Varga A, James D (2006) Real-time RT-PCR and SYBR Green I melting curve analysis for the
identification of Plum pox virus strains C, EA, and W: effect of amplicon size, melt rate, and dye
translocation. J Virol Methods 132:146–153
Vincent M, Xu Y, Kong H (2004) Helicase-dependent isothermal DNA amplification. EMBO Rep
5:795–800
Wade M (2015) High-throughput silencing using the CRISPR-Cas9 system: a review of the benefits
and challenges. J Biomol Screen 20:1027–1039
Walker GT, Fraiser MS, Schram JL, Little MC, Nadeau JG, Malinowski DP (1992a) Strand
displacement amplification – an isothermal, in vitro DNA amplification technique. Nucleic
Acids Res 20:1691–1696
Walker GT, Little MC, Nadeau JG, Shank DD (1992b) Isothermal in vitro amplification of DNA by
a restriction enzyme/DNA polymerase system. Proc Natl Acad Sci U S A 89:392–396
Walter NG, Strunk G (1994) Strand displacement amplification as an in vitro model for rolling-
circle replication: deletion formation and evolution during serial transfer. Proc Natl Acad Sci U
S A 91:7937–7941
Wang W, Chen K, Xu C (2006) DNA quantification using EvaGreen and a real-time PCR
instrument. Anal Biochem 356:303–305
Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev
Biochem 85:227–264
Wang Q, Zhang B, Xu X, Long F, Wang J (2018) CRISPR-typing PCR (ctPCR), a new Cas9-based
DNA detection method. Sci Rep 8:14126
Wang B, Wang R, Wang D, Wu J, Li J, Wang J, Liu H, Wang Y (2019) Cas12aVdet: a CRISPR/
Cas12a-based platform for rapid and visual nucleic acid detection. Anal Chem 91:12156–12161
96 M. Kunta et al.
Whitcombe D, Theaker J, Guy SP, Brown T, Little S (1999) Detection of PCR products using self-
probing amplicons and fluorescence. Nat Biotechnol 17:804–807
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal
RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR
protocols: a guide to methods and applications. Academic, pp 315–322
Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997) Continuous fluorescence monitoring
of rapid cycle DNA amplification. Biotechniques 22:130–138
Wu W, Zhang T, Han D, Fan H, Zhu G, Ding X, Wu C, You M, Qiu L, Li J et al (2018) Aligner-
mediated cleavage of nucleic acids and its application to isothermal exponential amplification.
Chem Sci 9:3050–3055
Xie S, Chai Y, Yuan Y, Bai L, Yuan R (2014) Development of an electrochemical method for
Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification. Biosens
Bioelectron 55:324–329
Yi MY, Ling L, Neogi SB, Sen FY, Tang DY, Yamasaki S, Shi L, Ye L (2014) Real time loop-
mediated isothermal amplification using a portable fluorescence scanner for rapid and simple
detection of Vibrio parahaemolyticus. Food Control 41:91–95
Yin P, Deng D, Yan C, Pan X, Xi JJ, Yan N, Shi Y (2012) Specific DNA-RNA hybrid recognition
by TAL effectors. Cell Rep 2:707–713
Zanoli LM, Spoto G (2013) Isothermal amplification methods for the detection of nucleic acids in
microfluidic devices. Biosensors 3:18–43
Zarco-Tejada PJ, Camino C, Beck PSA, Calderon R, Horner A, Hernández-Clemente R, Gonzalez-
Dugo V et al (2018) Previsual symptoms of Xylella fastidiosa infection revealed in spectral
plant-trait alterations. Nat Plants 4:432
Zeng L, Mukama O, Lu X, Cao S, Lin D (2019) Strand displacement amplification for multiplex
strand displacement amplification for multiplex detection of nucleic acids detection of nucleic
acids Lingwen. In: Singh A, Khan MW (eds) Modulating Gene Expression – Abridging the
RNAi and CRISPR-Cas9 Technologies. IntechOpen Ltd., London, pp 61–82
Zhang C, Xing D (2010) Single-molecule DNA amplification and analysis using microfluidics.
Chem Rev 110:4910–4947
Zhang X, Lowe SB, Gooding JJ (2014a) Brief review of monitoring methods for loop-mediated
isothermal amplification (LAMP). Biosens Bioelectron 61:491–499
Zhang Z, Xiao H, Xie F, Zhang H, Chen C, Xiao H, Yang Z, Wang D, Li Z, Wang G (2014b) High-
incidence of PTEN mutations in Chinese patients with primary small cell carcinoma of the
esophagus. BMC Cancer 14:19
Zhang P, Liu H, Ma S, Men S, Li Q, Yang X, Wang H, Zhang A (2016a) A label-free ultrasensitive
fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage
toehold strand-displacement-driven assembly of G-quadruplex DNA. Biosens Bioelectron
80:538–542
Zhang K, Deng R, Li Y, Zhang L, Li J (2016b) Cas9 cleavage assay for pre-screening of sgRNAs
using nicking triggered isothermal amplification. J Chem Sci 7:4951–4957
Zhang Y, Qian L, Wei W, Wang Y, Wang B, Lin P, Liu W, Xu L, Li X, Liu D, Cheng S, Li J, Ye Y,
Li H, Zhang X, Dong Y, Zhao X, Liu C, Zhang H, Ouyang Q, Lou C (2017) Paired design of
dCas9 as a systematic platform for the detection of featured nucleic acid sequences in patho-
genic strains. ACS Synth Biol 6:211–216
Zhang B, Wang Q, Xu X, Long F, Li W, Shui Y, Xia X, Wang J (2018) Detection of target DNA
with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique. Anal Bioanal Chem
410:2889–2900
Zhao Y, Chen F, Li Q, Wang L, Fan C (2015) Isothermal amplification of nucleic acids. Chem Rev
115:12491–12545
Zhu H, Chu B, Zhang C, Liu F, Jiang L, He Y (2017) Hyperspectral imaging for presymptomatic
detection of tobacco disease with successive projections algorithm and machine-learning
classifiers. Sci Rep 7:4125
Zischewski J, Fischer R, Bortesi L (2017) Detection of on-target and off-target mutations generated
by CRISPR/Cas9 and other sequence specific nucleases. Biotechnol Adv 35:95–104
Plant Virus Diagnostics: Traditional
to Recent and Emerging Advances 5
V. K. Baranwal, Sajad Un Nabi, and Manoj K. Yadav
Abstract
Viral diseases cause huge economic losses in agriculture systems globally and
their management is a big challenge to growers as well as researchers. For any
successful management of viral disease, detection and identification of plant virus
associated with the plant disease is of foremost importance. Specific, robust and
precise diagnostics of plant viruses is also essential to prevent the introduction of
viruses in a new region as free trade agreement in absence of virus diagnostics can
lead to the introduction of new viruses through transfer and exchange of planting
material. The advancements in molecular biology have led to major
breakthroughs in the form of newer, sensitive and efficient diagnostic techniques.
Several detection techniques have been developed in the last three decades, which
are broadly based on serological and molecular approaches for the detection of
viruses. Next-generation sequencing has been used to detect unknown or new
viruses in several crops. Several specific, simple, fast, farmer-friendly and sensi-
tive technologies as point-of-care diagnosis have also been developed. Plant virus
diagnostics, after integrating with portable devices, has a promising future for
on-site field diagnosis. Here, we review various methods of plant virus detection
including those methods that have been successfully used in field conditions.
Keywords
Plant viruses · Detection · Robust · Serological · Molecular
V. K. Baranwal (*) · S. Un Nabi · M. K. Yadav

Advanced Center of Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural
Research institute, New Delhi, India

https://doi.org/10.1007/978-981-15-6275-4_5
98 V. K. Baranwal et al.
5.1 Introduction
Viruses are catastrophic, tiny entities, which are visible only under a transmission
electron microscope. The genetic material of viruses comprises of either DNA or
RNA, encapsidated in coat protein. Being obligate in nature, viruses exclusively
utilize the metabolic machinery inside the host cell for its multiplication. Plant
viruses are ubiquitous and rank second among economically vital plant pathogens
(Vidaver and Lambrecht 2004). It is difficult to estimate crop loss due to plant viral
diseases as it is highly variable and depends on virus strain, region, time of infection
and cultivar (Sharma et al. 2014). However, it has been reported that economically
significant viral diseases cause economic losses more than several billion dollars
annually throughout the world (Hull 2002; Joo et al. 2014; Islam et al. 2017). Viruses
intervene by the allocation of resources produced through photosynthesis inside
plant cells and alter physiology, which results in symptoms like mosaic, necrosis,
mottling, curling and puckering. However, sometimes the phenotypic expression of
symptoms may not be visible due to latent infection of plant viruses (Van der Want
and Dijkstra 2006; Nabi et al. 2018). Additionally, plants can also express symptoms
similar to viral diseases in response to nutritional disorders, harsh climatic conditions
and other abiotic agents (Vander Want and Dijkstra 2006). Thus, symptom-based
diagnostics of virus disease is complex in comparison to other pathogens (Lievens
et al. 2005). The diagnostic methods as well as their application are influenced by
several factors (Islam et al. 2017). Many virus species from closely related families
show high mutation rates and exchange of genetic components, which can result in
recombination events between these related species, thus enhancing the chances of
genetic variability and aggressiveness in viral strains (García-Arenal et al. 2001).
Similarly, the mixed infection of more than two viruses infecting a single plant
results in either synergistic or antagonistic effects among the viruses (Syller 2012).
Also the increased globalization of trade as well as the climate change has consider-
ably lead to a rise in the plant virus movement. Consequently, viral disease preven-
tion and economic damage warrant the robust, accurate and specific methods for
detection. Several methods, including new high emergent throughput diagnostic
techniques, have been developed to detect plant viruses, viz., biological, physical,
morphological, immunological, molecular methods and point-of-care techniques
(Lopez et al. 2008; Yadav and Khurana 2016; Rani et al. 2019).
5.2 Detection and Diagnostic Methods
Management of any plant disease depends on proper identification of disease and its
causal agent. Therefore, detection and diagnosis is the most important aspect for
managing plant viruses, as the viral infection remains systemic throughout the life
cycle of the crop. Detection and identification of viruses is based on biological
properties, morphological properties and intrinsic properties of the viruses. An
overview of various methods suitable for plant virus detection is described in the
following sections.
5 Plant Virus Diagnostics: Traditional to Recent and Emerging Advances 99
5.2.1 Detection Based on Biological Properties
Biological indexing was an essential tool for the detection of a particular plant virus
followed by characterization. Biological detection is based on the symptom expres-
sion on the natural hosts or on susceptible herbaceous or woody indicator plants
mechanically inoculated by sap or grafting or budding. In the present era, the labour-
intensive and time-consuming biological indexing has little application and not
preferred for routine virus detection.
5.2.2 Detection Based on Morphological Properties
5.2.2.1 Electron Microscopy (EM)

Morphological properties like shape, size and other surface features of the virus
particle are a vital requirement for virus detection (Bernd and Gunther 2009). In
electron microscopy, a negative staining leaf-dip preparation technique is used
universally to detect differential filamentous and rod-shaped viruses rather than
isometric and other viruses. The EM aids in rapid and precise results, and, in the
majority of cases it is sufficient to determine the shape and size of virus particles
(Harris 2007; Wild 2008).
5.2.3 Detection Based on Viral Proteins
Methods are generally called as serological or immunoassays which use viral

proteins, mostly coat protein (CP) for virus detection. Numerous serological or
immunological tests have been developed and used for plant virus detection.
These approaches are based on the interaction between a protein or proteins (antigen)
in the virus particle with antibodies raised against them in vertebrates. The various
immunological tests are as under.
5.2.3.1 Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA is the plate-based immunoassay and most versatile due to its specificity,
simplicity and robustness. The test is primarily based on the specificity of the
antibody to interact with the antigen (coat protein of virus). The ELISA microtitre
plate is coated with primary antibody (DAS-ELISA) followed by infected plant sap,
which is further detected with the help of secondary antibody conjugated with the
reporter molecule (an enzyme). If the target virus is present in the sap, it will interact
first with the primary antibody, which is consequently detected by conjugate anti-
body in the presence of substrate. The plant virus detection is confirmed through the
production of the visible colour. The quantity of the colour product is estimated
through ELISA-Reader, which gives a quantitative estimation of viral load indi-
rectly. Similarly sometimes microtitre plate is precoated with plant sap
(DAC-ELISA), which is further detected by primary and secondary antibodies
(Clark and Adams 1977). It has been widely used for the detection of many
important viruses including Apple stem grooving virus (ASGV), Citrus tristeza virus
(CTV), Potato virus X (PVX), Potato virus Y (PVY) and Potato leafroll virus
(PLRV) (Sun et al. 2001; El-Araby et al. 2009; Nabi et al. 2018). Polyclonal
antibodies have been produced against a large number of plant viruses using their
recombinant proteins and have been successfully used for specific detection of
Grapevine leafroll virus-3, Grapevine leafroll virus-4, Garlic common latent
virus, Leek yellow stripe virus and Banana streak Mysore virus in India (Pramesh
et al. 2012; Kumar et al. 2015, 2018; Rai et al. 2018). A more detailed information on
serological detection of plant viruses in India has been reviewed by Bhat and
Maheshwari (2017).
5.2.3.2 Tissue Immunobinding Assay (TIBA)

The TIBA is similar to ELISA, in which an impression is made onto the nylon or
nitrocellulose by pressing plant tissue over it, which is further used to detect the virus
following similar procedures as in ELISA (Webster et al. 2004). Several plant
viruses have been detected using TIBA, which includes Alfalfa mosaic virus
(AMV), Capsicum chlorosis virus (CaCV), Bean leafroll virus (BLRV), Bean
yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Soybean dwarf
virus (SbDV) and Watermelon bud necrosis virus (WBNV) (Kumari et al. 2008).
5.2.3.3 Immunosorbent Electron Microscopy (ISEM)

It is a combination of electron microscopy with serological specificity for morpho-
logical observation of virus particles (Derrick 1973) and has been used for the
detection of a broad range of plant viruses (Rod and polyhedral) (Naidu and Hughes
2001). From samples, the virus particles are selectively entrapped on copper grids
coated with antiserum specific to the virus (Lima and Purcifull 1980). The grid is
treated with infected plant sap for 3–4 h at 25 C after washing off the excess of
antibody. Further, the grid is washed thrice and stained with 1.0% uranyl acetate in
50% ethanol and dried. The grid is finally observed under a transmission electron
microscope for the detection of virus particles (Lima and Purcifull 1980). Because of
its higher sensitivity and rapidity, it finds wide applicability in the detection of
viruses present in low titre in plants (Khurana 1990).
5.2.4 Detection Based on Viral Nucleic Acid
Molecular detection methods involving amplification of nucleic acids (DNA or

RNA) have been developed and revitalized day by day for the detection of most
important plant viruses. A broad range of molecular diagnostic techniques such as
polymerase chain reaction (PCR), reverse transcriptase-PCR, multiplex PCR and
quantitative PCR proved to be the most important approaches. These methods are
more advantageous over biological or serodiagnostic techniques, as any viral geno-
mic region can be targeted to develop the detection tools. As many of the viruses are
poor immunogens, nucleic acid-based detection method would be a more promising
approach for detecting such viruses. These methods are highly efficient, robust,
specific and sensitive in comparison to immunological assays and are suitable for
plant virus detection on routine basis accurately (Chen and Adams 2001; Zheng et al.
2010).
5.2.4.1 PCR and RT-PCR

Most important milestone in molecular biology was the invention of PCR (Mullis
and Faloona 1987). Based on the sequence information, PCR has been effectively
used for the detection and characterization of plant viruses (Yadav and Khan 2009).
It is a highly accurate and sensitive detection protocol, as it relies mostly on the
presence of distinctive sequences in the viral genome. In RNA viruses, the first
cDNA strand is synthesized with the help of reverse transcriptase (RT) enzyme,
which is further used as a template for amplification. The amplicons obtained in PCR
from genomic regions of the virus need to be identified by cloning and sequencing. It
has been used to detect several viruses such as Citrus yellow mosaic virus Papaya
ringspot virus (PRSV), Potato virus X (PVX), Cucumber mosaic virus (CMV),
Potato leafroll virus (PLRV), Apple stem grooving virus (ASGV), Prunus necrotic
ringspot virus (PNRSV), Apple mosaic virus (ApMV), Leek yellow stripe virus
(LYSV), Apple necrotic mosaic virus (ApNMV), and Bhendi yellow vein mosaic
virus (BYVMV) (Baranwal et al. 2003; Jain et al. 2004; Rana et al. 2011; Gupta et al.
2013; Roy et al. 2015; Nabi et al. 2020). The PCR or RT-PCR has been optimized
for a number of plant viruses in India (Sharma et al. 2017).
5.2.4.2 Multiplex PCR/RT-PCR

It is a procedure of simultaneous detection of two or more viruses/viroids in a single
reaction tube (Nassuth et al. 2000). This technique is helpful for simultaneous
detection of viruses in mixed infection in the same host. Many sets of primers are
used in the single reaction mixture, which target different regions in the genome of
multiple viruses. The oligonucleotides (primers) are designed in such a manner that
these lack complementarities and have the same annealing temperature for amplifi-
cation. It is advantageous by saving time and reagent costs against individual PCR
(Deb and Anderson 2008; Roy et al. 2005, Bertolini et al. 2003). Several viruses
such as Apple mosaic virus (ApMV), Apple chlorotic leafspot virus (ACLSV),
Prunus necrotic ringspot virus (PNRSV) and Plum pox virus (PPV) were detected
simultaneously in apple trees via multiplex-PCR (Menzel et al. 2002). Selvarajan
et al. 2011 developed a multiplex-reverse transcription-PCR for the detection of
BSMYV and Banana bunchy top virus (BBTV) simultaneously in banana samples.
Multiplex RT-PCR for 4 viruses in garlic and for 4 viruses and a fastidious greening
bacterium in citrus has been optimized in India (Majumder and Baranwal 2014;
Meena and Baranwal 2015).
5.2.4.3 Immunocapture PCR (IC-PCR)

This technique involves a combination of PCR and immunoassay to entrap virus
particles by antibodies, which is further amplified using specific primers. The virus is
first adsorbed on antibody-coated PCR tube which is further removed in the presence
of non-ionic surfactant Triton X-100 by heating, followed by amplification using

PCR. Entrapment of virus particle by antibody helps to purify the virions from the
plant sap, hence proves very successful for viruses which are present in low titre and
extracts having certain inhibitors which can hamper PCR. The IC-PCR has been
employed for the detection of several viruses such as Pepper mild mosaic virus
(PMMV), Cucumber mosaic virus (CMV), Citrus tristeza virus (CTV), Grapevine
fanleaf virus (GFLV) and Tomato spotted wilt virus (TSWV) (Narayanasamy 2011).
A duplex-immunocapture PCR protocol developed and optimised for simultaneous
detection of two viruses, namely SCSMV and SCMV (Subba Reddy et al. 2011). It
has also been optimized for Grapevine leafroll-associated virus-3 (Kumar 2013).
5.2.4.4 Real-Time PCR (qPCR)

It is an advanced version of molecular detection, which is accurate, sensitive and
specific over conventional PCR. The basic principle is the monitoring of accumula-
tion of amplicon in real-time by the labelling of primers or amplicon with fluorescent
dyes. Several dyes have been used which binds to any dsDNA. It is a simple and
economic way to quantify amplification. It avoids the downstream process like
agarose gel electrophoresis as a product at each cycle is detected by fluorogenic
molecules. It has wide application in plant virus diagnostics and quantification
(Schaad and Frederick 2002; Boonham et al. 2004). A dye-based one-step reverse
transcription-quantitative PCR (RT-qPCR) assay was developed for the robust and
easy detection of Cardamom mosaic virus (CdMV) and Banana bract mosaic virus
(BBrMV) which infect cardamom (Siljo et al. 2014), Piper yellow mottle virus
(PYMoV) and CMV infecting black pepper plants (Bhat and Siljo 2014).
5.2.4.5 Microarray
It is evolved from southern blotting, which instead of nitrocellulose membrane uses
glass as a support (Maskos and Southern 1992). It can detect virus-specific serotypes
with high accuracy using specific probes in a single assay (Nam et al. 2014). The
25 bp to 70 bp nucleotide single-stranded synthesized DNA probes are hybridized
with the viruses extracted from plant samples. It is capable to identify plant virus at
the genus level and can also differentiate related strains (Boonham et al. 2007). The
important demerit is cost, as it requires dust-free room, sophisticated machine for
spotting probes and reading reactions. This platform is efficient and specific for the
detection of viruses along with satellites like CMV, Tomato infectious chlorosis
virus, TSWV, Tomato mosaic virus, Pepino mosaic virus, PVX, PVA, PLRV, PVY,
PVM and PVS (Bystricka et al. 2005; Lee et al. 2003). A microarray chip has been
developed at the Advance Centre for Plant Virology, ICAR-IARI, New Delhi for
parallel detection of more than 1100 viruses and 30 viroids whose genomic
sequences are available in the GenBank (Unpublished).
5.2.4.6 Isothermal Amplification

These methods were a major breakthrough in molecular diagnostics, avoiding the
need for costly equipments (PCR) to amplify DNA or RNA. It depends on the
non-thermal separation of the dsDNA. In this assay, the sequence-specific primers
amplify the target DNA just like in conventional PCR (Boonham et al. 2014).
Several methods, viz., loop-mediated isothermal amplification (LAMP), rolling
circle amplification (RCA) and recombinase polymerase amplification (RPA) have
significantly been used in plant virus diagnostics (Zhao et al. 2015).
Loop-Mediated Isothermal Amplification

The limitations of PCR-based molecular methods, such as costly thermocycler
requirement and low specificity in some cases, consequently lead to the development
of LAMP. It can amplify target DNA at very low copy number in a short span of
time. In a LAMP reaction, specifically Bst DNA polymerase having high
processivity and strand-displacement activity along with three primer pairs (internal,
external and loop primers) complementary to six specific genomic regions in DNA
template are used. The reaction is set at a temperature range of 60–65 C for
30–60 min. The primers are designed from online software Primer Explorer V4
(https://primerexplorer.jp/elamp4.0.0/). It has been used to detect plant viruses such
as Plum pox virus, Tomato yellow leaf curl virus, Wheat yellow mosaic virus,
Banana bunchy top virus and Zucchini yellow mosaic virus (Kuan et al. 2014;
Fukuta et al. 2003; Varga and James 2006; Zhang et al. 2011; Peng et al. 2012;
Ahmadi et al. 2013).
Rolling Circle Amplification

The discovery of RCA has revolutionized the field of diagnostics especially in
begomoviruses, where it was first time used for cloning of a single-stranded circular
DNA (Haible et al. 2006; Inoue-Nagata et al. 2004). The procedure involves phi
29 polymerase at isothermal temperature for sequence-independent amplification. It
uses exoresistant random hexamer primers instead of specific primers that provide an
advantage of amplification of variants as well. The RCA-amplified product can be
characterized by restriction digestion using restriction enzymes and by direct
sequencing (Haible et al. 2006). It is highly sensitive as compared to PCR for the
detection of integrated and episomal viral sequences of Badnaviruses (James et al.
2011; Baranwal et al. 2013; Sharma et al. 2015).
5.2.5 Recent and Emerging Advances
With the demand of producers for access to the high yielding plant varieties, to
facilitate the movement of germplasm from country to country, while maintaining
phytosanitary values, the efficient, rapid, robust, onsite and cheap methods of
diagnostics are need of the hour (Varvara et al. 2018). With continued efforts of
plant virologists across globe, novel and emergent approaches have been identified
to detect the causal agent of new and unusual diseases. These new high-throughput
diagnostic approaches are classified as lab- and point-of-care-based.
5.2.5.1 Lab-Based Methods
Next-Generation Sequencing (NGS)

From last two decades advancement in sequencing technologies had led to the
development of new methods for the detection, identification and characterization
of viruses. The availability of NGS has revolutionized the discovery and ease at
which novel phytovirome have been reported from agricultural ecosystems during
the last decade (Villamor et al. 2019). The new approach referred as NGS, or deep
sequencing or high-throughput or in-parallel, is the sequencing of total nucleic acid
content in symptomatic/asymptomatic samples for subsequent identification of
pathogen(s) using bioinformatics tools (Qingfa et al. 2015). Millions to billions of
nucleotides can be sequenced in parallel, minimizes cloning of large number of
fragments, which are used in Sanger sequencing. It substantially yields more
throughputs. The NGS being sequence and culture-independent approach, hence
concurrently detects RNA/DNA viruses and viroids even if present in low titre. It is a
revolutionary technology for easy identification of novel and unknown viruses as
compared to traditional diagnostics which uniquely target a species/strain (Baranwal
et al. 2015). The virome of various plants like citrus, apple, grapevine, etc. (Rott
et al. 2017) has been explored using the NGS technology.
Droplet Digital PCR (ddPCR)

Droplet Digital PCR invented in 2011 is a recent technology based on water-oil
emulsion droplet technology to increase the amplification efficiency of standard
PCR. The ddPCR is accurate, robust, sensitive and precise, producing simple
readouts such as ‘YES’ or ‘NO’. It provides absolute quantification as it eliminates
the need for standard curves and normalization. It is useful, where virus titre is low
and has uneven distribution in infected plants (Selvaraj et al. 2018). Until now, it has
been used for the detection of Grapevine red blotch-associated virus (GRBaV)
(Voegel and Nelson 2018).
5.2.5.2 Point of Care (POC) Detection

These approaches are on-site detection procedures without the use of sophisticated
equipments and do not require expert personnel. The POC devices are advantageous,
due to their sensitivity portability, accuracy and robustness (Lau et al. 2016).
Lateral Flow Immunoassay (LFIA)

It is a rapid and widely applied immune-chromatographic technique for plant virus
detection, especially in horticultural crops (Salomone et al. 2004). Due to the use of
simple devices, it is easy to perform even by a non-expert person. It is quick and
simple, hence allows on-field detection of plant viruses. The LFD is a paper-based
platform spotted with the sap of infected plant tissue; the sample flows via it and
commences antigen-antibody interaction, which finally leads to chemical reaction
detectable within 5–25 min as chromogenic bar/line for test and control samples.
Several kits have been developed for the detection of plant viruses. It has been used
in the detection of several plant viruses either individually or in combination such as

TMV, PVM PVX, PVA, PVY, PVS and PVY (Drygin et al. 2011).
Recombinase Polymerase Amplification (RPA)

It is a recent isothermal approach based on enzymatic denaturation. It uses
recombinase, which forms primer-recombinase complex for initiation of DNA
amplification. It performs amplification at the temperature range of 37–42 C, but
sometimes give amplification also at room temperature. The reaction mixture
comprises of primers of length 32–36 nucleotides, with other buffers and three
enzymes (recombinase, single-stranded DNA binding protein (SSB) and strand
displacing polymerase). The process begins by scanning of double-stranded DNA
with recombinase enzyme to bind primers on cognate sites in presence of ATP
molecule and open the double-helical structure which in turn is stabilized by the SSB
protein. The disassembly of recombinase is facilitated by ATP hydrolysis followed
by the addition of complementary nucleotides into the primer sequence via strand
displacing polymerase to form a new strand of DNA. The technique has been used
for the detection of several viruses like Plum pox virus (PPV), Banana streak Mysore
virus (BSMYV) and CMV (Zhang et al. 2014: Kapoor et al. 2017; Nishant et al.
2019).
Antibody-Based Biosensors
The antibody-based biosensors mainly work on the ELISA principle for signal
generation after target antigen (Ag) capturing using immobilized antibodies (Abs)
placed on a solid surface. The complex formed by Ag-Ab can be divided using an
immuno-magnetic separator (Cho and Irudayaraj 2013) and signal strength depends
on several types of potent transducers (Pilolli et al. 2013). The bigger limitation of
this method is cost, cross-reactivity and longer time for antibody synthesis (Lau and
Botella 2017). These biosensors are generally applied in several diagnostic including
plant viruses, e.g., Citrus tristeza virus (Shojaei et al. 2016).
Aptamer-Based Biosensors (Aptasensors)

Aptamers are defined as single-stranded nucleic acid consisting of 30–32 bp length
or amino acid polymers which displays a high degree of affinity to the target
molecule (Bahadir and Sezginturk 2016). An aptamer-based method has significant
advantages over the antibodies use due to their cheapness and less production time
(2–3 days). Moreover, these show high stability, flexibility and versatile binding
abilities (Seok Kim et al. 2016). The aptasensors involve the selected aptamer
immobilization on a solid surface to arrest the target molecule, which further help
to convert the resultant signal into readout with the help of the transducer. To
enhance the sensitivity and specificity of aptasensors, several nanomaterials have
been used (Khedri et al. 2018). The first aptamer detection method was developed
against Apple stem pitting virus (ASPV) using the coat protein of virus (Balogh et al.
2010). However, its use is still limited.
Lab-on-Chip (LOC) Devices and Paper-Based Devices

It is a miniaturized microfluidic device, which integrates several laboratory methods
onto a single chip, which includes biochemical methods, DNA sequencing and
finally detection of the pathogen. The various modules of a LOC device include
processor (for sampling), amplifier (for signal amplification), transducer (to produce
a measurable signal) and software (for data analysis) (Luka et al. 2015). The major
challenge faced by LOC devices is the requisite for label-free assays and new stable
polymers which hinders their on-site pathogen detection. However, the microfluidics
paper-based analytical devices (μPADs) development with 2-dimensional and
3-dimensional potential have been able to overcome the demerits faced using
standard LOC devices with a permeable porous cellulose paper (pore size
1–10 μm) which helps to reduce the costs further. These LOC devices are simple,
cheap, disposable, portable and easy to handle. The main LOC body is designed with
four parts: the sample pad (to load the sample), an indicator pad (for the test and
control line), a conjugate pad (for sample binding and label) and an absorption pad
(for the absorption of leftover fluids) (Mahato et al. 2017). Initially, μPADs pro-
duced mostly colourimetric readouts but with the advancement in wireless connec-
tivity (Novarum, Android app, iPhone and iBG star) the target quantitation task can
be done with the help of a cell phone-based detection system (Syedmoradi et al.
2017). These techniques are restricted with few limitations, for instance, light
scattering, non-specific adsorption, variable sensitivity and non-uniform wicking.
However, further research in μPADs could potentially help in the development of a
‘POCKET SIZED’ diagnostic techniques for pathogens detection in nurseries
(Mahato et al. 2017). Although this method has been used for the detection of
several plant pathogens, it has to be still used for viruses.
Cell Phone (CP)-Based Devices

These devices are based on the integration of sensor technologies with modern
communication systems to capture images of the target, followed by comparison
with images of diseased plants already stored. The data thus generated with these
devices could be easily transmissible from one location to another. Similarly, some
applications developed for crop plants aid in detection of pathogens based on colour
intensity, texture, edge outline and number of spots of the infected leaves. The
Google app, Plantix was developed by an AgTech startup which contains more
than 55,000 images of plants and could able to successfully detect over 60 plant
diseases (http://www.fao.org/e-agriculture/news/plantix-app-detect-and-cure-your-
plantdiseases). These CP-based methods need further refinement and optimization
prior to their acceptance in plant virus diagnostics to substitute molecular and
serological-based techniques, particularly in terms of accuracy (Vashist et al. 2015).
5.3 Conclusion
Diagnostics is the basis for any disease management programme and plant virus
diagnostics is gaining significance, due to rapid spread and identification of novel
plant viruses and to facilitate enforcement of quarantine measures. The detection and
diagnostic methods mostly include a number of immunological and molecular
techniques based on intrinsic virus properties. Developments of isothermal amplifi-
cation methods are gaining high acceptance owing for their portability and appropri-
ateness for resource crunch laboratories. Additionally, they also show sensitivity,
cost-effective, rapid and robust methodologies to support onsite detection of plant
pathogens. Point of care detection techniques are comparatively simpler, can be
performed by non-expert persons and need little handling. However, the major
challenge for the development of onsite technologies remains cost-effectiveness
and affordability.
References
Ahmadi S, Almasi MA, Fatehi F, Struik PC, Maradi A (2013) Visual detection of potato leafroll
virus by one-step reverse transcription loop-mediated isothermal amplification of DNA with
hydroxynaphthol blue dye. J Phytopathol 161:120–124
Bahadir EB, Sezginturk MK (2016) A review on impedimetric biosensors. Artif Cells Nanomed
Balogh Z et al (2010) Selection and versatile application of virus-specific aptamers. FASEB J
24:4187–4195
Baranwal VK, Majumder S, Ahlawat YS, Singh RP (2003) Sodium sulphite yields improved DNA
of higher stability for PCR detection of Citrus yellow mosaic virus from citrus leaves. J Virol
Methods 112:153–156
Baranwal VK, Sharma SK, Khurana D, Varma R (2013) Sequence analysis of shorter than genome
length episomal banana streak OL virus like sequences isolated from banana in India. Virus
Genes 48:120–127
Baranwal VK, Jain P, Saritha RK, Jain RK (2015) Detection and partial characterization of Cowpea
mild mottle virus in mungbean and urdbean by deep sequencing and RT PCR. Crop Prot
75:77–79
Bernd Z, Günther Z (2009) Rapid diagnosis of plant virus diseases by transmission electron
microscopy. J Virol Methods 162:163–169
Bertolini E, Olmos A, Lopez MM, Cambra M (2003) Multiplex nested reverse-transcription
polymerase chain reaction in a single tube for sensitive and simultaneous detection of four
RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees. Phytopathology
93:286–292
Bhat AI, Siljo A (2014) Detection of viruses infecting black pepper by SYBR green-based real-time
PCR assay. J Plant Pathol 96:105–109
Bhat AI, Maheshwari YA (2017) Century of plant virology in India In: application of immuno-
diagnosis for plant viruses occurring in India. Springer, Singapore, pp 583–619
Boonham N, Perez LG, Mendez MS, Peralta EL, Blockley A, Walsh K, Barker I, Mumford RA
(2004) Development of a real-time RT-PCR assay for the detection of potato spindle tuber
viroid. J Virol Methods 116:139–146
Boonham N, Tomlinson J, Mumford RA (2007) Microarrays for rapid identification of plant
viruses. Annu Rev Phytopathol 45:307–328
Boonham N, Kreuzeb J, Winterc S, Vlugtd R, Bergervoetd J, Tomlinsona J, Mumforda R (2014)

Methods in virus diagnostics: from ELISA to next generation sequencing. Virus Res. https://doi.
org/10.1016/j.virusres.2013.12.007
Bystricka D, Lenza O, Mraza I, Piherovad L, Kmochd S, Sipc M (2005) Oligonucleotide-based
microarray: a new improvement in microarray detection of plant viruses. J Virol Methods
128:176–182
Chen J, Adams MJ (2001) A universal PCR primer to detect members of the Potyviridae and its use
to examine the taxonomic status of several members of the family. Arch Virol 146:757–766
Cho IH, Irudayaraj J (2013) In-situ immuno-gold nanoparticle network ELISA biosensors for
pathogen detection. Int J Food Microbiol 164:70–75
Clark MF, Adams AN (1977) Characteristics of the micro-plate method of enzyme linked immu-
nosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483
Deb M, Anderson JM (2008) Development of a multiplexed PCR detection method for barley and
cereal yellow dwarf viruses, wheat spindle streak virus, wheat streak mosaic virus and soil-
borne wheat mosaic virus. J Virol Methods 148:17–24
Derrick KS (1973) Quantitative assay for plant viruses using serologically specific electron
microscopy. Virology 56:652–653
Drygin YF, Blintsov AN, Grigorenko VG, Andreeva IP, Osipov AP, Varitzev YA, Uskov AI,
Kravchenko DV, Atabekov JG (2011) Highly sensitive field test lateral flow immunodiagnostics
of PVX infection. Appl Microbiol Biotechnol 93:179–189
El-Araby SW, Ibrahin AI, Hemeida AA, Mahmo A, Soliman MA, El-Attar KA, Mazyad MH
(2009) Biological, serological and molecular diagnosis of three major potato viruses in Egypt.
Int J Virol 5:77–88
Fukuta S, Kato S, Yoshida K, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y (2003)
Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction. J
Virol Methods 112:35–40
García-Arenal F, Fraile A, Malpica JM (2001) Variability and genetic structure of plant
viruspopulations. Annu Rev Phytopathol 39:157–186
Gupta N, Prabha K, Islam S, Baranwal VK (2013) First report of Leek yellow stripe virus in garlic
from India. J Plant Pathol 95:4
Haible D, Kober S, Jeske H (2006) Rolling circle amplification revolutionizes diagnosis and
genomics of geminiviruses. J Virol Methods 135:9–16
Harris JR (2007) Negative staining of thinly spread biological samples. In: Kuo J (ed) Methods in
molecular biology, electron microscopy, methods and protocols, vol 369. Humana Press,
Totowa, pp 107–142
Hull R (2002) Matthews’ plant virology, 4th edn. Academic, San Diego
Inoue-Nagata AK, Albuquerque LC, Rocha WB, Nagata T (2004) A simple method or cloning the
complete begomovirus genome using the bacteriophage ϕ29 DNA polymerase. J Virol Methods
116:209–211
Islam W, Zhang J, Adnan M, Noman A, Zaynab M, Wu Z (2017) Plant virus ecology: a glimpse of
recent accomplishments. Appl Ecol Environ Res 15:691–705
Jain RK, Sharma J, Sivakumar AS, Sharma PK, Byadgi AS, Verma AK, Varma A (2004)
Variability in the coat protein gene of Papaya ringspot virus isolates from multiple locations
in India. Arch Virol 149:2435–2442
James AP, Geijskes RJ, Dale JL, Harding RM (2011) Development of a novel rolling-circle
amplification technique to detect Banana streak virus that also discriminates between integrated
and episomal virus sequences. Plant Dis 95:57–62
Joo JJ, Ho j, Jaejong N (2014) A review of detection methods for the plant viruses. Res Plant Dis
20:173–181
Kapoor R, Srivastava N, Kumar S, Baranwal VK (2017) Development of recombinase polymerase
amplification assay for the diagnosis of Banana bunchy top virus in different banana cultivars.
Arch Virol 162:2791–2796
Khedri M et al (2018) Detection of food-born allergens with aptamer-based biosensors. Trac Trends
Anal Chem 103:126–136
Khurana SMP (1990) Modern approaches for detection and management of the potato viruses and
viroid. In: Grewal JS et al (eds) Current facets in potato research. IPA, CPRI, Simla, pp 98–108
Kuan CP, Deng TC, Huang HC, Chi HH, Lu YL (2014) Use of reverse transcription loop-mediated
isothermal amplification for the detection of Zucchini yellow mosaic virus. J Phytopathol
162:238–244
Kumar S (2013) Studies on virus (es) associated with grapevine leafroll disease in India. Ph.D.
thesis. Indian Agricultural Research Institute, New Delhi, India
Kumar S, Rai R, Baranwal VK (2015) Development of an immunocapture–reverse transcription–
polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the
detection of Grapevine leafroll-associated virus 3 (GLRaV-3). Phytoparasitica 43:311–316
Kumar S, Singh P, Rai R, Baranwal VK (2018) Development of immunodiagnostics for the
detection of Grapevine leafroll-associated virus 3 (GLRaV-3) in grapevine using in vitro
expression and purification of its coat protein gene. J Plant Biochem Biotechnol 27:425–434
Kumari SG, Makkouk KM, Loh MH, Negassi K, Tsegay S, Kidane R, Kibret A, Tesfatsion Y
(2008) Viral diseases affecting chickpea crops in Eritrea. Phytopathol Mediterr 47:42–4.9
Lau HY, Botella JR (2017) Advanced DNA-based point-of-care diagnostic methods for plant
diseases detection. Front Plant Sci 8
Lau HY et al (2016) Field demonstration of a multiplexed point-of-care diagnostic platform for
plant pathogens. Anal Chem 88:8074–8081
Lee GP, Min BE, Kim CS, Choi SH, Harn HH, Kim SU, Ryu KH (2003) Plant virus cDNA chip
hybridization for detection and differentiation of four cucurbit-infecting Tobamoviruses. J Virol
Methods 110:19–24
Lievens B, Grauwet TA, Cammue BPA, Thomma BPHJ (2005) Recent developments in
diagnostics of plant pathogens: a review. Recent Res Dev Microbiol 9:57–79
Lima JAA, Purcifull DE (1980) Immunochemical and microscopical techniques for detecting
blackeye cowpea mosaic and soybean mosaic viruses in hypocotyls of germinated seeds.
Phytopathology 70:142–147
Lopez MM, Llop P, Olmos A, Marco-Noales E, Cambra M, Bertolini E (2008) Are molecular tools
solving the challenges posed by detection of plant pathogenic bacteria and viruses? Curr Issues
Mol Biol 11:13–45
Luka G et al (2015) Microfluidics integrated biosensors: a leading technology towards Lab-on-a-
chip and sensing applications. Sensors 15:30011–30031
Mahato K, Srivastava A, Chandra P (2017) Paper based diagnostics for personalized health care:
emerging technologies and commercial aspects. Biosens Bioelectron 96:246–259
Majumder S, Baranwal VK (2014) Simultaneous detection of four garlic viruses by multiplex
reverse transcription PCR and their distribution in Indian garlic accessions. J Virol Methods
202:34–38
Maliogka VI, Angelantonio M, Pasquale S, Ana B (2018) Recent advances on detection and
characterization of fruit tree viruses using high-throughput sequencing technologies. Viruses
10:436
Maskos U, Southern EM (1992) Oligonucleotide hybridizations on glass supports: a novel linker for
oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.
Nucleic Acids Res 20:1679–1684
Meena RP, Baranwal VK (2015) Development of multiplex polymerase chain reaction assay for
simultaneous detection of clostero-, badna- and mandari-viruses along with huanglongbing
bacterium in citrus trees. J Virol Methods 235:58–64
Menzel W, Jelkmann W, Maiss E (2002) Detection of four apple viruses by multiplex RT-PCR
assays with coamplification of plant mRNA as internal control. J Virol Methods 99:81–92
Mullis K, Faloona F (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain
reaction. Methods Enzymol 155:335–350
Nabi SU, Mir JI, Sharma OC, Singh DB, Zaffer S, Sheikh MA (2018) Optimization of tissue and
time for rapid serological and molecular detection of Apple stem pitting virus and Apple stem
grooving virus in apple. Phytoparasitica 46:705–713
Nabi SU, Baranwal VK, Yadav MK et al (2020) Association of Apple necrotic mosaic virus
(ApNMV) with mosaic disease in commercially grown cultivars of apple (Malus domestica
Borkh) in India. 3 Biotech 10:122
Naidu RA, Hughes JA (2001) Methods for the detection of plant virus diseases. In: Hughes JA, Odu
BO (eds) Plant virology in Sub-Saharan Africa. Proceedings of a conference organized by IITA.
International Institute of Tropical Agriculture, Ibadan, pp 233–260
Nam M, Kim JS, Lim SM, Park CY, Kim JG, Choi FS, Lim H, Moon JS, Lee SH (2014)
Development of the large-scale oligonucleotide chip for the diagnosis of plant viruses and its
practical use. Plant Pathol J 30:51–57
Narayanasamy P (2011) Detection of virus and viroid pathogens in plants. Microbial plant
pathogens- detection and disease diagnosis. Springer, Dordrecht/
Heidelberg/London/New York, pp 7–220
Nassuth A, Pollari E, Helmeczy K, Stewart S, Kofalvi SA (2000) Improved RNA extraction and
one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in
plant extracts. J Virol Methods 90:37–49
Nishant S, Kapoor R, Kumar R, Kumar S, Saritha RK, Kumar S, Baranwal VK (2019) Rapid
diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time
isothermal reverse transcription-recombinase polymerase amplification assay. J Virol Methods
270:52–58
Peng J, Zhang J, Xiaa Z, Li Y, Huang J, Fan Z (2012) Rapid and sensitive detection of banana
bunchy top virus by loop-mediated isothermal amplification. J Virol Methods 185:254–258
Pilolli R, Monaci L, Visconti A (2013) Advances in biosensor development based on integrating
nanotechnology and applied to food-allergen management. TrAC Trends Anal Chem 47:12–26
Pramesh D, Islam S, Singh J, Baranwal VK (2012) Serological and PCR detection of Garlic
common latent virus associated with garlic accessions of India. Med Plant 4:17–22
Qingfa W, Shou WD, Yongjiang Z, Zhu S (2015) Identification of viruses and viroids by next-
generation sequencing and homology- dependent and homology-independent algorithms. Annu
Rev Phytopathol 53:425–444
Rai R, Khurana SMP, Kumar S, Gupta N, Baranwal VK (2018) Serological detection of Grapevine
leafroll-associated virus 4 in grapevine growing areas of India using polyclonal antiserum raised
against the recombinant coat protein. Crop Prot 109:128–135
Rana T, Negi A, Dhir S, Thockchom T, Chandel V, Walia Y, Singh RM, Ram R, Hallan V, Zaidi
AA (2011) Molecular diagnosis of apple virus and viroid pathogens from India. Arch
Phytopathol Plant Prot 44:505–512
Rani A, Nerida D, Nitin M (2019) The future of plant pathogen diagnostics in a nursery production
system. Biosens Bioelectron. https://doi.org/10.1016/j.bios.2019.111631
Rott M, Xiang Y, Boyes I, Belton M, Saaed H et al (2017) Applications of next generation
sequencing for diagnostic testing of tree fruit viruses and viroids. Plant Dis 101:1489–1499
Roy A, Fayad A, Barthe G, Brlansky RH (2005) A multiplex polymerase chain reaction method for
reliable, sensitive and simultaneous detection of multiple viruses in citrus trees. J Virol Methods
129:47–55
Roy B, Chakraborty B, Mitra A, Sultana S, Sherpa AR (2015) Natural occurrence of Bhendi yellow
vein mosaic virus on Litsea spp. in India. New Dis Rep 31:7
Salomone A, Mongelli M, Roggero P, Boscia D (2004) Reliability of detection of Citrus tristeza
virus by an immunochromatographic lateral flow assay in comparison with ELISA. J Plant
Pathol 86:43–48
Schaad NW, Frederick RD (2002) Real-time PCR and its application for rapid plant disease
diagnostics. Can J Plant Pathol 24:250–258
Selvaraj V et al (2018) Development of a duplex droplet digital PCR assay for absolute quantitative
detection of “Candidatus Liberibacter asiaticus”. PLoS One 13
Selvarajan R, Mary SM, Balasubramanian V (2011) Simultaneous detection of episomal Banana
streak Mysore virus and Banana bunchy top virus using multiplex RT-PCR. Curr Sci 100:31–34
Seok Kim Y, Ahmad Raston NH, Bock Gu M (2016) Aptamer-based nano biosensors. Biosens
Bioelectron 76:2–19
Sharma SK, Vignesh Kumar P, Baranwal VK (2014) Immunodiagnosis of episomal Banana streak
MY virus using polyclonal antibodies to an expressed putative coat protein. J Virol Methods
207:86–94
Sharma SK, Vignesh Kumar P, Geetanjali AS, Pun KB, Baranwal VK (2015) Subpopulation level
variation of banana streak viruses in India and common evolution of banana and sugarcane
badnaviruses. Virus Genes 50:450–465
Sharma SK, Meena RP, Devana P et al (2017) Development of nucleic-acid based techniques for
fine diagnosis of plant viruses in India. In: A century of plant virology in India. Springer,
Singapore
Shojaei TR et al (2016) Detection of Citrus tristeza virus by using fluorescence resonance energy
transfer-based biosensor. Spectrochim Acta A Mol Biomol Spectrosc 169:216–222
Siljo A, Bhat AI, Biju CN (2014) Detection of Cardamom mosaic virus and Banana bract mosaic
virus in cardamom using SYBR Green based reverse transcription-quantitative PCR. Virus Dis
25:137–141
Subba Reddy Ch V, Sreenivasulu P, Sekhar G (2011) Duplex-immunocapture-RT-PCR for detec-
tion and discrimination of two distinct potyviruses naturally infecting sugarcane (Saccharum
spp. hybrids). Indian J Exp Biol 49:68–73
Sun W, Jiao K, Zhang S, Zhang C, Zhang Z (2001) Electrochemical detection for horseradish
peroxidase- based enzyme immunoassay using p-aminophenol as substrate and its application to
detection of plant virus. Anal Chim Acta 434:43–50
Syedmoradi L et al (2017) Point of care testing: the impact of nanotechnology. Biosens Bioelectron
87:373–387
Syller J (2012) Facilitative and antagonistic interactions between plant viruses in mixed infections.
Mol Plant Pathol 13:204–216
van der Want JPH, Dijkstra J (2006) A history of plant virology. Arch Virol 151:1467–1498
Varga A, James D (2006) Use of reverse transcription loop-mediated isothermal amplification for
the detection of Plum pox virus. J Virol Methods 138:184–190
Vashist SK et al (2015) Emerging technologies for next-generation point-of-car testing. Trends
Vidaver AK, Lambrecht PA (2004) Bacteria as plant pathogens. The Plant Health Instructor. www.
apsnet.org
Villamor D, Ho T, Rwahnih M, Martin R, Zanetakis E (2019) High through put sequencing for
plant virus detection and discovery. Phytopathology 109:716–725
Voegel T, Nelson L (2018) Quantification of Agrobacterium vitis from grapevine nursery stock and
vineyard soil using droplet digital PCR. Plant Dis 102:2136–2141
Webster CG, Wylie SJ, Jones MGK (2004) Diagnosis of plant viral pathogens. Curr Sci
86:1604–1607
Wild P (2008) Electron microscopy of viruses and virus–cell interactions. Methods Cell Biol
88:497–524
Yadav N, Khan JA (2009) Over expression of Narcissus potyvirus coat protein in E. coli. Indian J
Virol 21:44
Yadav N, Khurana SMP (2016) Plant virus detection and diagnosis: progress and challenges. In:
Shukla P (ed) Frontier discoveries and innovations in interdisciplinary microbiology. Springer,
New Delhi
Zhang ZY, Liu XJ, Li DW, Yu JL, Han CG (2011) Rapid detection of wheat yellow mosaic virus by
reverse transcription loop-mediated isothermal amplification. Virol J 8:550
Zhang S, Ravelonandro M, Russell P, McOwen N, Briard P, Bohannon S, Vrient A (2014) Rapid
diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using
reverse transcription-recombinase polymerase amplification. J Virol Methods 207:114–120
Zhao Y, Chen F, Li Q, Wang L, Fan A (2015) Isothermal amplification of nucleic acids. Chem Rev
115(22):12491–12545
Zheng L, Rodoni BC, Gibbs MJ, Gibbs AJ (2010) A novel pair of universal primers for the
detection of potyviruses. Plant Pathol 59:211–220
Epidemiology and Management of Potato
Virus Y 6
Tyler D. B. MacKenzie, Xianzhou Nie, and Mathuresh Singh
Abstract
Potato virus Y (PVY) is an economically important disease agent in potatoes with

worldwide distribution. It is mainly transmitted vegetatively by tubers and
between plants by an aphid vector. PVY management is primarily through
reducing viral inoculum in seed tubers and other sources and controlling trans-
mission by aphids. Seed certification programs are designed to comprehensively
test and restrict levels of PVY within a region, enforcing low levels of PVY in
marketed seed lots and allowing growers informed choice to plant low-PVY
crops. Sanitation techniques on the farm can also reduce volunteer, weed, and
neighboring field sources of PVY. Major techniques to reduce aphid-mediated
PVY spread include mineral oil foliar sprays, especially combined with
insecticides. Spray timing is important, starting early after first plants emerge,
continuing weekly through season to vine-kill, with additional sprays during
rapid growth and periods of aphid abundance. Other practices for PVY reduction
include avoiding mechanical transmission by field equipment, planting crop
borders or intercropping as a barrier or sink for PVY, breeding PVY-resistant
potato varieties, and roguing symptomatic plants from fields. Major remaining
challenges in combatting PVY are management complacency, proliferation of
PVY strains circumventing resistance and roguing, and informal trade of untested
potatoes.
T. D. B. MacKenzie · M. Singh (*)

Agricultural Certification Services Inc., Fredericton, NB, Canada
e-mail: msingh@potatoesnb.com
X. Nie
Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton,
NB, Canada
e-mail: xianzhou.nie@canada.ca

https://doi.org/10.1007/978-981-15-6275-4_6
114 T. D. B. MacKenzie et al.
Keywords
PVY · Foliar spray · Seed certification · Crop management · Crop protection ·

PVY strains · Mechanical transmission · Aphids
6.1 Introduction
Potato virus Y (PVY; genus Potyvirus, family Potyviridae) is a viral pathogen of

potato crops that causes significant economic impact in all major potato-producing
regions. Potato is the fourth largest staple food crop in the world, behind corn, wheat,
and rice, and the largest non-grain crop. PVY is considered the most important virus
of potatoes because of its effect on potato yield and quality and because of its
worldwide distribution. Economic costs associated with PVY are not only from
reductions in yield and tuber quality, however, but also through the substantial costs
incurred from in-field management, potato seed certification, and breeding programs
to develop tolerant and resistant varieties (Lacomme and Jacquot 2017). Though
primarily a threat to potato crops, it can also impact other important crops such as
tomato, tobacco, pepper, and other solanaceous species (Karasev and Gray 2013a).
PVY is a positive-sense single-stranded RNA virus with a genome of ca. 9700
nucleotides (Singh and Singh 1996), encoding ten multifunctional proteins. It causes
a mosaic disease in potato, characterized by a mottled yellow-green coloration of the
foliage and often accompanied by wrinkling and stunting of the leaves and reduced
growth vigor of the whole plant. PVY is primarily spread by an aphid vector, which
acquires and transmits the virus during feeding and flights between plants, and
secondarily by residing dormant in overwintering tubers and emerging in subsequent
plants grown from these tubers.
PVY is thought to have originated and co-evolved with wild potato species in the
Andes region of South America and was transported around the world subsequent to
European colonization and adoption of potato as a food crop (Bellstedt et al. 2017).
Differences in symptomology, particularly evident in different host species, lead to
the recognition of different strains of PVY in the early twentieth century. Today,
PVY occurs as a complex of many strains originating from mutation and genetic
recombination, which have diversified rapidly around the world in recent decades,
continuing to this day (Karasev and Gray 2013b; Green et al. 2018). Currently, the
main strains infecting crops in major potato-producing regions exhibit varying
symptomology and yield effects, complicating management schemes designed to
control PVY.
Despite considerable efforts to control its spread, PVY remains an ongoing and
evolving threat needing vigilant, continuous management (Gray et al. 2010). Man-
agement practices also must evolve in step with the changing challenges associated
with PVY, being reassessed on an ongoing basis to best cope with continuing
changes in PVY strain evolution, varietal selection of producers, and changes in
environment and aphid vectors through time (Davidson et al. 2013). This chapter
represents a brief overview of the epidemiology of PVY, peculiarities of its biology
6 Epidemiology and Management of Potato Virus Y 115
and recent evolution, which present challenges to potato producers, and reviews
diverse management practices with examples of current research to minimize its
spread in the field.
6.2 Epidemiology of PVY
6.2.1 Sources and Modes of PVY Transmission
6.2.1.1 Vertical Transmission and Sources of PVY in the Field

Generally, potato is a vegetatively propagated food plant, with the vast majority of
potato crops being planted with seed tubers harvested from field-grown plants
exposed to PVY in previous growing seasons. Relatively few potato plants are
grown from true potato seed, tissue culture, or lab-grown tubers unexposed to
PVY. PVY cannot be transmitted in pollen or through true potato seed, and
phytosanitary procedures can eliminate it from tissue culture; thus in the laboratory
and through breeding programs, potato plantlets and mini-tubers can be produced
free from PVY (Lacomme et al. 2017). Vegetative production of potentially infected
tubers dominates the seed trade, however, both to maintain varietal characteristics of
the crop over the seasons and because of the required scale necessary to supply the
seed requirements of industry (Frost et al. 2013).
PVY travels systemically in the plant following infection; thus tubers developing
from infected plants can also harbor PVY and carry the virus into subsequently
planted crops – termed “vertical PVY transmission”. Not all tubers of an aphid- or
mechanically infected plant (called “primary infection”) necessarily develop PVY,
though plants grown from infected tubers will necessarily carry that infection
throughout the daughter plant (called “secondary infection”) (Nolte et al. 2004).
PVY burden of harvested seed lots destined for the seed trade can be readily
quantified by molecular testing or winter grow-out assay of a sample of the harvest.
As this seed is used to plant subsequent crops, it is the most direct source of PVY
inoculum in the crop and of greatest interest to potato producers. Thus, management
of vertical transmission of PVY in the seed trade is the focus of strict regulation in
seed certification programs, which are discussed further in Sect. 6.3.1.2.
Seed tubers, however, are not the only source of PVY in the field. Volunteers,
unintended potato plants growing from tubers not harvested or destroyed by cultiva-
tion or overwintering in the field from a previous crop, can be a significant source of
PVY (Jones et al. 1996). Also, neighboring fields with different, more susceptible, or
PVY-tolerant varieties, or ware crops not requiring low PVY for certification, can
represent PVY inoculum that can be transported into more valuable low-PVY seed
crops by flying aphid vectors (Dupuis et al. 2017).
PVY also has a broad enough host range to include several weed species
frequently found in or around potato fields in Eurasia and the Americas, including
solanaceous weeds such as several species of nightshade (Solanum sarrachoides,
S. nigrum, and S. dulcamara) and Physalis floridana, and non-solanaceous weeds
like lamb’s quarters (Chenopodium album; Amaranthaceae), stork’s bill and crane’s
bill (Erodium cicutarium, Geranium pusillum; Geraniaciae), milk thistle (Lactuca

serriola; Asteriaceae), and purple deadnettle (Lamium purpureum; Lamiaceae),
among others (Kaliciak and Syller 2009; Cervantes and Alvarez 2011; Nanayakkara
et al. 2012). Indeed, no fewer than 43 non-crop weed species have been identified as
hosts for PVY (Dupuis et al. 2017). Combined with potential threats from neighbor-
ing PVY-susceptible crops such as tobacco, pepper, tomato, and other potatoes,
these weed reservoirs may represent a source of PVY inoculum that could be
transported into the field by aphid flights. The actual impact of weed reservoirs of
PVY in the field, however, has not been conclusively established (Gray and Power
2018). PVY is likely eliminated yearly with the death of annual weeds (i.e., it is self-
limiting) as it has not been observed to transmit vertically via seeds, and the
efficiency of aphids to transmit PVY from weeds to potato in the field is unknown
and limited to a few lab-based demonstrations (e.g., Cervantes and Alvarez 2011). If
weeds are present within the field, preemergent herbicides can be applied to elimi-
nate early weeds and volunteer potatoes before the emergence of the planted potato
crop, and regular cultivation and later row closure by the crop would reduce the
threat of PVY in later growing in-field weeds.
6.2.1.2 Plant-to-Plant Transmission of PVY by Aphids

PVY is primarily transmitted between plants by an aphid vector. Aphids acquire
PVY during probing of the plant epidermis as part of its feeding activities. PVY is
drawn into the stylet of the aphid and is retained through complex interactions
between the coat protein and the helper-CP protein of the virus and proteins in the
acrostyle near the tip of the aphid stylet (Valli et al. 2018). PVY transmission by
aphids is considered “nonpersistent.” Once acquired, viruliferous aphids only remain
able to infect plants briefly (minutes to hours) and to only a few other plants, as the
virus is quickly eliminated from the aphid stylet after leaving an infected plant
(Pirone and Perry 2002).
This mechanism of acquisition and binding of the virus to the aphid stylet is not
species specific, as viral particles can be acquired this way from a number of host
plants of PVY and by a wide range of aphid species. Indeed, no fewer than 65 species
of field-collected aphids, including both known potato-colonizing and
non-colonizing aphids, have been found naturally able to carry PVY in their stylets
(Pelletier et al. 2012).While many aphids are capable of transmitting PVY, they do
so with measurably different efficiencies. The “type species” widely considered most
efficient at PVY transmission is the potato-colonizing green peach or peach-potato
aphid (Myzus persicae), which is also used to calculate the relative efficiency factor
(REF) for PVY transmission of other aphid species. Many common aphid species
have had REFs calculated for several common strains of PVY (Halbert et al. 2003;
Verbeek et al. 2010), and these REFs can be used in combination with aphid
monitoring programs to calculate near-real-time cumulative vector pressure infor-
mation in a potato-producing region (Fenton et al. 2012).
Given the importance of aphids in the spread of PVY, several regional efforts
have been undertaken to quantitatively link aphid flights to PVY in commercial
potato fields. Aphid abundance was strongly correlated to local PVY spread when
counted weekly from pan traps placed in study fields (MacKenzie et al. 2014) or at
more regional scales using aphid abundance data collected and published by gov-
ernment sources (MacKenzie et al. 2016). Similar modeling studies have been used
as the basis for designing regional notification services in a number of countries. For
example, the TuberPro models produced since the 1990s in Switzerland employed
regional enumeration of a number of aphid species with known REFs regularly
during the growing season and forecasted a PVY risk level communicated to
growers in a weekly newsletter. This model, however, has been simplified consider-
ably more recently, to produce more realistically reliable forecasts (Steinger et al.
2015). The ambitious European EXAMINE program spearheaded by the National
Federation of Seed Potato Growers of France has also worked on forecasting PVY
risk originally through aphid monitoring, but more recently with additional data
inputs relating to climate and geography. While useful as an alert service to growers,
it has been recognized that the ultimate goal of accurate prediction of post-harvest
PVY would be very complex and require more specific data on the planted inoculum
and field-scale knowledge of the cultural practices of the growers (Lacomme et al.
2017). There have been challenges maintaining the applicability of these notification
services continuously over time, due to more fundamental biological causes as well
as bureaucratic reasons of funding and personnel (Radcliffe et al. 2008). Changes
over time in aphid species assemblages and expertise in identifying them, types or
locations of traps used to capture aphids, new or revised REF values of aphid
species, weather-related differences in activity levels across aphid species, or
changes in populations of PVY strains (with their varying intrinsic rates of spread)
can all affect model predictions of cumulative vector pressure or PVY spread based
upon aphid abundance measurements. Many of these factors, and case studies of
different aphid monitoring programs around the world, are reviewed in Lacomme
et al. (2017).
6.2.1.3 Mechanical Transmission of PVY

Relatively little is known about the mechanical transmission of PVY in the field.
“Mechanical transmission” generally includes any artificial means of transmitting
PVY between plants through infected plant sap on equipment, such as plant-to-plant
transmission by tractor or other traffic in the field or transmission between tubers
during seed cutting or handling. PVY is known to remain viable in infected plant sap
on plastic, metal, and rubber surfaces for up to 2 days and in fresh water at least
1 week, and artificial transmission through sap transfer or abrasion between plant
vines occurs readily (Mehle et al. 2014; Coutts and Jones 2015; Fageria et al. 2015).
Recent experimental results show a very large increase in PVY transmission in
commercial potato fields under normal industry management in New Brunswick
(NB), Canada (MacKenzie et al. 2018a). In this study, replicated over six fields in
two crop seasons, known PVY-infected tubers of three major PVY strains (PVYO,
PVYN:O, and PVYNTN) were planted in sprayer track rows where plants were
routinely crushed, abraded, or otherwise disturbed from repeated tractor traffic
through the season, compared to nearby control rows without such traffic.
Subsequent PVY spread from planted inoculum tubers was two to seven times
greater (majority being PVYNTN) in the tractor traffic rows than in the control rows,
and the spatial distribution – in terms of greater along-row distance from inoculum to
new infection and a significant frequency of infection closely matching the circum-
ference of the growers’ tractor tires – strongly suggested that the tractor equipment
was transporting PVY through the field. An earlier in-field study in the same potato-
producing region did not show convincing evidence of heightened PVY transmis-
sion, however, but this was solely based on a lack of an expected spatial patterning of
PVY-infected plants observed in fields with natural assemblages of PVY (Sturz et al.
2000).
Handling of infected seed tubers, particularly cutting tubers prior to planting, is a
concern in the industry for multiplying PVY inoculum when planting the field.
Several studies, however, show that cutting known-infected seed tubers did not
transmit PVY to subsequently cut PVY-free tubers at any measurable rate (Sturz
et al. 2000; Fageria et al. 2015). This includes when tubers were infected with novel
recombinant PVY strains that show evidence of greater rates of plant-to-plant spread
via aphids (Fageria et al. 2015). These several studies, however, only investigated
the potential for transmission from tuber cutting in three potato varieties, and it
remains possible that more PVY-susceptible varieties could show some significant
rate of transmission through this little studied route.
6.2.2 Biotic and Abiotic Effects on Rate of PVY Spread
6.2.2.1 PVY Strains Spread at Different Rates

PVY exists as a complex of at least 36 strains distinguished by genome sequencing,
including the nonrecombinant parental strains PVYO, PVYC, and PVYN first
differentiated about a century ago based on symptomological effects on host plants,
and many recombinant strains containing genomes consisting of various mixed
sections of the parental strains that have evolved more recently (Green et al.
2018). Many strains, including the parental PVYC and PVYN, are now only seen
rarely in some localities, while the recombinant PVYNTN, PVYN-Wi, and PVYN:O
strains and to a far lesser extent the nonrecombinant PVYO are more common in
most potato-growing regions currently. This diversification of PVY strains is of
global importance to the potato industry, because these novel strains exhibit
different – often cryptic – symptoms than more traditional strains like PVYO with
which industry has long experience. These new strains also have different capacity to
spread, including evasion of traditional resistance factors in some varieties, and in
particular PVYNTN can produce damaging tuber necrosis (Karasev and Gray 2013b;
Gray and Power 2018; MacKenzie et al. 2019). Strain populations vary with locality
and change over time, though often several strains co-occur within defined potato-
growing regions and even within the same crop field. Examples of recently changing
populations of major strains in western and eastern Canada and the USA are shown
in Fig. 6.1. There are several proposed mechanisms likely responsible for explaining
the proliferation of these novel recombinant strains over the original nonrecombinant
strains, including intrinsically greater rates of plant-to-plant transmission
Fig. 6.1 Changing (A)

populations of PVY strains in 80
(a) eastern Canada, (b)
western Canada and (c) the 60
USA (predominantly
northwestern states). PVY
strains are indicated as PVYO 40
(solid line, black symbol),
PVYN:O/N-Wi (dashed line, 20
gray symbol), and PVYNTN
% of each PVY strain in collected PVY samples
(alternating line, white
symbol) (Data in [a] and (B)
[b] adapted from MacKenzie 80
et al. 2019 and Nanayakkara
et al. 2012; [c] from Gray and
60
Power 2018, with permission)
40
20
0
(C)
80
60
40
20
0
2004-06 2010 2012 2014 2016
(MacKenzie et al. 2018b; Funke et al. 2017), failure to identify and rogue them from
fields due to more cryptic symptoms in foliage (MacKenzie et al. 2019), and
selective resistance of some potato varieties for older strains (Gray and Power
2018). These proposed mechanisms explaining their rise to prominence are also
the primary issues complicating management of these novel PVY strains.
In a specific example comparing spread of several PVY strains, the authors
conducted a series of experimental trials in NB and Manitoba, Canada, testing
different management strategies for PVY, as well as simultaneously studying spread
of three locally important PVY strains, the traditionally prevalent PVYO and novel
recombinant PVYN:O and PVYNTN strains. In five large, replicated experimental
trials, MacKenzie et al. (2018b) showed that on average the recombinant strains
spread more effectively than PVYO. These differences in virus spread, expressed as
the spread of PVY strains to harvested tubers of initially virus-free plants in a field
inoculated with a known number and strain identity of infected tubers, were variety
specific, however. The rate of spread of PVYN:O and PVYNTN in cv. Goldrush trials
averaged 2.5 and 7.7 times greater than PVYO, though only 3.1 times greater in
PVYNTN and slightly less spread (0.8 times) of PVYN:O in cv. Russet Burbank crops.
Varietal differences in relative spread of PVY strains may be due to differences in
selective resistance to PVY infection, i.e., hypersensitive resistance (HR) in
Goldrush against PVYO infection.
Likelihood of mechanical transmission of PVY between plants also seems to be
strain-dependent. In a field study of mechanical versus aphid transmission of PVY
(MacKenzie et al. 2018a), the ratios of transmission of PVYO, PVYN:O, and
PVYNTN strains were consistent across six trial fields regardless of potato variety,
and most importantly those ratios were nearly the same whether measured in high-
transmission tractor traffic rows (mechanically dominated) or control rows far from
tractor traffic (aphid-dominated). These results suggest the susceptibility of the
recipient plant to infection is likely the selective agent for differential spread of the
strains, not selection by the aphid vector or during injury or transport of the virus
ex vivo on farm equipment.
6.2.2.2 PVY Resistance in Potato Varieties

A range of PVY-resistant potato varieties exist and can generally be categorized into
three forms: extreme resistance (ER) which do not allow replication of PVY within
the plant, hypersensitive resistance (HR) in which newly infected tissues experience
rapid cell death to halt systemic spread of the virus, and PVY tolerance, in which
plants show relatively little symptomological or yield-affecting impacts despite PVY
infection and replication (Dupuis et al. 2017). Only the former two are truly resistant
to PVY and can halt the replication of the virus in the field, whereas tolerant varieties
allow continued proliferation of PVY despite it having little impact on the crop of
that variety. Ideally for disease management, a critical density of truly resistant
varieties could be planted in a region to reduce the available hosts for PVY over a
number of years and ultimately eradicate it in a process similar to vaccination herd
immunity and isolation in human viral diseases. However, the availability of resis-
tant varieties with other commercially competitive characteristics is limited. Particu-
larly in North America, breeding of commercially successful tolerant varieties like
Russet Norkotah and Red LaSoda has allowed continued virus spread without
consequence for growers of these varieties (Gray et al. 2010), or focus has been on
intensive PVY management in fields of high-yielding but susceptible varieties like
Russet Burbank. Thus far, market pressures and possibly grower familiarity seem to
support continued use of commercially successful but nonresistant varieties, over the
slow, costly development of potentially less economically competitive resistant
varieties.
The genetics of HR- and ER-type resistance in potato varieties has been well
studied, and its specificity for different PVY strains has also enjoyed considerable
study. Many varieties show complex viral strain-specific PVY resistance,
complicating their utility in industry. For example, the varieties Alturas, Umatilla
Russet, and Ranger Russet show HR resistance slowing transmission of PVYO in a
greenhouse experiment, but the recombinant strains PVYN-Wi and PVYNTN evaded
the HR response and were transmitted at two to three times greater rate (Funke et al.
2017). With current changes in PVY strain populations and availabilities of resistant
potato varieties in different production regions, careful variety selection, crop
management, and PVY testing are necessary to successfully exploit PVY resistance
in the field. A deeper review of the genetics of PVY resistance and characteristics of
resistant varieties is a complex subject outside the scope of this chapter, but is well
reviewed in Valkonen et al. (2017).
PVY translocation within the potato plant generally slows with age across potato
varieties, a phenomenon termed “mature plant resistance” (Beemster 1972). Most
important to industry is the degree of PVY translocation to progeny tubers after
primary infection of plants. PVY translocation through the phloem sap to roots and
later to developing tubers is rapid in the first ~4 weeks after plant emergence and
then progressively slows to rates insufficient to produce viral particles and carry
them to tubers from new PVY infections after 8–10 weeks of further plant growth
(Dupuis 2017). The expression of mature plant resistance also seems to be
PVY-strain dependent, with nonrecombinant PVYN and recombinant PVYNTN and
PVYN-Wi translocating to tubers more effectively than PVYO after infection of older
plants (Basky and Almási 2005; Dupuis 2017).
6.2.2.3 Abiotic Factors Affecting Spread

It is not uncommon that commercial growers contacted by this chapter’s authors
express opinions that PVY spread is largely under the control of weather and
abundance of aphids. These weather and aphid factors are intimately related and
not under control of the grower, and thus the pressure to expend the effort and costs
of combatting PVY spread is lessened in a sense of resignation (M. Singh pers. obs.).
In our own studies, however, weather is far less correlated with PVY spread than
other factors directly under the control of the grower, such as planting low-PVY seed
and managing in-field spread of PVY with foliar sprays (MacKenzie et al. 2014;
MacKenzie et al. 2016). From these studies, the greatest climatological predictor of
PVY spread was found to be the departure of previous winter temperatures from
long-term average, with higher than normal winter temperatures associated with
higher following-season PVY spread; temperature and precipitation values during
the season, surprisingly, were less so or not correlated at all with PVY spread
(MacKenzie et al. 2016). The link between warm winter temperatures and PVY is
likely due to increased overwintering survival of aphids. Klueken et al. (2009)
quantified specific temperature thresholds in spring conditions (e.g., 10–13 C)
under which particular aphid species would not fly from their winter host plants.
Also, in studies of aphid flights in mountainous Switzerland, altitude and average
wind speed of fields was strongly correlated with aphid abundance and could serve
as a planning tool for growers deciding where to site valuable PVY-susceptible seed
crops (Steinger et al. 2014). While not directly under the control of growers,
however, information on previous winter conditions, prevailing temperature, and
wind conditions or early forecasts nearing temperature thresholds for specific aphid
activities would be known early enough to inform grower decisions on planting and
in-field management of PVY at the beginning of the growing season.
6.3 Management of PVY
Many factors – biological, physical, and cultural – affect the spread of PVY in the
potato crop, several of which described above that are under little control of the
potato grower. There are, however, management practices available to effectively
reduce the impact of PVY in the crop, which can generally be categorized into two
approaches: (1) minimizing PVY inoculum planted within or from a neighboring
field, and (2) reducing PVY spread during the growing season within the field.
6.3.1 PVY in Planted Seed and Seed Certification Programs
6.3.1.1 Efficacy of Reducing PVY in Planted Seed

The most effective source of PVY inoculum in the potato field is that which is
planted into it with the potato seed. While there are several potential sources of PVY
to infect a potato field, many of them can be managed and reduced. Infected
volunteer potatoes and weeds can be reduced by tillage, herbicide application, and
crop rotation, while impact of PVY from neighboring fields can be reduced by
distance, vigilant scheduling of crops in nearby fields, and border crops. Strict testing
and selection of low- to zero-PVY seed is the only way to reduce the amount of PVY
imported to the field at planting time.
In a series of studies over the past decade observing commercial potato produc-
tion fields and through conducting experimental plantings in NB, Canada, the strong
and proportional influence of planted PVY on subsequent virus spread was clear
(MacKenzie et al. 2014, 2016, 2017). Even when PVY was planted at relatively low
and commercially acceptable levels (i.e., <3%), the degree of resulting PVY spread
through the field during the growing season was substantial and only partly
mitigated by significant and costly application of foliar mineral oil and insecticide
sprays after planting.
While a detailed cost-benefit analysis was not done as part of these studies, it was
evident that the increased cost of sourcing lower PVY seed would be minor
compared to the impact on the crop in terms of reducing PVY spread in seed
production fields. These savings would not be primarily through marginal increases
in yield with lower PVY in the field, but in the far greater reduction of risk of the
field surpassing the strict regulatory caps on PVY to be rejected as a seed crop and
reduced input costs of needed oil and insecticide sprays to manage PVY spread
during the growing season. Processing and table stock fields, though, are not under
the same pressure to maintain very low-PVY rates in the harvested crop, and the
yield difference between, for example, 2% and 5% PVY at harvest may not justify
the cost of sourcing very-low- to zero-PVY seed. Considerations other than minor
yield reduction should be taken even in these crops, however, including the
increased possibility of frequent tuber necrosis from the now-common PVYNTN
strain causing loss of the whole crop due to market rejection, or local buildup of PVY
inoculum for growers or neighbors simultaneously growing processing and seed
crops.
6.3.1.2 Seed Certification and PVY Distribution Across Seed Classes

Seed certification is a systematic program in regional potato industries to control
aspects of the commercial seed trade, specifically variety purity, seed class, prove-
nance, quality and labeling, and, of particular focus in this chapter, disease burden.
Some aspects of organized potato seed certification have been in place in Europe and
North America for about 200 years, and other potato-producing developed nations
such as Australia and New Zealand for about a century (Dupuis et al. 2017). These
programs are of great value to local potato producers and exporters, and the
comprehensive third-party testing and monitoring of potato seed has allowed
government-imposed and market-driven reductions and eradications of disease
agents in many major production regions around the world. The economic return
to growers participating in seed certification programs has also been assessed
(Gildemacher et al. 2011) and has shown clearly that the output of high-yielding,
high-quality, and low-disease potato crops from these programs is one of the most
cost-effective strategies in seed potato management.
In terms of PVY management, seed certification traditionally relied on visual
inspection in the field or from a winter “grow-out” of a portion of the harvested crop.
More recently, however, molecular testing with ELISA or traditional or real-time
RT-PCR has become standard. These molecular techniques have the advantage of
allowing rapid and high volume testing, without the expense and time required to
transport tubers and grow plants in distant warm regions during winter grow-out
tests. Nor do they require expertise in recognizing symptomatic plants, especially
with latent infections in tolerant potato varieties and cryptic infections with increas-
ingly common recombinant PVY strains. Standardized, quantitative results from
comprehensive PVY testing within a producing region thus allow identification and
disposal of problem seed lots, predictable government regulation of disease limits,
and market forces to reward production of clean potato seed.
A clear result of seed certification data is that PVY is not evenly distributed in
local potato-producing regions, but instead it tends to be concentrated in a minority
of problem seed lots, retained at higher levels in some varieties, and concentrated in
later generation seed. This relatively small number of seed lots can thus be targeted
for intensive management or elimination by ranking into identifiable categories such
as seed class (generations grown in the field), potato variety, or growers’ practices.
Figure 6.2 shows the relative distribution of PVY incidences in seed lots from a 2008
to 2015 survey in a US seed certification program (from Gray and Power 2018).
Quickly apparent is the stubborn stability of high PVY levels – though still within
regulatory thresholds for planting – in a minority of seed lots (i.e., only 8% to 21% of
tested seed lots contain PVY at >2% incidence). Calculated differently, however,
the total impact of those relatively few high-PVY seed lots is more clear. Factoring
together the number of seed lots and degree of PVY contamination, these relatively
few poor seed lots carried about 50–70% of the annual burden of PVY industry-wide
during these years. Identifying the underlying causes for these high-PVY seed lots
and targeting them for special management attention would be a most cost-effective
means to expend management effort at reducing PVY impact on a local industry
scale.
Fig. 6.2 Distribution of PVY 8

15 12
in seed lots from a major 19 18 18 21 20
potato-producing state of the 18
USA. Values are percentages 15
22 21 18
of lots tested in each indicated
21 18
category of PVY 28 14 10
contamination from all lots 9
13 11 8 11
tested each year (Adapted
from Gray and Power 2018, 12
with permission)
61 62
50 50 55 50 51
41
2008 2009 2010 2011 2012 2013 2014 2015

0% PVY 0%<PVY<0.5% 0.5%<PVY<2% PVY>2%
The number of generations that a seed crop is grown in the field (seed class)
determines the amount of exposure a crop has to PVY infection and spread. Though
it varies with the background inoculum level, aphid pressure, and management of the
crop, PVY incidence increases exponentially with seed class within a given region.
In NB, Canada, despite a great overall reduction in PVY incidence averaged across
the certified seed lots of the region over recent years, later generation seed still shows
relatively high levels of PVY. As an example, 2018 seed certification in NB showed
that E4 seed lots averaged 2.16% PVY compared to only 0.99% in all other seed
classes (M. Singh, unpub.). Indeed, even though the number of lots and total acreage
of this older-generation E4 seed is small, representing only 7.5% of 453 tested lots
and 13.3% of seed acreage in 2018, the relatively high levels of PVY in those fields
factored together with their acreage means that they contained 32% of the total PVY
in the NB seed industry that year and are thus disproportionate contributing to the
region’s total PVY burden. Also, growers planting this seed class took on a signifi-
cantly increased risk of failing certification, with nearly 14.7% of E4 seed lots
surpassing the regulatory PVY cap to allow sale (4% PVY at harvest in 2018),
compared to only 5.4% of lots in all other seed classes which failed. Though
comprehensive PVY data do not exist for processing and table stock potato fields
in the region, which represent about four times the acreage of the seed producers, the
same buildup of PVY in the generations can be assumed to occur. Thus, especially if
applied on an industry-wide scale, tighter grower marketing and acceptance or
government-mandated restrictions on sale of later generation potato seed could
remove a large component of the PVY from the industry at a minimum cost of lost
potato production.
Lastly, results of seed certification can be used to identify particular growers
within a region who produce atypically high-PVY seed. While singling out individ-
ual potato producers in a local industry could be controversial, the potential benefit
to industry of focusing on such growers could be substantial. Two helpful
approaches from such grower-focused data analyses would be (1) to identify specific
issues with their management practices and advise on practical ways of improving
PVY management individually and (2) using a behavioral economics approach to
“nudge” individual growers into more effective and competitive PVY management
strategies. Identifying of PVY-problem fields, surveying management techniques,
and tailoring industry-appropriate best management practices have proven very
effective in the potato industry of NB, Canada (MacKenzie et al. 2016). Little
information is available on practical behavioral economics approaches (i.e.,
“nudge theory,” Thaler and Sunstein 2009) to disease management in the potato
industry. However, these sort of “big data” approaches to identify and target
individuals with specific messaging are increasingly and successfully being used
in diverse fields, from affecting domestic energy usage (Sudarshan 2017), to
increasing state tax compliance (Christian and Alm 2014), to reducing infectious
and noncommunicable disease incidence in humans (Bond and Nolan 2011; Hansen
et al. 2016). Such approaches could be easily and economically applied to managing
PVY in a local industry with existing data from seed certification and marketing
programs, such as informing growers of their relative performance or ranking in
PVY control compared to anonymous neighboring competitors (social proof nudg-
ing) or personalized estimates of economic gain from better PVY control (economic
messaging).
6.3.1.3 Informal Seed Trade as a PVY Source

Unregulated potato seed can also be a significant source of PVY inoculum and can
originate from a wide range of sources described collectively as the informal seed
trade. Examples of such are otherwise unmarketable seed crops planted in small
personal gardens (i.e., “farm-saved” seed), small-batch seed sets sold at the retail
level in the usually unregulated garden supply trade, or larger-scale unregulated
trade more typical occurring in the developing world. In many developing countries,
particularly in Africa, Asia, and South America, as much as 94% of potato seed is
unregulated and of uncertain PVY status, because testing and seed certification
infrastructure is not in place (Thomas-Sharma et al. 2016). Coupled with limited
resources to manage PVY spread in these regions, PVY incidence and its
consequences on yield and quality in the potato crop is often severe. However,
because of the lack of certification programs in such regions, export of high-PVY
seed tubers to low-PVY countries is generally blocked. Other than the obvious
impacts on the local industry, one possible global concern of existence of such
unregulated, high-PVY regions is that they may serve as incubators for rapid virus
evolution and recombination, producing new PVY strains with novel characteristics
that could increasingly challenge potato crop management worldwide.
On the smaller scale, farm-saved seed and the retail garden trade have been
implicated in spread of PVY. A recent survey of potato seed available at garden
centers in northwestern USA between 2016 and 2018 (Inglis et al. 2019) found
widespread PVY contamination of retail-size sample batches (26% to 47% over the
three annual surveys). PVY was present as PVYO, PVYN-Wi, and PVYNTN strains or
as mixed infections of multiple strains. Grown-out samples from these retail batches
exhibited significant tuber quality deficits, particularly growth cracks highly
associated with PVYN-Wi. While retail of potato seed at any commercial level could
be regulated with the same strict seed certification standards as applied to large-scale
industry, discouraging farm-saved seed or other informal noncommercial trade may
need more creative approaches and local education efforts. As an example, in a high-
value seed production area outside the main potato production region of NB,
Canada, the local industrial producer freely provides small quantities of high-quality
PVY-free seed potatoes to local residents with garden plots over a generous
surrounding area (M. Singh pers. obs.). This service, at moderate cost, provides
quality potatoes to residents and engenders local engagement, goodwill, and educa-
tion while ensuring a substantial PVY-free buffer zone around the sensitive indus-
trial production fields.
6.3.2 Management Factors Effective for Reducing In-Field PVY

Spread
Once PVY inoculum is identified in or nearby the potato field, there are several
management approaches to minimize virus spread in the crop during the growing
season. Generally, these can be categorized into practices to directly combat trans-
mission of PVY between plants by aphids, managing timing of crop planting,
harvesting, and spraying to reduce unprotected exposure to aphid vectors, resistant
variety selection, and other practices to reduce emergent PVY inoculum or spread.
6.3.2.1 Foliar Spraying of Mineral Oil and Insecticides Targeting Aphid

Vectors of PVY
Regular foliar spraying of potato crops with agents to reduce plant-to-plant trans-
mission of PVY during the growing season is a widely used practice to control
in-field spread of the virus. Central to this strategy for the past half-century is the
spraying of mineral oil-water emulsions onto growing plants to interfere with proper
feeding or PVY acquisition by aphids (Bradley et al. 1966). Many studies have
measured the effectiveness of mineral oils alone and in combination with other
practices for reducing PVY spread (examples are Boiteau et al. 2009, Steinger et al.
2014, MacKenzie et al. 2016, 2017). The proposed mechanisms of action vary,
including effects on retention of PVY in the aphid stylet, changes in aphid feeding
behavior, priming of the natural defense mechanisms of the plant, and more. Many
examples of the effect of mineral oils on PVY spread, and studies on these varied
mechanisms of action, have been well reviewed elsewhere (Al-Mrabeh et al. 2010;
Dupuis et al. 2017).
More controversial, the spraying of foliar contact insecticides targeting aphids has
received considerable attention in recent years. This practice is questioned because
of inconsistent early studies investigating the efficacy of foliar spraying of
insecticides and the widespread opinion that insecticides act too slowly compared
to the mere seconds required for an aphid to probe a susceptible plant with its stylet
mouthparts and infect the plant (Al-Mrabeh et al. 2010).
40
30
PVY spread (%)
20
10
0
2010 2011 2012 2013 2014
Fig. 6.3 In-field PVY spread grouped by foliar spray management strategy. Blank bars indicate
fields with no foliar spraying of mineral oil or insecticides, light gray bars are fields with nine or
fewer mineral oil and three or fewer insecticide sprays, and dark gray bars are fields with >9 mineral
oil and >3insecticide sprays applied through the growing season. Values are means SEM from
56 study fields measured between 2010 and 2014 in NB, Canada (Adapted from MacKenzie et al.
2016, with permission)
Recent survey and observational studies and controlled experimental field trials
by the authors’ research group in NB, Canada, have produced substantial evidence
supporting the use of mineral oils and specific insecticides in combination with
mineral oil sprays. This Canadian province supports an intense, technically
advanced local potato industry that produces the majority of agricultural revenues
for the province; commercial growers here operate with significant government
oversight, including a strict and comprehensive seed certification program and
ongoing applied research to maintain and improve crop quality. From 2010 to
2014, an epidemiological study of PVY in commercial potato fields in the region
was undertaken to elucidate the main factors associated with PVY spread and
quantify the efficacy of management practices to control it locally. During the
epidemiological study period, PVY spread was closely followed on 16 participating
growers’ farms, including 56 separate crop fields representing 13 locally important
potato varieties (MacKenzie et al. 2016). The approach of this wide-ranging study
was to select a range of experienced commercial growers and quantify on-farm PVY
spread as a function of PVY inoculum level in the seed planted, aphid abundance and
climate factors, and the effectiveness of in-field management practices, specifically
the timing and number of foliar mineral oil and insecticide sprays. Through the years
of this study, the growers of the region – including those participating in the study –
were updated on the results of this and other local research and informed of
developing best management practices. Over the course of the 5-year study, average
PVY spread in the study fields dropped substantially, particularly in fields managed
with the most intense foliar spraying programs; average PVY spread in these fields
dropped nearly tenfold from 2010 to 2014 (Fig. 6.3).
The data gathered showed that not only the number of foliar spray applications
but also timing and composition of sprays, as well as initial inoculum of PVY
planted in the field, were correlated with in-field PVY spread (MacKenzie et al.
2016). Over the course of the study, growers who used approximately weekly
simultaneous (tank-mixed) water emulsions of mineral oil and insecticide for most
or all of their foliar spraying showed greatest reductions of in-field PVY spread. This
degree of reduction in PVY spread was greater than with mineral oil-only sprays in
other fields in the same season and increased over the years along with the industry-
wide increase in use of simultaneous mineral oil-insecticide sprays. Later planting
dates and shorter time intervals between planting and first spray application after
crop emergence were also correlated with reduced PVY spread. Other factors, such
as level of PVY inoculum in the planted seed, described in Sect. 6.3.1.1 above, and
the abundance of aphids each year were unsurprisingly correlated with greater
in-field spread of PVY.
6.3.2.2 Timing and Composition of Foliar Sprays to Maximize PVY

Protection
Early application of foliar sprays to protect the emerging crop is critical, though in
the local industry it has been difficult to convince growers of this necessity
(M. Singh pers. obs.). Common opinion has been that prior to majority emergence
of potato plants and significant plant growth, most sprayed mineral oil and insecti-
cide is “wasted” as it lands on bare soil rather than plant leaves. Also, it is thought
that if the potato crop is not substantially emerged, there is not a source of PVY for
aphids to acquire and thus transmit the virus, and retrospective aphid counts from
pan traps necessarily cannot alert growers to early aphid flights until they are counted
(weekly starting in mid-June in this region). Given this information, potato crops are
thus often left unprotected at a critical early stage, when aphid flights typically peak
in this region, long before any mature plant resistance to PVY develops in the plant,
and when winged aphids trapped in potato fields have been demonstrated to carry
PVY in their stylets (Fig. 6.4, Pelletier et al. 2012). It is critical that this interval of
time in which plants are unprotected from PVY-carrying aphids, from emergence to
initiation of regular foliar spraying, is as short as possible – in our recommendations
beginning at approximately 30% crop emergence.
Careful focus on the timing of crop-protecting sprays should keep both (1) periods
of maximal aphid flight activity, and (2) changes in the susceptibility of the crop to
infection, in mind. For example, in these NB, Canada, studies, aphid activity was
maximum early in the growing season, with abundance in a 2-week period spanning
late June to early July being best correlated with PVY outcome at harvest. Coupled
with generally maximal susceptibility prior to development of mature plant resis-
tance, a rapid growth of new unprotected leaves between sprays, intense and
frequent foliar spraying of the crop was warranted. Many growers using this
approach relaxed the frequency of insecticides somewhat later on, when plants
mature and aphid numbers lessened, without apparent compromise on PVY spread.
From this and other research, increased attention to early frequent spraying due to the
dynamics of mineral oil coverage in rapidly growing crops has been recommended
140 14
Planting
Crop emergence
% tested aphids positive for PVY

120 12
aphids captured (per day)
100 10
80 8
60 6
40 4
First spray
20 2
0 0
Fig. 6.4 Aphids captured from a network of yellow-bowl traps in the potato-growing region of
NB, Canada, during the cropping season of 2010. Samples were collected approximately every
3 days, then averaged here into weekly values of total aphid abundance (filled symbols, solid line)
and % of aphids tested by RT-PCR for presence of PVY in their mouthparts (open symbols, dashed
line). Shaded regions indicate typical time periods locally for potato planting, range of time for
emergence of the crop, and times of first foliar spraying of field; foliar spraying in this region
usually continues at ~weekly intervals until late August/early September (Adapted from Pelletier
et al. 2012, with permission)
and adopted in major potato-growing regions in North America (Fageria et al. 2014;
MacKenzie et al. 2016) and Europe (Dupuis et al. 2017).
Late-season protection, however, also needs attention. In most years in NB,
Canada, as an example, a second peak of aphid flights is measured in August well
before vine-killing in potatoes (MacKenzie et al. 2016), which may be associated
with local timing of grain harvesting. Also, while the most efficient known aphid
vector of PVY, M. persicae, is relatively less common than other species in the
region, it exclusively appears near season end and sometimes in very large numbers
locally. Early vine-kill could avoid numerous, late, and effective vectors of PVY, as
has been suggested elsewhere. Steinger et al. (2014) reported that under certain
vector conditions at season end in their study fields, delaying vine-kill could increase
likelihood of PVY infection by up to 3.5% per day of delay.
Changes with the types of insecticide chemistries used during the NB, Canada,
studies were also associated with reduced PVY spread. Specifically, use of lambda-
cyhalothrin (trade names Silencer®, Matador®) and flonicamid (Beleaf®) in sprays
was strongly correlated with reduced PVY spread, while no significant correlation
was found with many other insecticide chemistries. While controversy persists over
the general efficacy of insecticides for reducing PVY spread, some particular classes
of insecticides may show general utility as they have in our studies (MacKenzie et al.
2014, 2016, 2017). Synthetic pyrethroids as a group, including lambda-cyhalothrin,
have demonstrated a very rapid knockdown effect that has been shown elsewhere to
slow aphid probing behavior (Collar et al. 1997) and reduce PVY spread by aphids
(Perring et al. 1999; Boquel et al. 2015). Similarly, flonicamid has been
demonstrated to produce a rapid anti-feedant behavior impact on aphids, which
should slow acquisition of PVY from plants (Morita et al. 2007).
Another important consideration for using insecticides with specific and
pharmacologically narrow mechanisms of action is the phenomenon of insecticide
resistance in the aphid vector. Resistance mechanisms vary from genetic mutation of
insecticide-targeted enzymes or their upregulation to overcome toxicity of the agent,
increased metabolism of insecticide agents, or simple behavioral change to avoid
exposure (Criniti et al. 2008). Surveying aphid DNA for known resistance mutations
is the simplest approach to quantify heritable genetic insecticide resistance and has
shown very high rates (up to beyond 75%) of genetically conferred resistance to
pyrethroids in M. persicae and other aphid populations in limited surveys in North
America (MacKenzie et al. 2018c) and the UK (Foster et al. 2014). Similar surveys
have also found high population rates of mutations conferring resistance elsewhere
around the world to this and other insecticides (e.g., Criniti et al. 2008; Slater et al.
2012). Other approaches to chemical control of aphids and PVY spread are through
using synthetic analogues of pheromones to alter aphid behavior or elicitors which
provoke natural defense responses in potato to reduce virus replication or transloca-
tion in the plant (Dupuis et al. 2017). The efficacy of these approaches has not been
studied as well as more traditional chemical insecticides, though they may represent
more environmentally benign treatments less likely to select for resistance in aphid
populations than would specific insecticidal chemicals.
6.3.2.3 Modeling and Experimental Approaches to Elucidate Factors

Associated with PVY Spread
Considering the multiple varying factors potentially affecting in-field PVY spread
described above, several research groups including our own have attempted statisti-
cal modeling approaches to determine the significant factors associated with PVY
spread, their relative effect size, and the efficacy of quantifiable management
practices. Typically, a multiple logistic regression approach is used to model the
likelihood of PVY infection during the season (or more simply the rate of PVY
infection at harvest), a statistical approach commonly used in epidemiological
studies (Hosmer and Lemeshow 2000) including in potato disease (e.g., Johnson
et al. 1998; Martín-López et al. 2006). As part of the authors’ research in the NB
industry, a complex statistical modeling study was undertaken within our larger
5-year epidemiological study, to better quantify the interactions of various factors on
PVY spread (MacKenzie et al. 2014). In-field PVY spread was very strongly related
to PVY inoculum planted in the field, specifically to a compounded factor of planted
PVY Aphid abundance in the beginning of the growing season. Indeed, this PVY
Aphid interaction term representing the available virus inoculum together with early-
season vector abundance was the single most significant factor determining in-field
PVY spread in this modeling study. Clear from these studies was that the most
effective, low-cost, and simple practice for reducing PVY spread in the NB potato
Fig. 6.5 In-field PVY spread

as a function of initial PVY 30
inoculum in the field and
PVY spread (%)

foliar spray management
(fields with foliar spraying,
20
gray columns; fields without
spray, white columns). Values
are means measured from
56 study fields in NB, Canada, 10
2010–2014 (Adapted from
MacKenzie et al. 2016, with
permission) 0
PVY=0 PVY>0
Inoculum PVY planted in field
industry was to plant potato seed certified with low or zero PVY. While later labor-
intensive and costly management of PVY spread was feasible, it was not as effective
as simply planting low-PVY seed. Figure 6.5 shows this point clearly; over the
5-year study, fields with no detected PVY in seed averaged less PVY spread during
the season even without a foliar spray program, than fields planted with >0% PVY
in seed but managed with foliar sprays to reduce aphid-vectored PVY spread.
After accounting for planted PVY inoculum, the most statistically significant
factor reducing PVY spread in the field was the number of combined mineral oil and
insecticide sprays applied to the crop. Still significant, but of lower magnitude in
effect, was the number of mineral oil-only (not simultaneous with insecticide)
sprays. Interestingly, despite the wide range of mineral oil application rates, and
opinions on it in the local industry, there was no statistically resolvable difference in
PVY spread across the range of 2 to 5 L/hectare mineral oil application in each spray.
Also worth consideration is that it may be safer to use more frequent, but lower areal
rates of mineral oil spray rather than fewer more concentrated sprays to avoid
potential phytotoxicity of the oils in the crop (Kirchner et al. 2014). As found
more generally in the 5-year study, only insecticides of the lambda-cyahalothrin
and flonicamid chemistries showed strong and significant reducing effect on PVY,
while other chemistries (e.g., other older pyrethroids or organophosphates) did not.
While the planting date and time from planting to first spraying significantly
correlated with PVY spread over the larger and more varied data set of the 5-year
study (MacKenzie et al. 2016), these could not be resolved into a statistically
significant effect of quantifiable size in the smaller and more focused modeling
study (MacKenzie et al. 2014).
From the early results of these observational epidemiological studies in coopera-
tion with local industry partners, our research group also undertook large-scale
experimental field trials. Among the more controversial findings of our studies
within the operating industry (MacKenzie et al. 2014, 2016) was the apparent
success of simultaneous mineral oil and insecticide foliar spray to reduce PVY
spread. Thus our controlled and replicated field trials were primarily designed to
rigorously test and compare the efficacy of regular foliar sprays of mineral oil-only,
Fig. 6.6 PVY incidence in 30 Low aphid year

harvested tubers from field (mean 14.6 aphids/trap/week; peak 49 on 30 June)
plots managed under ten
different foliar spray 20
treatments. Fields were treated
2014
identically, planted with
PVY-free seed and hand-
10
PVY % in tubers at harvest

planted PVY inoculum tubers
˚ ˚ * * *
to 3% initial level, in
relatively low- and high-aphid
years (2014 and 2015, 0
High aphid year
respectively) in NB, Canada. (mean 27.4 aphids/trap/week; peak 99 on 7 July)
Values are means of PVY in
100 tubers from four replicate 20
plots in each experiment; error
2015
is SEM, * indicates significant * * * *

difference from no-spray
10
control at p < 0.05, and
indicates marginal
significance at 0.05 < p < 0.1
(Adapted from MacKenzie 0
oil-low ONLY
oil-high ONLY
5 insecticide
& oil-low
5 insecticide
& oil-high
11 insecticide
& oil-low (Silencer)
11 insecticide
& oil-low (Fulfill)
11 insecticide
& oil-low (Concept)
5 insecticide ONLY
11 insecticide ONLY
no-spray control
et al. 2017, with permission)
insecticide-only, and combined mineral oil and insecticide to reduce in-field PVY
spread. These trials clearly concluded that frequent (weekly) mineral oil spraying
can reduce PVY spread compared to unsprayed control plots and that combined
mineral oil and insecticide sprays reduce PVY spread even further (Fig. 6.6). An
additional important result to come from these trials was that insecticide-only sprays
(without simultaneous application of mineral oil) had no significant effect on PVY
spread in treatment plots. This result came to light only because the standard practice
in the NB industry is to tank-mix mineral oil and insecticides to minimize labor in the
field, and it may address some of the controversy in the literature about the
effectiveness of insecticides in combatting PVY spread. One proposed mechanism
to explain the synergistic effect of mineral oil and insecticide is that the oil-soluble
insecticides may be carried into the leaf tissue after spraying, as these horticultural
mineral oils have been shown to be rapidly absorbed into the interior of the leaf
(Fageria et al. 2014), and thus be protected from loss due to washing by dew or rain
or degradation from UV or air exposure. Another important result from these trials
was that the degree of PVY protection by these foliar sprays was lower in a high-
aphid than low-aphid pressure year (Fig. 6.6, comparing 2015, 2014); furthermore,
when under higher aphid pressure, mineral oil-only spray treatments lost more of
their protective function, which was retained in combined mineral oil and insecticide
sprayed treatments. In a multiyear study in the UK, Dawson et al. (reported in
Dupuis et al. 2017), however, found the opposite to be true, with mineral oil sprays
conferring more protection against PVY spread in relatively high vector pressure
years; year-to-year changes in aphid species proportions were suspected to contrib-
ute to the variability in relative PVY reduction. With more study on aphid species-
specific response to oil sprays, decisions on the intensity or frequency of such
combined spray treatments will be better informed by timely information on relative
abundance of aphid flights in the area, which are monitored and reported by local
government agencies in many potato-producing regions.
6.3.2.4 Mid-Season PVY Testing as a Predictor of Crop Outcome

Testing individual potato crops in mid-season for measuring early PVY spread has a
significant predictive power for estimating the ultimate outcome of PVY at harvest.
As part of our survey (MacKenzie et al. 2016) and modeling studies (MacKenzie
et al. 2014), a routine ELISA leaf test of PVY in ~100 plants was made from each
study field in mid- to late July, about 1 month after full emergence. Increase in PVY
from initial inoculum level to that mid-season test was strongly predictive of whether
PVY spread would exceed the government regulatory threshold, which at that time
was set at 5% PVY for sale of seed potatoes (Fig. 6.7). Across all fields, years, and
varieties in this study, 86% of fields showing no increase from inoculum to
mid-season (0% early PVY spread) remained at or below 5% spread at harvest and
63% of fields showing up to 1% early spread stayed below this level. Of fields
showing higher early season spread, only 25% of fields showing 2% and no fields
showing 3% or higher early PVY spread remained below 5% by the end of season
(Fig.6.7c). This predictive capacity of a simple mid-season leaf test, available for
<$200 and with data reporting within ~3 days of sampling, could be economical for
growers to consider if producing a large and valuable seed lot.
Our modeling studies also showed that, in addition to knowledge of initial PVY
level planted in the field and local aphid abundance, adding a mid-season PVY leaf
test to models greatly increased the model’s predictive power (correlation of
predicted and actual PVY at harvest increased from r2 ¼ 0.18 to r2 ¼ 0.86 with
addition of mid-season test, MacKenzie et al. 2014). Most commercially important,
the rate of false negatives from these predictions, where the model predicted a field
remaining below 5% PVY spread but it actually exceeded that regulatory threshold,
fell from three of 19 modeled fields without the mid-season test to only a single false
negative when mid-season PVY testing was added. Also important with this predic-
tive modeling was that the impact of management, specifically the type and number
of costly mineral oil and insecticide sprays, could be quantitatively assessed,
allowing a powerful cost-benefit decision-making tool.
6.3.2.5 Roguing, Crop Borders, and Other Field Practices

Roguing is the inspection, identification, and removal of potato plants that are
visually different from the majority of the crop to reduce disease inoculum in the
field (Kerlan et al. 1987). Unusual looking plants may occur because they are a
different variety or physiologically older (indicative of volunteers at greater disease
risk) or show clear mosaic symptoms in leaves characteristic of PVY infection.
Roguing, however, is very labor intensive, requiring walking rows and surveying all
Season-long PVY spread (%)

80 (A)
60
40
20
0
0 2 4 6 8 10 12 14
PVY spread measured at mid-season
Season-long PVY spread (%)
60 100
Likelihood remaining <5%

(B) (C)
80
40
60
40
20
20
0 0
0% 1% 2-3% >3% 0% 1% 2-3% >3%
PVY spread measured at mid-season
Fig. 6.7 In-field PVY spread predicted by mid-season ELISA leaf test of PVY in 56 study fields in
NB, Canada, 2010–2014. (a) Correlations of season-long PVY spread with mid-season test in fields
managed with foliar spray (filled symbols, solid line) and unsprayed fields (open symbols, dashed
line). Alternating dashed line shows correlation of unsprayed fields matches that in sprayed fields
almost precisely when two outlier fields >6% mid-season test are ignored. (b) Mean SEM
season-long PVY spread as function of mid-season test in categories. (c) Likelihood that season-
long PVY spread would remain below regulatory limit of 5% as a function of mid-season test result;
values are % of all study fields in each category remaining within 5% PVY (Adapted from
MacKenzie et al. 2016, with permission)
plants in the field, often multiple times per season, with personnel experienced in
recognizing problem plants. In the field, different varieties show varying symptom
expressions at different growth stages or time since PVY infection. As well, many
varieties are cryptic when infected with some strains of PVY, particularly recombi-
nant strains such as PVYNTN, which are proliferating more rapidly in recent years in
many potato-producing regions (MacKenzie et al. 2019; Karasev and Gray 2013b).
Roguing may also cause disturbance of aphids during inspection of the crop, or high-
contrast bare patches from removed plants serving to visually cue aphids, and thus
may actually increase PVY spread in a crop (Dupuis et al. 2017). Thus, the efficacy
and economics of roguing specifically for PVY management is increasingly
questionable and indeed may promote a false sense of security with some of the
PVY-tolerant varieties or cryptic variety-strain combinations.
A number of other cultural practices have been investigated for reducing PVY
spread into or within the potato crop, such as crop borders, mulching, or
intercropping. Many of these approaches have been used in combination with
other techniques to reduce PVY spread or have been employed to combat other
disease organisms in addition to PVY (Dupuis et al. 2017). Crop borders use a
narrow sacrificial crop surrounding a susceptible primary potato crop; it could be
different crop such as a fast growing and tall grain or a tolerant or PVY-resistant
potato variety that need not be harvested for marketing as seed. The border can serve
as both a physical barrier and a virus sink, which can slow viruliferous aphids
entering the field and physically clean viral particles from their mouthparts if it is
a plant they would probe for feeding (Dupuis et al. 2017). Experimental studies have
shown widely varying success rates at reducing PVY spread in bordered plots versus
non-bordered ones. Relative reductions in PVY spread reached 27–60% with a very
large (24 m wide) soybean border in a study by Difonzo et al. (1996). Boiteau et al.
(2009), however, attained 20–60% reductions over 3 trial years with narrower (4 m)
borders of grass or the resistant potato variety Kennebec, similar to the
PVY-protective effect of regular mineral oil spraying without crop borders. Major
considerations for using crop borders, which may limit their adoption by industry,
are properly balancing the size and composition of a border to be sufficiently
protective, yet not wasting valuable field area for regular production. Also important
is the added management complexity of sourcing seed, maintaining and separate
harvesting of both the primary and border crop, and care isolating tubers during
harvest if an alternate potato variety is employed as a border.
Mulching, piling of dry plant materials such as straw from grain harvesting
between potato rows, and intercropping, growing other crops between potato rows,
have also been investigated for reducing PVY spread. Mulching is thought to reduce
contrast and thus visual cues for aphids to land on potato plants and potentially infect
them with PVY. In one multiyear study, relative reductions in PVY spread of
27–48% compared to non-mulched controls were realized (Saucke and Döring
2004). Similarly, intercropping may reduce visual cuing of aphids, but also may
serve as a virus sink during aphid feeding, like crop borders. In a study comparing
mineral oil spraying, straw mulching, intercropping with oats, and combined
treatments (described in Dupuis et al. 2017), nearly 90% reduction in PVY was
attained with combined mineral oil and intercropping treatments, about 70–80%
reduction with all three combined or paired combined mineral oil and mulching, and
somewhat less protection when each of the three were used alone. Several
advantages of these techniques are that they are generally more environmentally
friendly than more standard chemical foliar sprays, can help mitigate soil erosion,
and add organic matter to field soil. However, the mulch may introduce other
pathogenic organisms and requires a large source of grain straw or similar material
early in the season and specialized equipment and labor to deploy. Intercropping also
requires extra labor and equipment, cost of intercropping seed, and careful planning
of crop type, planting, and harvest of intercrop to minimize nutrient, light, or space
competition with the potato crop.
6.4 Conclusions and Continuing Challenges
In recent decades, major advances have been made in understanding the evolution-
ary diversity, host interactions, and mechanisms of transmission of PVY, which have
served the design of management practices to control PVY’s impact in potato
industries around the world. Despite this, PVY remains the most common virus in
industrial potato production, causing a significant economic impact on these
industries through crop yield and quality reduction and rejection of tubers from
local and export markets.
Control of PVY has been presented with many new and continuing challenges,
which remain important focal points for research and vigilant implementation in the
field. These center on aspects of potato breeding, PVY detection and certification,
in-field chemical control, continuous grower education and discipline, and the
changing biology of PVY itself. Though several PVY-resistant varieties have been
commercialized in recent decades, their often less-than-preferred performance, lim-
ited availability, or simple novelty in a sometimes conservative industry has limited
their broad appeal in the market. Renewing efforts to produce high-yielding varieties
resistant to all PVY strains while maintaining characteristics of locally important
PVY-susceptible varieties should be prioritized, along with education and marketing
efforts to encourage their uptake. Instituting seed certification programs in regions
without them and more aggressive virus incidence targets under existing programs
could rapidly force down PVY levels in the local seed supply. With the varying
symptomologies of diverse PVY strains, molecular technologies are increasingly
important for accurate assessment of PVY incidence in seed lots; many such tests
(ELISA, RT-PCR) have been available for decades. A growing number of different
molecular detection technologies, while some are relatively obscure and
underutilized, may allow for faster, cheaper, and field-deployable testing to assist
growers in rapid and informed decision-making during the growing season.
Particularly in Europe and North America, frequent insecticide limitations may
restrict use of the few chemical controls which show some effectiveness at reducing
aphid-mediated transmission. Also, mounting resistance in aphid populations to
common insecticide chemistries like pyrethroids and neonicotinoids progressively
reduces their effectiveness. Development and testing of novel, effective chemical
controls should continue, particularly including biological agents that are more
environmentally acceptable and less likely to generate resistance. Also, while
many research groups like ours have developed locally relevant, economical, and
effective best management practice recommendations to control PVY on-farm, these
practices need to be validated in different regions, with different aphid species and
abundances, potato varieties, climate, and labor and input cost structures. As well, in
regions which have gained substantial control of PVY, continuous discipline in
monitoring, control, and engagement between growers, industry oversight bodies,

and researchers is needed to prevent complacency and resurgence of the virus.
Finally, changes in the virus itself – namely, the rapid emergence and spread of
novel recombinant PVY strains – present an extra layer of challenge and require all
existing and future management practices to be reassessed in light of strain-specific
responses of the virus. Vigilance surrounding the continued efficacy of management
techniques, detection technologies and identification of PVY and its effects on the
crop will be critical to coping with PVY at field, industry, and global scales into the
future.
References
Al-Mrabeh A, Anderson E, Torrance L, Evans A, Fenton B (2010) A literature review of insecticide
and mineral oil use in preventing the spread of non-persistent viruses in potato crops. Agricul-
ture and Horticulture Development Board, UK
Basky Z, Almási A (2005) Differences in aphid transmissibility and translocation between PVYN
and PVYO isolates. J Pest Sci 78:67–75
Beemster ABR (1972) Virus translocation in potato plants and mature-plant resistance. In: Viruses
of potatoes and seed-potato production. Centre for Agricultural Publishing and Documentation
(PUDOC), Wageningen, pp 144–151
Bellstedt DU, Glais L, Davie K, Lacomme C (2017) Evolution and Origin of PVY. In: Lacomme C
et al (eds) Potato virus Y: biodiversity, pathogenicity, epidemiology and management. Springer,
Cham, pp 77–101
Boiteau G, Singh M, Lavoie J (2009) Crop border and mineral oil sprays used in combination as
physical control methods of the aphid-transmitted potato virus Y in potato. Pest Manag Sci
65:255–259
Bond L, Nolan T (2011) Making sense of perceptions of risk of diseases and vaccinations: a
qualitative study combining models of health beliefs, decision-making and risk perception.
BMC Public Health 11:943
Boquel S, Zhang J, Goyer C, Giguère MA, Clark C, Pelletier Y (2015) Effect of insecticide-treated
potato plants on aphid behavior and potato virus Y acquisition. Pest Manag Sci 71:1106–1112
Bradley RHE, Moore CA, Pond DD (1966) Spread of potato virus Y curtailed by oil. Nature
209:1370–1371
Cervantes FA, Alvarez JM (2011) Within plant distribution of potato virus Y in hairy nightshade
(Solanum sarrachoides): an inoculum source affecting PVY aphid transmission. Virus Res
159:194–200
Christian RC, Alm J (2014) Empathy, sympathy, and tax compliance. J Econ Psychol 40:62–82
Collar JL, Avilla C, Duque M, Fereres A (1997) Behavioral response and virus vector ability of
Myzus persicae (Homoptera: Aphididae) probing on pepper plants treated with aphicides. J
Econ Entomol 90:1628–1634
Coutts BA, Jones RAC (2015) Potato virus Y: contact transmission, stability, inactivation, and
infection sources. Plant Dis 99:387–394
Criniti A, Mazzoni E, Cassanelli S, Cravedi P, Tondelli A, Bizzaro D, Manicardi GC (2008)
Biochemical and molecular diagnosis of insecticide resistance conferred by esterase, MACE,
kdr and super-kdr based mechanisms in Italian strains of the peach potato aphid, Myzus persicae
(Sulzer). Pestic Biochem Physiol 90:168–174
Davidson RD, Houser AJ, Sather K, Haslar R (2013) Controlling PVY in seed: what works and
what does not. Am J Potato Res 90:28–32
DiFonzo CD, Ragsdale DW, Radcliffe EB, Gudmestad NC, Secor GA (1996) Crop borders reduce
potato virus Y incidence in seed potato. Ann Appl Biol 129:289–302
Dupuis B (2017) The movement of potato virus Y (PVY) in the vascular system of potato plants.
Eur J Plant Pathol 147:365–373
Dupuis B, Bragard C, Carnegie S, Kerr J, Glais L, Singh M, Nolte P, Rolot JL, Demeulemeester K,
Lacomme C (2017) Potato virus Y: Control, Management and Seed Certification Programmes.
In: Lacomme C et al (eds) Potato virus Y: biodiversity, pathogenicity, epidemiology and
management. Springer, Cham, pp 177–206
Fageria MS, Boquel S, Leclair G, Pelletier Y (2014) The use of mineral oil in potato protection:
dynamics in the plant and effect on potato virus Y spread. Am J Potato Res 91:476–484
Fageria M, Nie X, Gallagher A, Singh M (2015) Mechanical transmission of potato virus Y (PVY)
through seed cutting and plant wounding. Am J Potato Res 92:143–147
Fenton B, Lacomme C, Collins L, Fox A, Pickup J, Evans A, Northing P, Anderson E (2012)
Aphids and virus transmission in seed crops. Potato Council and Agriculture and Horticulture
Development Board (AHDB, UK) project R428 final report
Foster SP, Paul VL, Slater R, Warren A, Denholm I, Field LM, Williamson MS (2014) A mutation
(L1014F) in the voltage-gated sodium channel of the grain aphid, Sitobion avenae, is associated
with resistance to pyrethroid insecticides. Pest Manag Sci 70:1249–1253
Frost KE, Groves RL, Charkowski AO (2013) Integrated control of potato pathogens through seed
potato certification and provision of clean seed potatoes. Plant Dis 97:1268–1280
Funke CN, Nikolaeva OV, Green KJ, Tran LT, Chikh-Ali M, Quintero-Ferrer A, Cating RA, Frost
KE, Hamm PB, Olsen N, Pavek MJ, Gray SM, Crosslin JM, Karasev AV (2017) Strain-specific
resistance to potato virus Y (PVY) in potato and its effect on the relative abundance of PVY
strains in commercial potato fields. Plant Dis 101:20–28
Gildemacher PR, Schulte-Geldermann E, Borus D, Demo P, Kinyae P, Mundia P, Struik PC (2011)
Seed potato quality improvement through positive selection by smallholder farmers in Kenya.
Potato Res 54:253
Gray SM, Power AG (2018) Anthropogenic influences on emergence of vector-borne plant viruses:
the persistent problem of potato virus Y. Curr Opin Virol 33:177–183
Gray S, De Boer S, Lorenzen J, Karasev A, Whitworth J, Nolte P, Singh RP, Boucher A, Xu H
(2010) Potato virus Y: an evolving concern for potato crops in the United States and Canada.
Plant Dis 94:1384–1397
Green KJ, Brown CJ, Karasev AV (2018) Genetic diversity of potato virus Y (PVY): sequence
analyses reveal ten novel PVY recombinant structures. Arch Virol 163:23–32
Halbert SE, Corsini DL, Wiebe MA (2003) Potato virus Y transmission efficiency for some
common aphids in Idaho. Am J Potato Res 80:87–91
Hansen PG, Skov LR, Skov KL (2016) Making healthy choices easier: regulation versus nudging.
Annu Rev Public Health 37:237–251
Hosmer DW, Lemeshow S (2000) Applied logistic regression, 2nd edn. Wiley, New York
Inglis DA, Gundersen B, Beissinger A, Benedict C, Karasev AV (2019) Potato virus Y in seed
potatoes sold at garden stores in Western Washington: prevalence and strain composition. Am J
Potato Res 96:235–243
Johnson DA, Alldredge JR, Hamm PB (1998) Expansion of potato late blight forecasting models
for the Columbia Basin of Washington and Oregon. Plant Dis 82:642–645
Jones DAC, Woodford JAT, Main SC, Pallett D, Barker H (1996) The role of volunteer potatoes in
the spread of potato virus YN in ware crops of cv. Record. Ann Appl Biol 129:471–478
Kaliciak A, Syller J (2009) New hosts of potato virus Y (PVY) among common wild plants in
Europe. Eur J Plant Pathol 124:707–713
Karasev AV, Gray SM (2013a) Continuous and emerging challenges of potato virus Y in potato.
Annu Rev Phytopathol l51:571–586
Karasev AV, Gray SM (2013b) Genetic diversity of potato virus Y complex. Am J Potato Res
90:7–13
Kerlan C, Robert Y, Perennec P, Guillery E (1987) Survey of the level of infection by PVYO and
control methods developed in France for potato seed production. Potato Res 30:651–667
Kirchner SM, Hiltunen LH, Santala J, Döring TF, Ketola J, Kankaala A, Wirtanen E, Valkonen JPT
(2014) Comparison of straw mulch, insecticides, mineral oil, and birch extract for control of
transmission of potato virus Y in seed potato crops. Potato Res 57:59–75
Klueken AM, Hau B, Ulber B, Poehling HM (2009) Forecasting migration of cereal aphids
(Hemiptera: Aphididae) in autumn and spring. J Appl Entomol 133:328–344
Lacomme C, Jacquot E (2017) General characteristics of potato virus Y (PVY) and its impact on
potato production: An overview. In: Lacomme C et al (eds) Potato virus Y: biodiversity,
pathogenicity, epidemiology and management. Springer, Cham, pp 1–19
Lacomme C, Pickup J, Fox A, Glais L, Dupuis B, Steinger T, Rolot JL, Vakonen JPT, Kruger K,
Nie X, Modic S, Mehle N, Ravnikar M, Hullé M (2017) Transmission and epidemiology of
potato virus. In: n Lacomme c Y et al (eds) Potato virus Y: biodiversity, pathogenicity,
epidemiology and management. Springer, Cham, pp 141–176
MacKenzie TD, Fageria MS, Nie X, Singh M (2014) Effects of crop management practices on
current-season spread of potato virus Y. Plant Dis 98:213–222
MacKenzie TD, Nie X, Singh M (2016) Crop management practices and reduction of on-farm
spread of potato virus Y: a 5-year study in commercial potato fields in New Brunswick, Canada.
Am J Potato Res 93:552–563
MacKenzie TD, Lavoie J, Nie X, Singh M (2017) Effectiveness of combined use of mineral oil and
insecticide spray in reducing potato virus Y (PVY) spread under field conditions in New
Brunswick, Canada. Am J Potato Res 94:70–80
MacKenzie TD, ArjuI GA, Nie X, Singh M (2018a) Evidence of potato virus Y spread through
post-emergence management practices in commercial potato fields. Am J Potato Res
95:720–728
MacKenzie TD, Lavoie J, Nie X, Singh M (2018b) Differential spread of potato virus Y (PVY)
strains O, N:O and NTN in the field: implications for the rise of recombinant PVY strains in
New Brunswick, Canada. Am J Potato Res 95:301–310
MacKenzie TD, Arju I, Poirier R, Singh M (2018c) A genetic survey of pyrethroid insecticide
resistance in aphids in New Brunswick, Canada, with particular emphasis on aphids as vectors
of potato virus Y. J Econ Entomol 111:1361–1368
MacKenzie TD, Nie X, Bisht V, Singh M (2019) Proliferation of recombinant PVY strains in two
potato producing regions of Canada, and symptom expression in 30 important potato varieties
with different PVY strains. Plant Dis. https://doi.org/10.1094/PDIS-09-18-1564-RE
Martín-López B, Varela I, Marnotes S, Cabaleiro C (2006) Use of oils combined with low doses of
insecticide for the control of Myzus persicae and PVY epidemics. Pest Manag Sci 62:372–378
Mehle N, Gutiérrez-Aguirre I, Prezelj N, Delić D, Vidic U, Ravnikar M (2014) Survival and
transmission of potato virus Y, Pepino mosaic virus, and potato spindle tuber viroid in water.
Morita M, Ueda T, Yoneda T, Koyanagi T, Haga T (2007) Flonicamid, a novel insecticide with a
rapid inhibitory effect on aphid feeding. Pest Manag Sci 63:969–973
Nanayakkara U, Nie X, Giguère MA, Zhang J, Pelletier Y (2012) Chenopodium album L. as a host
for potato virus Y (PVY) in New Brunswick, Canada. Am J Potato Res 89:245–247
Nolte P, Whitworth JL, Thornton MK, McIntosh CS (2004) Effect of seedborne potato virus Y on
performance of russet Burbank, russet Norkotah, and Shepody potato. Plant Dis 88:248–252
Pelletier Y, Nie X, Giguère MA, Nanayakkara U, Maw E, Foottit R (2012) A new approach for the
identification of aphid vectors (Hemiptera: Aphididae) of potato virus Y. J Econ Entomol
105:1909–1914
Perring TM, Gruenhagen NM, Farrar CA (1999) Management of plant viral diseases through
chemical control of insect vectors. Annu Rev Entomol 44:457–481
Pirone TP, Perry KL (2002) Aphids: non-persistent transmission. Adv Bot Res 36:1–20
Radcliffe EB, Ragsdale DW, Suranyi RA, Difonzo CD, Hladilek EE (2008) Aphid alert: how it
came to be, what it achieved and why it proved unsustainable. In: Koul O et al (eds) Areawide
pest management: Theory and implementation. CABI Publishing, pp 244–260
Saucke H, Döring TF (2004) Potato virus Y reduction by straw mulch in organic potatoes. Ann
Appl Biol 144:347–355
Singh M, Singh RP (1996) Nucleotide sequence and genome organization of a Canadian isolate of
the common strain of potato virus Y (PVYO). Can J Plant Pathol 18:209–224
Slater R, Paul VL, Andrews M, Garbay M, Camblin P (2012) Identifying the presence of
neonicotinoid resistant peach-potato aphid (Myzus persicae) in the peach-growing regions of
southern France and northern Spain. Pest Manag Sci 68:634–638
Steinger T, Gilliand H, Hebeisen T (2014) Epidemiological analysis of risk factors for the spread of
potato viruses in Switzerland. Ann Appl Biol 164:200–207
Steinger T, Goy G, Gilliand H, Hebeisen T, Derron J (2015) Forecasting virus disease in seed
potatoes using flight activity data of aphid vectors. Ann Appl Biol 166:410–419
Sturz AV, Stewart JG, McRae KB, Diamond JF, Lu X, Singh RP (2000) Assessment of the
importance of seed cutting, in-season cultivation, and the passage of row equipment in the
spread of PVY in potatoes. Can J Plant Pathol 22:166–173
Sudarshan A (2017) Nudges in the marketplace: the response of household electricity consumption
to information and monetary incentives. J Econ Behav Organ 134:320–335
Thaler RH, Sunstein CR (2009) Nudge: improving decisions about health, wealth, and happiness.
Yale University Press, New Haven and London
Thomas-Sharma S, Abdurahman A, AliS A-PJL, Bao S, Charkowski AO, Crook D, Kadian M,
Kromann P, Struik PC, Torrance L, Garrett KA, Forbes GA (2016) Seed degeneration in potato:
the need for an integrated seed health strategy to mitigate the problem in developing countries.
Plant Pathol 65:3–16
Valkonen JP, Gebhardt C, Zimnoch-Guzowska E, Watanabe KN (2017) Resistance to Potato virus
Y in Potato. In: Lacomme C et al (eds) Potato virus Y: biodiversity, pathogenicity, epidemiol-
ogy and management. Springer, Cham, pp 207–241
Valli AA, Gallo A, Rodamilans B, López-Moya JJ, García JA (2018) The HCPro from the
Potyviridae family: an enviable multitasking helper component that every virus would like to
have. Mol Plant Pathol 19:744–763
Verbeek M, Piron PGM, Dullemans AM, Cuperus C, Van Der Vlugt RAA (2010) Determination of
aphid transmission efficiencies for N, NTN and Wilga strains of potato virus Y. Ann Appl Biol
156:39–49
Major Fungal French Bean Diseases:
Epidemiology and Management 7
S. K. Gupta and Guervinder Singh
Abstract
French bean (Phaseolus vulgaris L.) is an important leguminous vegetable crop,

grown throughout the world for its green pods as well as dry beans. During
cultivation these crops are infected with several diseases of fungal, bacterial and
viral nature which not only reduces the yield but also impair the quality of the
produce. In this chapter, major fungal diseases like root rot and web blight
(Rhizoctonia solani), angular leaf spot (Phaeoisariopsis griseola), anthracnose
(Colletotrichum lindemuthianum) and rust (Uromyces appendiculatus) infecting
this crop are discussed in light of their epidemiology and management. The
management practices include cultural, host resistance, biological and chemical
control alone and their integration. It was also discussed how number of sprays of
fungicides can be reduced by using epidemiological parameters.
Keywords
Phaseolus vulgaris · Fungal diseases · Epidemiology · Management
7.1 Introduction
French bean (Phaseolus vulgaris L.) is one of the most important leguminous
vegetable crops grown throughout the world for its green pods as well as dry
beans (rajmah). Various types of beans are grown throughout the world belonging
S. K. Gupta (*)
School of Agriculture, Shoolini University of Biotechnology and Management Sciences, Solan,
India
e-mail: satishgupta@shooliniuniversity.com
G. Singh
Department of Plant Pathology, Dr. Y.S. Parmar University of Horticulture and Forestry, Solan,
India

https://doi.org/10.1007/978-981-15-6275-4_7
142 S. K. Gupta and G. Singh
to different genera of the family Leguminosae. The important members of broad

group termed as beans are common bean or French bean (Phaseolus vulgaris L.),
lima bean (P. lunatus L.), scarlet bean (P. multiflorus Wild.), tepary bean (Mucuna
sp.), field bean or lablab (Dolichos lablab), sword bean (Canavalia ensiformis DC.)
and cluster bean (Cyamopsis tetragonoloba (L.) DC.); these are grown as grain
crops, as vegetables for green pods and as green manure crops for incorporating into
the soil. It has gained increasing popularity due to its quality proteins and nutritional
balance besides certain medicinal properties. The dry seeds are a rich source of
proteins, calcium and iron and contain a large amount of Vitamin B1 (Stiebeling and
Clark 1939). At present French bean is grown throughout the cooler tropics but not
in the semi-arid or wet humid regions (Chatterjee and Bhattacharyya 1986).
French bean is a traditional crop of temperate region and it occupies an important
position among various kharif vegetable crops and pulses grown in temperate hills of
India at an altitude ranging from 900 to 2500 m above mean sea level (Sharma and
Sohi 1989). In India, the crop is largely grown in Himachal Pradesh, Jammu and
Kashmir, Uttarakhand, North Eastern Hills, Darjeeling, South Plateau, South Indian
Hills (Nilgiri and Palni hills), Mahabaleshwar, Ratnagiri (Maharashtra) and
Chickmangalore (Karnataka), having mild climate with humid environmental
conditions. In India, it is cultivated in an area of 1,98,000 ha with a production of
2,012,000 MT (NHB 2017).
Successful cultivation of this crop is hindered due to the attack of various diseases
of fungal, bacterial and viral in nature of which fungal diseases are very important.
Under favourable environmental conditions, epiphytotics of these diseases have
often reduced the yields considerably. Fungal diseases are either monocyclic or
polycyclic in nature. Monocyclic diseases are mostly soil-borne diseases like wilts
and root rots and they complete their one disease cycle in a year. In these diseases
mostly disease incidence is recorded. In case of polycyclic diseases, the inoculum
(s) is both soil- and air-borne and pathogen complete many cycles in a single season
and can cause epidemic under favourable weather conditions. Examples are of leaf
spots, rusts and anthracnose. In these diseases, disease severity is recorded in most of
the cases.
The introduction of high yielding cultivars like pencil beans coupled with higher
doses of fertilizers, assured irrigation and intensive agriculture has resulted in the
higher susceptibility of the plants to diseases causing severe qualitative and quanti-
tative losses thus making the produce less competitive in the national and interna-
tional markets. These problems are further aggravated by the changing climatic
conditions. A number of new diseases have appeared in different areas and many
diseases which were of minor importance have become a major threat under this
system. In favourable weather conditions, the cumulative losses have been estimated
to 26–40% and even complete crop failure of the crop in certain cases which
necessitate the role of plant protection. In this chapter, epidemiological parameters
of major fungal diseases of French bean along with their management by including
cultural, host resistance, biological and chemical methods alone and their integration
have been discussed.
7 Major Fungal French Bean Diseases: Epidemiology and Management 143
Epidemic is the “Change in disease intensity in a host population over time and
space” whereas Epidemiology is the science of populations of pathogens in
populations of host plants, and the diseases resulting there from under the influence
of the environment and human interferences (Vander Plank 1963). Elements of
epidemic are very well described by disease triangle.
Elements of epidemic are susceptible host, virulent pathogen and favourable
environment. Interaction of these three components describes the disease develop-
ment. Disease development is also affected by time and human activity. Each of
these components affects the development of epidemic. Host affects the develop-
ment of epidemic in many ways like genetic resistance or susceptibility, degree of
genetic uniformity among host plants in a particular field, age of the host plants and
types of crop (annual or perennial). Similarly, epidemics also depend on the viru-
lence of the pathogen, type of reproduction like monocyclic, polycyclic or polyetic
and also depend on the ecology and mode of spread of the pathogen. Environment
plays an important role in the development of epidemic. Environmental factors like
rain, dew, high humidity, temperature, wind velocity, etc. affect the diseases devel-
opment and pathogen cycles. Time factors like season of the year, duration and
frequency of favourable temperature and rain also affect the disease development.
Human interferences also help in epidemic development in many ways like site
selection and preparation, selection of the planting material, cultural practices
followed and disease control measures used.
To study the epidemiology of a particular disease, one has to carry out two types
of study: one is under control conditions, i.e. under glasshouse having controlled
conditions and other under natural epiphytotic conditions. In glasshouse, the studies
like the effect of temperature, humidity, leaf wetness and intermittent leaf wetness
can be carried out. Under field conditions, mainly the role of environmental factors
like temperature, relative humidity rainfall, wind speed, etc. are recorded under
natural epiphytotic conditions. Disease severity of each interval is collected. Meteo-
rological data is generally collected from the Meteorological sections of the Univer-
sity if the meteorological instruments have not been installed in the field where the
experiment is laid out. Correlation and regression coefficients are calculated by
following the procedures as described by Gomez and Gomez (1984) and the
regression equation is developed. Besides this, infection rate (Vander Plank 1963)
and area under disease progress curve (Shanner and Finney 1977) are also
calculated.
Diurnal periodicity of spore release is generally conducted in the diseases like leaf
spots and rusts where the inoculums are wind-borne. Studies on spores release in
relation to climatic factors and diurnal periodicity of spore density in air are
conducted in the experimental fields. For this purpose, the field air at the
mid-height of crop canopy is periodically assessed for the presence of spores by a
Burkard Air Sampler (Burkard Manufacturing Co. Ltd., Hertfordshire, England)
which sucks in air at the rate of 10 l/min. The diurnal periodicity of the spore density
in air is measured at every 4 h interval in the crop season for a week by counting the
number of spores deposited on the slides. Simultaneously, the periodicity of
predominant weather parameters, viz., temperature, relative humidity and wind

speed were also recorded and compared.
7.2 Epidemiology and Management of Major Fungal Diseases

of French Bean
The information on epidemiology and management of important fungal diseases is

given in this chapter.
7.2.1 Root Rot and Web Blight
This is an important disease of this crop world over. Root rot phase of this disease
was first reported from New Jersey in the year 1924 (Cook 1924) while web blight
was first described as a destructive disease of beans in Puerto Rico (Matz 1921). In
India, root rot disease of French bean was recorded for the first time in 1980 from
Bangalore (Sharma and Sohi 1981). Later, Paul and Sharma (1990) reported the
occurrence of root rot from the Solan area of Himachal Pradesh while web blight was
recorded during the 1994 crop season (Mathew and Gupta 1996) from the same
locality. Rajnauth (1987) reported 25–100% yield losses in Trinidad due to this
disease while whole bean crop has been destroyed within 48–72 h in Brazil
(Sartorato 1988). In India, around 15% incidence of root rot in fields around
Bangalore has been recorded (Sharma and Sohi 1981) while yield losses in green
pods at different stages of plant growth varied from 8.4% to 64.6% (Sharma and Sohi
1980). The disease is caused by Rhizoctonia solani Kuhn. The basidial stage of the
fungus (Thanatephorus cucumeris) also occurs but very rarely.
7.2.1.1 Symptoms
The disease occurs on roots, stem, leaves, pods and seed (Mathew and Gupta 1996).
Sanchez and Cardenaz (1988) reported that root rot affects plant populations from
emergence to the first 30 days of crop growth. The distinguishing symptoms of the
disease occur in two phases and are described below:
Root Rot
Symptoms appear on roots and stem above and below the surface of the soil as
reddish brown cankers. The lesions enlarge rapidly and engirdle the stem at the
collar region, extending longitudinally downward to the roots. Roots are affected in
advanced stages of infection leading to partial or complete rotting of the root system.
Web Blight
The symptoms on leaves appear as small, circular, water-soaked spots. These spots
increase rapidly in size and cover extensive areas of the leaf blade. Initially, the spots
are much lighter in colour than the surrounding unaffected area. At later stages, they
become tan brown and surrounded by dark borders. The spots coalesce to form larger
areas on the leaf blade. Light tan hyphae develop on both surfaces of the infected leaf
and at later stages are studded with minute sclerotia of the fungus. Defoliation occurs
in advanced stages of infection. The pods are attacked at all stages of growth. On
young pods, circular slimy water-soaked spots appear which are light tan in colour.
On mature pods, the spots are dark brown, more or less circular, slightly zonate and
definitely sunken. Spots coalesce to cover the entire pod. The distal end of the pods is
the most affected. In favourable weather conditions, runner hyphae of the fungus are
seen spreading from the point of infection to healthy parts, which later become
studded with sclerotia. Tan brown to reddish brown discoloration is observed on
infected seed, located immediately below the spots on the affected pods.
7.2.1.2 Epidemiology
The pathogen perennates as mycelium or sclerotia in the soil on plant debris. The
germination of sclerotia gives rise to infective propagules, which are sufficient to
cause infection. The fungus spreads with rain, irrigation or floodwater, tools and with
infected or contaminated propagation material. The association of R. solani with
bean seed has also been reported (Nitshe and Cafati 1985). Seed transmission up to
46% in cultivars like Kentucky Wonder and Contender (Gupta et al. 2000a) has been
reported. Shama and Shama (1989) indicated that seed penetration of R. solani
occurs directly through intact seed coat or through the hilum tracheids and then
the pathogen is transferred from infected seed to the soil. The infected seeds after
germination either rots in the soil itself or give rise to plants that later damped off.
The soil-borne inoculum is splashed by rain to the foliage that causes web blight. On
some plants or plant parts the pathogen can enter only through wounds or through
openings in the epidermis while in others, it thrives in the soil or in mud splashed on
to the plants until it can enter through healthy tissue. In favourable weather, runner
hyphae of the pathogen are seen spreading from the point of infection to healthy
parts, which later become studded with sclerotia.
Root rot is favoured by high soil temperature and soil moisture while web blight
is more prevalent in the wet season. Rhizoctonia diseases of roots are most severe
under moist soil conditions (Tu et al. 1992). Bruggen et al. (1986) reported a positive
correlation between lesion size and soil moisture. If the moisture is sufficient enough
or there is frequent drizzle, pronounced blight symptoms will appear. During humid
periods fan-shaped whitish hyphae spread from the point of infection and produce
the conspicuous and destructive phase of the disease. Diaz Plaza et al. (1991)
reported that disease incidence and severity increased after rain. Continuous leaf
wetness for at least 6 h was essential for disease initiation and increase in leaf
wetness durations from 6 to 12 h showed a corresponding decrease in incubation
period from 36 to 26 h whereas further increase in leaf wetness did not exert any
effect on incubation period (Upmanyu and Gupta 2005). High rainfall and soil
moisture coupled with high relative humidity and soil temperature (23–25 C)
were found to be favourable for web blight (Mathew and Gupta 1996) development.
A high soil moisture (80%) and a temperature of 25 C were the most favourable for
root rot development, while web blight development was optimum at >85% relative
humidity coupled with 25 C temperature (Upmanyu and Gupta 2005).
7.2.1.3 Management
Since the causal fungus persists in the soil either as mycelium or sclerotia, therefore,
an integrated approach of disease management combining the use of cultural,
biological and chemical methods will be required to keep the disease in check.
Cultural Practices
Cultural practices, viz. planting disease-free seed, mulching, adjustment of planting
date, medium (30–60 15 cm) plant spacing, crop rotation and soil solarization
have been recommended to keep the disease under check (Sartarato et al. 1988; Tu
1992; Sharma and Gupta 2003). Deep tillage (30 cm sub-soiling) can reduce soil
compaction and thereby, root rot severity in addition to increased yield (Tan and Tu
1995). Voland and Epstein (1994) reported the efficacy of fresh manure over
composted manure in reducing bean hypocotyl lesions while soil amendment with
cotton, mustard and neem cakes was found effective in reducing root rot incidence
and increased yields (Upmanyu et al. 2002). The effectiveness of these amendments
may be due to enhanced populations of non-parasitic microorganisms exhibiting
antagonistic effects on the pathogen thereby inhibiting the mycelial growth and
sclerotia production of the pathogen. Soil amendments with low soil C:N ratio
increased incidence of R. solani whereas high C:N ratio amendment did not interfere
with the incidence while the incidence was independent of soil moisture conditions
(Fenille and Souzo 1999). Poultry manure, composted Urtica sp. and composted
Lantana camara treatments were superior to other composts in suppressing Rhizoc-
tonia root rot (Joshi et al. 2009).
Host Resistance
Prasad and Weigle (1976) observed that dark seeded PI accessions of French bean,
viz. 109859, 163583, 174908, 226895 and 300665 were resistant to R. solani while
most of the white seeded cultivars were susceptible to both seed infection and
pre-emergence damping off. However, Sharma and Sohi (1981) reported all the
three French bean cultivars, viz. “Contender”, “Premier” and “Giant Stringless” to be
equally susceptible irrespective of their colour. Bush type cultivars/lines are more
susceptible to the disease than pole type ones. The cultivars/lines, viz. Wisconsin
RRR 46, BAT 477, BAT 332, BAT 1753, RIZ 30, EMP 81, A 300, ICA Pijao and
Jackson Wonder have been reported to be highly resistant (Hagedorn and Rand
1980; Tu and Park 1993; Muyolo et al. 1993), while PLB-43, EC-18834, IC-18593,
PI-249554, Phagu Chitra, Pole wall-I, Pole wall-II and K-13522 were found moder-
ately susceptible to the disease (Gupta et al. 1997). Cvs./lines ET 8396 and IPR 96-4
were found resistant both under natural epiphytotic and in vitro conditions that can
be used in breeding programmes as a source of resistance (Upmanyu et al. 2004;
Khati and Hooda 2006).
Biological Control
Exploitation of the populations of bioagents may prove useful to combat the ravages
of soil-borne diseases. Biological treatment of soil under glasshouse conditions with
Trichoderma viride showed results similar to chemical control (Dumitras and Sesan
1990). Elad et al. (1980) found that in naturally infested soils, wheat bran
preparations of Trichoderma harzianum significantly decreased the Rhizoctonia
disease of beans. Tu and Vaartaja (1981) reported that Gliocladium virens, a
hyperparasite of R. solani, inhibited production of sclerotia. Management of this
disease by seed/soil treatment and foliar sprays with T. harzianum, T. virens and
T. viride has also been reported (Mathew and Gupta 1998; Hazarika and Das 1998;
Upmanyu et al. 2002). Inhibition of mycelial growth and sclerotial germination of
this pathogen by T. harzianum and T. viride culture filtrate has also been observed
(Hazarika and Das 1998). Roberti et al. (1993) indicated that T. virde produced
non-volatile compounds while T. harzianum produced coils and hook pincer-shaped
structures, which prevented the growth of R. solani. The efficacy of potential
bacterial antagonists, viz. fluorescent pseudomonads and Bacillus spp. against root
rot has also been reported (Sanchez et al. 1994; Wolk and Sarkar 1994, 1995). An
endophytic Pseudomonas florescens carrying Serratia marcescens Chi A gene either
on the plasmid or integrated into the chromosome controlled effectively the phyto-
pathogenic fungus Rhizoctonia solani on bean seedlings under plant growth cham-
ber conditions (Downing and Thomson 2000). Coinoculation of seed with Bacillus
subtilis MB1600 (Epic) and Rhizobium tropici significantly reduced root rot severity
and enhanced yield (Estevez-de-Jensen et al. 2002). Efficacy of seed treatment and
soil application of B. subtilis in the reduction of pre- and post-emergence root rot
have also been reported (Sharma and Gupta 2003). The water extracts obtained from
green parts of potato, tomato and rape and Trichoderma PBG-1 showed marked
antagonistic activity against Rhizoctonia solani (Smolinska and Kowalska 2006).
Out of 60 isolates of Trichoderma, 7 isolates significantly reduced the incidence of
root rot compared to the control. T. harzianum isolates Tr-34 and Tr-14 were the
most effective, resulting in only 20.8% and 23.4% root-rot incidence, respectively
(Joshi et al. 2008).
Chemical Control
Different fungicides have been reported to be effective for the management of this
disease. Seed treatment with fungicides, viz. benomyl, captan, thiram, thiophanate-
methyl, carbendazim, carboxin, terrachlor, pencycuron, iprodione + thiram (Raffat
1992; Maringoni et al. 1992) gave maximum protection against pre- and post-
emergence mortality and also significantly increased the yield of green pods. How-
ever, seed treatment followed by foliar sprays with carbendazim or bitertanol gave
best results against web blight (Thakur et al. 1991). Gupta et al. (1999) reported good
efficacy of Bavistin (0.2%), Celest (0.1%), Topsin-M (0.2%) and Raxil (0.2%)
against pre-emergence damping off of beans. Good control of web blight phase of
this disease was achieved by seed treatment with Celest (0.2%) or Raxil (0.2%) or
Bavistin (0.2%) followed by foliar sprays of Bavistin (0.05%) or tebuconazole
(0.05%) (Mathew and Gupta 1996; Upmanyu et al. 2002).
Integrated Approach
Integration of seed treatment with bioagents and fungicides has been found to be
more effective to manage soil-borne pathogens. Tu and Zheng (1993) studied the
compatibility of seed treatment with Bacillus subtilis, Pseudomonas fluorescence or

Gliocladium virens and subsequently dressed with diazinon + captan + thiophanate-
methyl (DCT). They observed that fungicide treated seed showed inhibition of
bioagents, which was transient, and the growth and sporulation of the bioagents
resumed as the effect of the fungicides diminished. They further suggested that
bioagents such as B. subtilis and P. fluorescence are more mobile in the rhizosphere
of seedlings grown from treated seed and hence, their incorporation together with
fungicides can extend the effect of the chemical to give protection against root rot.
Upmanyu et al. (2002) also reported the tolerance in T. viride to carboxin,
tebuconazole and carbendazim. The combined seed treatment of French bean with
Contaf (0.025%) and Gliocladium virens (0.1%) resulted in higher seed germination,
lower incidence of the disease and higher grain yield (Mukherjee et al. 2001).
Combined treatment of seed (soaking in Trichoderma harzianum, 1 107
conidia/ml) and soil (20 and 100 kg/calcium and micronized sulphur (40% micron
sulphur), respectively) before sowing reduced the root rot incidence and increased
green pod yield (Ziedan and Mahamoud 2002). Soil amendment with mustard cake
along with carbendazim seed treatment and foliar sprays were found highly effective
in reducing the pre-emergence and post-emergence root rot and web blight severity
(Upmanyu et al. 2002). A combination treatment including soil solarization (SS) for
50 days + soil amendment (mustard cake) + combination of Allium sativum ethanol
extract and Bacillus subtilis was found effective in reducing pre- and post-
emergence damping-off while web blight was best contained by SS + soil amend-
ment + A. sativum (ST + FS) and increased yields (Sharma and Gupta 2003).
7.2.2 Angular Leaf Spot
It is an important disease of French beans particularly in mid-hill conditions where

moderate temperatures with high humidity conditions favour the development of this
disease. The losses due to angular leaf spot are of two types. Firstly, there is
reduction in photosynthetic capacity because of the heavy leaf spotting which drop
down prematurely those results in subsequent poor pod setting and secondly, the
green pod infection makes the pods unfit for market. Up to 50% yield losses under
epiphytotic condition have been reported from the USA whereas in Mexico, it has
been considered as a principal fungal disease where up to 90% incidence has been
recorded causing significant losses (Campos and Fucikovsky 1981). In India, the
total loss including damaged and unmarketable pods has been estimated to be about
40–70% due to this disease (Singh and Saini 1980) whereas up to 20–25% loss in
grain yield every year has been recorded in Sikkam (Srivastava and Gupta 1994).
The disease is caused by Phaeoisariopsis griseola (Sacc.) Ferr.
7.2.2.1 Symptoms
The pathogen infects all green parts of the plants, viz. leaves, petioles, stems, pods
and seed, producing varied symptoms. On cotyledonary leaves, the symptoms
appear as circular spots while on true leaves, 3–5 angled spots appear in between
veins and veinlets (Fig. 7.1). The spots are dark greyish on the upper surface and
light grey on the lower surface of the leaves but with the passage of time, change to
reddish brown and finally attain dark brown colour. On the lower surface, light
reddish discolouration appears on the veins and veinlets, which delimit such spots.
Grey mouldy coating covers the spots on the lower surface of the leaves. On close
observation, the spots reveal the presence of coremia bearing a large number of
spores. Such severely infected leaves show upward curling and defoliate prema-
turely. Elongated dark brown spots appear both on petioles and stems. Under severe
conditions, complete defoliation occurs resulting in premature death of the plants.
Pods are infected at all the developmental stages. Lesions on the pods are smooth,
usually circular and rarely elongated (Fig. 7.2). At first, these spots are superficial
having reddish brown centre with well-defined ashy black borders but with the
passage of time, deeper tissues are involved. In the central portion reddish brown,
coremia and spores are produced. Severely infected pods either bear no seeds or
produce only shrivelled seeds. In late infections, i.e. at maturity stage, the seeds
underneath the pod lesions show yellowish brown discolouration which is more
prominent on hilum region but can occur anywhere on the seed surface.
Fig. 7.1 Symptoms of angular leaf spot on leaves

Fig. 7.2 Symptoms of

angular leaf spot on pods
The pathogen (P. griseola) overwinters both on plant debris and in infected seed
(Sohi and Sharma 1974; Sindhan and Bose 1980). The viability of conidia on plant
debris is 6 and 8 months in laboratory and field, respectively (Sindhan and Bose
1980), thus it serves as a source of primary inoculum. Other source of primary
inoculum is an infected seed. Though the infestation site has been identified in the
hilum of seed but in some cultivars both hilum and seed coat seem to be infected
(Saettler and Corea 1988). While disease can initially result from the planting of
infected seed causing spots on cotyledonary leaves, the primary route in most bean
growing areas involves the rain splashed conidia from plant debris or windblown
conidia with soil particles attacking leaves, pods and stems. Infected leaves become
inoculum source for secondary spread of the disease. However, the rate and degree to
which secondary spread occurs is dependent on the interactions and influences of all
factors involved with the disease and its spread such as climate, pathogen and the
host. Moderate temperatures and moist conditions favour the arrival of viable
inoculum and successful field infections. Optimum temperature for growth and
sporulation, spore germination and development of the disease is 21–24 C,
18–24 C and 24 C (Sindhan and Bose 1980; Sarotorato 1988), while optimum
RH for sporulation, spore germination and development of symptoms has been
reported as >90%.
Mathew et al. (1998) studied the effect of leaf wetness durations at optimum
temperature and reported that leaf wetness of at least 12 h duration coupled with
moderate temperature (25 C) exhibited minimum incubation period of 9 days
(Table 7.1).
The inoculum concentration from 1 104 to 5 104 conidia of Phaeoisariopsis
griseola, leaf wetness durations from 3 to 9 h showed a progressive decline of the
incubation period of angular leaf spot on French bean. Intermittent wetness up to
3 cycles (12 h dry and 12 h wet) was found effective to increase the number of
Table 7.1 Effect of different leaf wetness durations on number of spots per leaf and incubation
period
Leaf Wetness (h) Average spots/leaf (No.) Incubation period (days)
3 9.22 12
6 24.25 12
9 33.25 9
12 38.33 9
18 40.50 9
24 49.25 9
48 46.25 9
LSD( 0.05) 1.5
Evaluated at temperature 24 1 C
lesions per leaf. Gupta et al. (2005) reported that an increase in relative humidity
from 85.7% to 89.9% coupled with moderate temperatures of 25 C resulted in a
sharp increase in disease development. These workers also studied the role of
environmental factors on disease development and reported the consistent effect of
rainfall, soil moisture and relative humidity on the disease development. Simple
correlation coefficients between disease severity and rainfall (0.7596), soil moisture
(0.7070) and relative humidity (0.5253) were found to be highly significant and
positive. But Chhetry et al. (1994) reported non-significant linear correlation and
regression coefficients of disease incidence and severity with microclimatic factors.
7.2.2.3 Diurnal Periodicity of Conidial Release of P. griseola

The observations pertaining to conidial density were recorded at 4 h intervals starting
from midnight and the release of conidia was continuous and that the air over the
field was never free of conidia of P. griseola. It revealed a distinct rhythm at different
times of the day with maximum conidial density being observed between midnight
and early morning hours (Gupta et al. 2005). Conidial formation and release was
favoured by temperature (23–24 C), high relative humidity (>90%), prevalence of
dew (20.00–8.00 h) and dark period.
7.2.2.4 Management
Cultural Practices
Various cultural practices, viz. use of disease-free seed and removal of diseased
leaves and seedlings (Trutmann and Kayitare 1991); 3–4 year crop rotation (Correa-
Victoria and Saetler 1987); destruction of diseased plant material; deep ploughing
(Hocking 1967); wider spacing (Navarro et al. 1981; Gupta et al. 2000d) and altering
of sowing dates (Bhardwaj et al. 1994; Gupta et al. 2000d) and staking of pole type
varieties (Gupta et al. 2000d) have been recommended from time to time for the
control of this disease.
Moreno (1977) studied the effect of different cropping systems (bean grown
alone or in combination with maize, sweet potato or Cassava) on the severity of
angular leaf spot and reported that severity was maximum when maize was included
and lowest with sweet potato and Cassava. However, during a multi-location trial in
Tanzania, Kikoka et al. (1989) found intercropping with maize effective to reduce
the severity of the disease. Boudreau (1993) and Gupta et al. (2000d) reported
similar observations from Kenya and India, respectively. Poultry manure, composted
Urtica sp. and composted Lantana camara treatments were superior to other
composts in suppressing angular leaf spot (Joshi et al. 2009).
Host Resistance
French bean cultivars are known to possess a varying degree of resistance against the
pathogen (Buruchara et al. 1988). Various cultivars/lines like Albama No.1, Café,
California Small White, Case Knife, Epicure, Mexico blank, McKastan, Navy bean,
Negro Costa Rica, Scotia Rojo Chico, Mexico 11, Mexico 12 and Cauca
27, Carpoata (Santos Filho et al. 1978); Negro Tacana (Lopez et al. 1997) and
Manteiga Maravilha (Paula et al. 1998) are reported as resistant. Araiyo et al. (1989)
reported that non-black cv. Ouro and black coloured cvs. Rio Tibagi and Milionario
1732 possess resistance to P. griseola.
In India, cvs./lines, viz. EC 38921, EC 44621, PLB 148, Kentucky Wonder,
Canadian Round, Canadian Long Red, IC 47651-6, EP 146 (BAT-482), HPR-54,
HPR-63, HPR-92, HPR-93, HPR-111, HPR-232, HPR-260, HPR-299, HPR-332,
HPR-326, Hans, Nagar Local, SVM-1, JK-8, Him-12, IIHR 42-2, IIHR 901, EC
97830, EC 44758 and NIC 14402 were reported to be resistant against this disease
(Singh and Sharma 1976; Bhardwaj et al. 1992; Srivastava et al. 1995; Mohan et al.
1995; Gupta et al. 2000c).
The effectiveness of supplementing local bean mixture with 25% or more resis-
tant lines (BAT 76 and A 285) was evaluated and it resulted in a significant reduction
of angular leaf spot severity in Africa (Pyndji and Trutmann 1992; Trutman and
Pyndji 1994). Of the 68 French bean genotypes/lines screened for resistance to
Phaeoisariopsis griseola during 2002 and 2003 at two locations namely, Shalimar
and Wadoora in Kashmir, India, 9 genotypes/lines, viz. SL-4-PL-1, HIM-1, Bounti-
ful, JKRO-3, L-27, L-28, L-8 and Local Big Beans, exhibited highly resistant
reaction on pods. On leaves, 6 genotypes, viz. Uri-Red, EC-285559, Medur Tral,
Local Red, Local Big Beans and SAW/GH/TA/41A at Wadoora location, and
4 genotypes, viz. Gurez Local, Big Bean Black, EC39855 and Local Big Beans at
Shalimar location showed a highly resistant reaction, while the genotype Shalimar
French Bean-1 gave a highly susceptible response on leaves at both locations (Bashir
et al. 2006).
Chemical Control
Various fungicides have been reported to give good control of this disease in India
and other countries. However, during continuous rains which are essential both for
good growth of bean plants and rapid development and spread of the disease, routine
application of fungicides is sometimes delayed which often results in a heavy
buildup of the disease. Proper timing of initial sprays is of prime importance to
achieve a desirable level of disease control.
Attempts have been made to control the disease by both seed treatment with
mercurial and other fungicides and foliar sprays with various fungicides. Sprays of
fungicides like wettable sulphur, mancozeb, Bordeaux mixture, zineb, carbendazim,

benomyl, triforine, triademorph (Fortugno 1977; Sindhan 1984; Issa et al. 1982;
Hidalgo and Araya 1993; Singh et al. 1995), chlorothalonil, TPTH, TPTA (Castro
et al. 1991; Vierra et al. 1998) and mancozeb (Issa et al. 1982; Castro et al. 1991;
Vierra et al. 1998) have been reported effective to reduce disease intensity and
increase yield. Oliveira et al. (1992) reported stannic triphenyl (fentin) acetate,
bitertanol and tebuconazole as more effective when compared to Bavistin while,
Srivastava and Gupta (1994) reported captan, thiophanate methyl, carbendazim,
ziram and captafol to be approximately equally superior to mancozeb, copper
oxychloride and tridemorph. Combi fungicide sprays like benomyl + captafol,
chlorothalonil + copper oxychloride (Issa et al. 1982) and thiophanate methyl +
thiram (Rodriques et al. 1987) have also been found effective. Combination
treatments of carbendazim (seed treatment + foliar spray) and bitertanol (seed
treatment + foliar spray) were also found effective in reducing the severity and
increasing the green pod yields than seed treatment or foliar spray alone (Mathew
et al. 1998).
Various EBI fungicides have longer post-infection and eradicant activity being
systemic in nature. Mathew et al. (1998) studied the mode of action of EBIs like
hexaconazole, penconazole and bitertanol along with conventional fungicides like
mancozeb and carbendazim by providing artificial weather conditions like temperature,
relative humidity and leaf wetness for infection recorded in epidemiological studies.
Protective Activity
Potted French bean plants sprayed with test fungicides and subsequently inoculated
with the conidial suspension (5 104 spores/ml) after 24, 48, 72, 96, 120, 144,
168 and 192 h of fungicide spray. Inoculated plants were kept in saturated humidity
for 12 h and then transferred to the net house. Humidity >75% and temperature
25 1 C 25 were maintained inside the net house for 9 days of incubation.
Carbendazim provided 100% control of the disease up to 120 h while mancozeb
up to 96 h EBIs exhibited poor protective activity only up to 72 h confirming the
increased protective activity of mancozeb (protectant) and carbendazim (systemic).
Post-infection Activity
Spray inoculated potted French bean plants were kept in moist chambers for 24, 48,
72 and 96 h and subsequently sprayed with a dilute suspension of test fungicides.
EBI fungicides demonstrated post-infection activity up to 72 h while it was only 24 h
in case of carbendazim and mancozeb.
Pre-symptom Activity
Spray inoculated potted French bean plants were kept in the moist chamber at 20–25 C
for 12 h before being returned to the net house for normal maintenance. Fungicide
suspension was sprayed on the seventh and eighth day of inoculation. Upon drying
plants were maintained in nethouse and the data was recorded 14 days after treatment.
Mancozeb when applied as pre-symptom spray it had little effect and produced
sporulating spots while EBIs and carbendazim produced reddish brown
non-sporulating spots when applied seventh and eighth day after inoculation (Fig. 7.2).
Post-symptom Activity
To evaluate anti-sporulant activity 15 naturally infected bean pods (5 pod/plant)
having 5–10 spots each (one treatment) were tagged and subjected to 5 fungicide
spray. After 3, 5 and 10 days of single spray, five tagged pods were removed from
each treatment and one lesion (5 mm2) was cut from each pod with the help of cork
borer and conidial concentration was determined using a haemocytometer.
Carbendazim, hexaconazole, flusilazole and bitertanol had high level of
antisporulant activity and their residue persisted for more than 10 days. These
properties of EBI fungicides can be utilized in planning different spray programmes
for effective control under different situations.
Comparative Efficacy of Protective Versus Post-infection Spray Programme

A Thermohygrograph was placed in the field to record hours of RH, i.e. >80%.
Continuous occurrence of RH >80% for 12 h was considered as one infection
period. Short intervening periods of (up to 3 h) of RH >80% in between the periods
of high humidity (>80% RH) was also considered while calculating infection
period. Sprays were usually applied at various times depending on weather
conditions but always within 72 h of initiation of infection period. No spray was
repeated within 9 days even if additional infection period has occurred.
In all 25 infection periods occurred. Post-infection sprays (Table 7.2) of bitertanol
and hexaconazole within 72 h of predicted infection period and protective sprays of
carbendazim were equally to manage the disease. Two less number of sprays were
required in post-infection spray programme in comparison to 7 sprays under protec-
tive spray programme.
Spray schedules involving the strobilurin (azoxystrobin) and triazoles
(tebuconazole, propiconazole) alone and their combinations were also found effec-
tive in controlling angular leaf spot (Oliviera and Oliveira 2003).
Integrated Approach
Fungicidal treatments coupled with careful selection of varieties and sowing dates
proved highly effective to reduce disease severity and increased the yield of the crop
(Bhardwaj et al. 1994). Effect of two mulches (Pine needles and Eucalyptus leaves)
alone and in combination with carbendazim (0.1%) sprays on mulch and foliage on
Table 7.2 Comparative efficacy of protective and post-infection (curative) spray programme
against angular leaf spot of French bean
Treatment Dose (μg/ml) Disease severity (%) Disease control
Protective spray programmea
Carbendazim 500 6.00 90.21
Mancozeb 2500 11.73 80.87
Curative spray programmeb
Bitertanol 500 8.66 85.87
Hexaconazole 500 7.33 88.04
a
No. of sprays ¼ 7
b
No. of sprays ¼ 5
the disease severity and pod yields has also been studied by Mathew et al. (1998).
Sprays of carbendazim on mulches and foliage were highly effective in reducing the
disease severity and increasing green pod yields. Sprays of carbendazim on mulches
alone were also found quite effective and reduced the severity and increased green
pod yields significantly with an additional advantage of residue-free pods.
7.2.3 Anthracnose
Anthracnose is an important disease of French bean world over but it is more severe
in tropical and subtropical regions (Pastor-Corrales et al. 1994). Scribner (1888) was
the first to use the name anthracnose for this disease, which is still generally used.
The term anthracnose is derived from the Greek word meaning ulcer and is appro-
priate because of the ulcer like depressed lesions appear on the pods. Though this
disease occur world over including tropical and subtropical regions, it causes greater
losses in the temperate zones than it does in tropics. In India, its occurrence was first
noticed in Nilgiri hills during 1915 (Hutchinson and Ram Ayyar 1915). Since then
the disease has been recorded in the entire bean growing areas of the country, which
have cool and moist weather during the growing season. Losses from this disease can
approach up to 100% when highly contaminated seed is planted under favourable
weather conditions for disease development. Yield losses of 90–100% have been
reported in many countries throughout tropical America and Africa (Pastor-Corrales
and Tu 1989; Lenne 1992). In Himachal Pradesh, India, the incidence of this disease
has been reported to range from 5.0% to 65.0% in different localities leading to
considerable yield losses in certain years (Sharma et al. 1994; Padder and Sharma
2010). The fungus, Colletotrichum lindemuthianum (Sacc. and Magn.) Briosi and
Cav. is responsible for this disease with Glomerella lindemuthiana Shear (Shear and
Wood 1913), but now it has been renamed as G. cingulata (Kimati and Galli 1970)
as its perfect stage.
7.2.3.1 Symptoms
The disease may appear on any above-ground plant part depending upon time of
infection and source of inoculum but most striking symptoms appear on immature
pods. Initial symptoms appear on the cotyledonary leaves as small, dark brown to
black lesions. Conidia and hyphae then may be disseminated by rain or dew to the
developing hypocotyl where rust coloured specks develop. The specks gradually
enlarge lengthwise along and partially around the hypocotyl and young stems,
forming a sunken lesion. On leaves, the infection may occur on both sides but an
early sign of infection usually appear on the under leaf surface as blackened dead
portions of the veins. These may extend to limited adjoining areas. Later, such spots
also appear on the upper leaf surface. Lesions are also formed on petioles and stems.
On pods, black sunken spots with lighter or grey central area are seen (Fig. 7.3). The
central portion of the spots shows pinkish masses of spores of the fungus, especially
in wet weather. Later, the sides of these spots appear raised. The seeds obtained from
Fig. 7.3 Symptoms of bean

anthracnose on pods
heavily infected pods show brown to light chocolate coloured sunken cankers on the
seed coats.
The pathogen overwinters in infected seeds and plant debris. Ravi et al. (1999)
reported that in seed the pathogen was mostly present in seed coats and cotyledons
and rarely in embryonic axes. Conidia and/or dormant mycelia in the infected seeds
and/or infected plant debris germinate and infect the young seedlings. It can remain
viable in infected seed for several years (Tu 1983). When infected seed germinates,
lesions appearing on cotyledons serve as the source of secondary inoculum. The
spores are almost entirely water-borne. Primary leaves and the hypocotyl are foci of
secondary infections. Borucka and Marcinkowska (2001) reported the importance of
wet seasons at early growing period besides availability of a source of inoculum for
the initiation and occurrence of this disease. The pathogen is very sensitive to
changes in temperature and humidity. It develops most abundantly in cool, wet
weather and largely disappears under hot and dry conditions. A relative humidity of
92% and above is essential for infection, the optimum being close to 100%. The
fungus requires about 10 mm of rain to establish initial infection (Tu 1981). The
optimum temperature for disease development ranges from 18 to 27 C with
maximum intensity at 21 C (Sindhan 1983) and is markedly reduced at 13 C.
Heavy and frequent rains with moderate temperatures (19–25 C) and high relative
humidity (>70%) favoured the progress of the disease in terms of vertical and
horizontal spread of the disease (Kumar et al. 1999). Moderate rainfalls at frequent
intervals are also essential for the local dissemination of conidia present in water-
soluble gelatinous matrix and the development of severe anthracnose epidemics. The
movement of insects, animals and man may spread conidia particularly when foliage
is moist.
7.2.3.3 Management
Various management strategies have been studied to keep this disease in check,
which are based on use of fungicides, resistant cultivars, cultural practices and
biocontrol of seed-borne infection. It is always better to integrate all available
disease management methods to reduce the losses caused by this disease to bare
minimum.
Cultural Practices
Use of healthy seed, clean cultivation and three-year crop rotation has been
recommended for the management of this disease (Sharma and Sohi 1989).
Disease-free seed can be produced either by surface irrigation in semi-arid regions
where conditions of high temperature and low humidity prevent infection of this
fungus, or under a pedigreed seed programme, in which seed plots were isolated and
subjected to strict inspection for disease-free seed. Best sowing time of French bean
in hill regions is in between mid-April to mid-May for maximum yield and minimum
anthracnose incidence.
Host Resistance
The most promising strategy to control this disease is the use of resistant cultivars.
However, the diversity and the high variability of the fungus with the continuous
emergence of new races pose problems in breeding programme. Although cultivars
with new resistance genes are resistant to majority of pathotypes, this resistance can
be broken when such variety is planted widely (Lenne 1992). Anderson et al. (1963)
reported that the resistance in cv. Charlevoix against alpha and beta races of
C. lindemuthianum is conferred by a single independent dominant gene. A glyco-
protein inhibitor in the cell wall of resistant varieties imparts resistance (Sharma et al.
1986). Due to the presence of much pathogenic variability in the pathogen, non-race-
specific resistance has been used in breeding for anthracnose. Cultivar IAPAR-Rio
Negro-8 is resistant to all races of the pathogen under field conditions (Alberini et al.
1987) while Menezes and Dianese (1988) have identified varieties (Aroana
80, Ayso, Carioca 80, Iguaco, Moruna 80, Riuo Piqiuri, Rio Vermelho, Mulatao,
Olho-de-Pombo and Vermelhao) which were resistant to at least 7 of the 9 races of
the pathogen and no race was virulent to all the 70 cvs. screened in Brazil.
Chakrabarty and Shyam (1990) demonstrated resistance in cvs./lines VL 60, VL
63, Jawala, HPR 33, B 4 B 6 and P 48 against eight isolates of the pathogen.
However, Jawala later exhibited highly susceptible reaction, which may be due to
the development of new pathotypes. Kumar et al. (1997) screened 60 cultivars/lines
of kidney bean against ten races, viz. beta, gamma, Ind I, Ind II, Ind II, Ind IV, Ind V,
Ind VI (alpha-Brazil), Ind VII and Ind VIII of C. lindemuthianum under field and
artificial epiphytotic conditions and found two accessions (AB-136 and G 2333) as
highly resistant to all the above races prevalent in Himachal Pradesh, India, while
cultivars Cornell 49-242, EC 43036, EC 57080, KRC 5, PI 207-262 and Widusa
exhibited resistance to more than five races whereas rest were susceptible and these
can be utilized in the breeding of race-specific resistant varieties. Later Gonalves-
Vidigal et al. (2001) reported that a single dominant gene controls the resistance of
cv. AB 136 to both races 31 and 69 and the symbol Co-6 was assigned to the gene. In
addition, linkage analysis using random amplified polymorphic DNA marker
indicated that Co-6 also controls the resistance of this cultivar to other races of the
pathogen, or that different genes are present in the same linkage block.
Few cvs./lines/land races, viz. Negro Otomi (Acosta-Gallegos et al. 2001), SRC
74, SRC 89A, SRC 90, SRC 95, SRC 201, PDR 14 (Sud and Sood 2002) and
Morden 003 (Mundel et al. 2004) and HUR-5, HUR-137, IPR 96-4, VL-63,
PDR-14, HUR-385 (Khati and Hooda 2006) have been reported resistant to this
disease. Some exotic accessions like G 2333, Cornell 49242, PI 207262, Mexique
222, TO, Perry Marrow, Kaboon and Widusa were resistant to more than five Indian
races, whereas two Indian accessions KRC-5 and Hans showed resistance to six and
four races, respectively (Pathania et al. 2006).
Common bean germplasm lines comprising of 65 indigenous and 34 exotic were
evaluated against four races, viz. 3, 515, 529 and 598 of C. lindemuthianum under
laboratory conditions and four indigenous accessions namely 47, 34, 32 and
18 showed resistant reaction to race3, 515, 598 and 529, respectively, whereas in
exotic accessions 16, 22, 18 and 11 exhibited resistance to these races (Sharma et al.
2012). However, accessions like IC-328537, IC-328538, IC-448888, IC313294,
IC-27823, IC-339645, IC-341862 (Indigenous), EC-169813, EC-398530 and EC
500226 (Exotic) were found resistant to all races evaluated.
Biocontrol
Limited work on this aspect in this disease has been carried out like in vitro
evaluation of the antagonists against this pathogen (Gupta et al. 1991) or the
phenomenon of cross-protection by inoculating a less virulent strain of the fungus
to reduce the severity from severe strains (Sutton 1979). Infected seed when soaked
in culture filtrate (10%) or treated with talc formulation (0.4%) of Trichoderma
viride recorded minimum seed infection and maximum seed germination (Ravi et al.
1999). Treatment with P. chlororaphis PCL1391 resulted in best biocontrol of
anthracnose, while P. fluorescens WCS365 showed no significant difference com-
pared to the positive control (Bardas et al. 2009). Padder et al. (2010) evaluated three
bioagents (Trichoderma viride, T. harzianum and Gliocladum virens) and five
biopesticides (Achook, Neemgold, Wannis, Spictaf and Neemazal) under in vitro
and in vivo conditions against this disease. All the three antagonists caused signifi-
cant mycelial growth inhibition, maximum being with T. viride (69.21%) followed
by T. harzianum (64.20%). Among the biopesticides tested at four concentrations,
Wanis applied at 1000μl/ml caused maximum inhibition of 82.12% followed by
Spictaf (52.85%). T. viride and Wanis at 1000μl/ml were most effective in reducing
the seed-borne infection.
Chemical Control
Various systemic and non-systemic fungicides such as captan, thiram, carbendazim,
etc. have been used by various workers to reduce seed infections (Sindhan and Bose
1981; Trutman et al. 1992). Foliar sprays of carbendazim, benomyl, zineb, maneb
and mancozeb have been recommended for its control (Chakrabarty and Shyam
1988). Benlate and Bavistin persisted in the plant up to 15–20 days after seed
treatment (Sindhan and Bose 1981). EBI fungicides like triadimefon and triforine
were effective against the pathogen under in vitro conditions but were phytotoxic to
P. vulgaris seedlings when used as a seed treatment (Chakrabarty and Shyam 1988).
Carbendazim alone or in combination with thiram as seed treatment followed by two
or three sprays of mancozeb at 45, 60–75 days after sowing effectively managed this
malady (Sharma and Sugha 1995). A combination of seed treatment with
tebuconazole (0.1%) and foliar sprays of tricyclazole (0.03%) reduced the disease
severity both on leaves and pods while maximum seed yield was recorded in case of
seed treatment and foliar sprays of carbendazim (Gupta et al. 2000b).
Continuous use of systemic fungicides has been shown to be associated with the
development of resistant biotypes (Tu and McNaughton 1980). Maringoni et al.
(2002) reported that all isolates of C. lindemuthianum collected from P. vulgaris
fields located at Paranpanema Valley, Brazil were insensitive to benomyl,
carbendazim and thiophanate methyl showing the occurrence of cross-resistance to
different benzimidazole fungicides while all isolates were sensitive to chlorothalonil.
Recently, Oliveira and Oliveira (2003) reported that strobilurin fungicides like
azoxystrobin and trifloxystrobin efficiently controlled this disease while chemicals
like carbendazim and fluquinconazole applied without rotation were least efficient
treatments. However, a combination of systemic fungicides with non-systemics and
rotation of fungicides can be an effective approach for management of this disease
instead of constant use of one systemic fungicide.
Plant Origin Pesticides

Under greenhouse conditions, the extracts of M. argyrophylla and O. vulgare caused
maximum reductions (41.82% and 37.65%, respectively) in disease severity when a
local effect assay was carried out (Pinto et al. 2010). Antimicrobial activity of
cascalote phenolics against phytopathogenic fungus C. lindemuthianum was carried
out which indicated that cascalote phenolics have fungistatic activity against races
R-0 and R-1472 of the pathogen under in vitro conditions. Under in vitro conditions,
spore germination and cellulase and polygalacturonase activities in this fungus have
been inhibited. In vivo assays with cascalote phenolics under greenhouse conditions
using susceptible cv. PI 206272 of common bean resulted in a good protection
against anthracnose severity especially as a preventive treatment (Garcia et al. 2010).
7.2.4 Rust
Rust is a polycyclic disease can cause significant losses under favourable environ-
mental conditions by causing premature defoliation, decrease in number of pods/
plant and their reduced weight. The disease has been reported from the entire bean
growing areas of the world and it was first described from Germany in 1795 (Persoon
1795). Since then it has been reported from many bean growing countries of the
world (Gupta and Thind 2018). In India, the disease was reported as early as in 1918
(Butler 1918). Now the occurrence of this disease has been reported from Karnataka
(Sharma 1989), Tamil Nadu, Uttarakhand (Sharma 1998), Meghalaya (Bhat et al.
1999) and Sikkim (Bhat 2002). Recently, the occurrence of this disease has been
reported on pencil beans in Solan area of Himachal Pradesh (Gupta et al. 2008).
Several workers have reported varying degree of losses due to this disease ranging
from 25% to 100% (Grafton et al. 1985; Wagacha et al. 2007). In India, losses in
green pod yield due to this disease ranged in between 4.7% and 69.0% (Sharma
1989; Devi et al. 2019). The disease is caused by Uromyces phaseoli var. typica
Arth. and U. appendiculatus. Being autoecious rust, all its spore stages are produced
on bean plant but pycnial and aecial stages are not commonly encountered in nature.
The pathogen has limited host range. Besides French bean (Phaseolus vulgaris), it
attacks P. lunatus, Dolichos lablab, D. biflorus, Vigna chinensis, etc.
7.2.4.1 Symptoms
Under favourable environmental conditions, all above-ground parts of the plant are
infected but leaves and pods are the principal plant parts attacked by the disease. On
leaves, the disease may appear on both the surfaces, but is more common on the
abaxial surface. Pustules of the disease usually appear first as small and slightly
raised spots that are almost white in colour. These pustules enlarge by forming
reddish brown sori, up to 2 mm in diameter, containing the urediniospores (Fig. 7.4).
After coalescing, they may occupy larger areas. A ring of secondary sori may
develop around the original infection on susceptible cultivars. Minute elongated
and raised spots also appear on petioles and stems when the severity of this disease is
more on leaves. Reddish brown, elongated raised pustules also appear on pods. The
affected leaves may turn yellow and dry or fall prematurely. Wherever telial stage
appears, they are dark brown to black in colour and linear.
Being autoceious rust, i.e. it has its life cycle confined to a single host. The aecia are
rare in nature but have been observed in bean fields in Oregon (Eastman 1944). The
Urediniospores are produced in great numbers in sori on the upper and lower
Fig. 7.4 Symptoms of

bean rust
surfaces of the leaf. These urediniospores are air-borne and blown to long distances
and cause severe epidemics. The urediniospores germinate as soon as they are
mature. One or two germ tubes developed from germinated urediniospores (Xin
et al. 2000). The penetration of the germtube occurs mainly through the stomata but
occasionally penetrated directly through the epidermal cell. A well-defined appres-
sorium is formed after penetration by the pathogen. The fungus forms a sub-stomatal
vesicle when it penetrates through the stomata and a round inflated body usually
occurs. One or two primary hyphae then develop from the inflated body. Once within
the bean leaf, the fungus grows intercellularly and the penetration to the leaf cell
occurs after a haustorium mother cell is formed at the hyphal tip in contact with a
host cell. The branches emerge from behind the site of the primary hyphae where a
haustorium mother cell develops. The secondary hyphae spread intercellularly.
Obligate parasitism and autoecious nature of rust pathogen helps it to complete
the entire life cycle on bean plants. It is not seed-borne. Davison and Vaughan (1963)
suggested that rust overwinters in or as urediniospores in trash and on trellis poles
because 16% urediniospores of U. phaseoli race 33 germinated even after storage
at –18 C for >600 days and produced abundant infection on bean leaves. In cooler
regions, survival is ensured through plant debris in the form of teliospores while in
other areas through the continuous growing of crop including other hosts. Cloudy
and humid days, allowing dew deposition on leaves for some time in the morning,
favour germination of spores and subsequent infection. Optimum infection takes
place at 17 C (Harter et al. 1935; Mendes and Bergamin-Filho 1989) or 18–21 C
(Shands and Schein 1962) Singh and Gupta (2019) studied the effect of different
temperature regimes on the disease development under glasshouse conditions
(Fig. 7.5) and reported that maximum disease severity (82.99%) and number of
pustules/cm2 (69.40) were recorded at 20 C followed by 25 C where 72.3% disease
severity and 63.20 pustules/cm2 were recorded while least number of pustules/cm2
90.00 Disease severity (%) No. of pustule/ cm2

80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
15 20 25 30 35
Temperature degree Celsius (±1)
Fig. 7.5 Effect of temperature on bean rust development under glasshouse conditions
Disease severity (%) No. of pustule/cm2

100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
75 80 85 90 95 100
Per cent relative humidity
Fig. 7.6 Effect of different relative humidity levels on bean rust development under glasshouse
conditions
(1.60) and disease severity (12.44%) was recorded at 30 C, however, no disease

developed at 35 ºC.
Harter et al. (1935) obtained high level of rust infection when plants were
exposed to 96% or more RH. Maximum percentage of bean rust infection was
observed when plants were exposed to 11 h or more to 100% RH (Rey and Lozono
1961). Mendes and Bergamin-Filho (1989) attained maximum infection (Number of
sporulating pustules/leaf) after 22 h of high RH. Singh and Gupta (2019) reported
that there was a corresponding increase in the disease severity and number of
pustules as humidity increased from 75% to 100% (Fig. 7.6) with maximum disease
severity (81. 92%) and number of pustules/cm2 (70.50) at 100% RH level followed
by 95% and 90% RH levels on which 78.90% and 75.58% disease severity was
recorded while it was minimum (55.63%) at 75% RH level. Sharma (1977) also
studied the effect of number irrigations on the disease development and observed
that irrigation given at 3 days intervals favoured disease development than with
18-day intervals.
Effect of duration of leaf wetness also has a significant effect on disease develop-
ment under field conditions which is mostly provided by rain and dew deposition
under field conditions. Singh and Gupta (2019) also carried out experiments to see
the effect of leaf wetness (Fig. 7.7) and intermittent leaf wetness (Table 7.3)
durations on disease development under glasshouse conditions and reported that
no disease developed when plants were exposed to 3 h leaf wetness whereas
continuous leaf wetness of 6 h was sufficient to initiate infection by rust pathogen.
They further reported that when leaf wetness durations were increased from 6 to
24 h, there was a corresponding decrease in the incubation period from 243 to 168 h
with the increase in the disease severity and number of pustules from 30.0% to
83.6% and 6.0 to 72.3/cm2 of leaf, respectively. However, further increase in leaf
wetness did not exert any effect on the incubation period.
Fig. 7.7 Effect of duration of leaf wetness on initiation and development of bean rust under
glasshouse conditions
Table 7.3 Effect of intermittent leaf wetness on development of the disease under glasshouse
conditions
Total leaf Wet/Dry sequence (hr) Disease No. of
wetness severity Pustules/
(h) Wet Dry Wet Dry Wet Dry Wet Dry (%) cm2
12 12 12 – – – – – – 68.28 57.33
(55.71)
24 12 12 12 12 – – – – 82.49 72.50
(65.31)
36 12 12 12 12 12 12 – – 78.90 72.17
(62.67)
48 12 12 12 12 12 12 12 12 76.75 69.83
(61.19)
LSD0.05 (2.44) 8.28
SE(m) (0.82) 2.79
Number of pustules and disease severity increased with the interruption of leaf
wetness with dry periods up to two cycles (72.50 pustules/cm2 and 82.49% disease
severity) and further extension of wet and dry periods resulted in a non-significant
decrease in number of pustules and disease severity (Table 7.3).
Sharma (1998) analysed the meteorological data for 8 years (1986–1993) and
showed that excessive precipitation was deleterious for disease development, which
explains the occurrence of severe infection during periods of low rainfall. Singh and
Gupta (2019) studied the effect of different meteorological factors on disease
progress under natural epiphytotic conditions and observed that the disease was
initiated in the second week of August and reached at peak in mid-September.
Multiple correlation coefficients between disease severity and these environmental
factors suggested that 92.36% rust severity was due to mean temperature, average
relative humidity and cumulative rainfall while rest of the variation may be attributed
to the factors not included in the investigations. The multiple linear regression
equation was fitted to the data and the equation arrived for all the weather parameters
was Y ¼ 52.4852 + 1.2535 X1 + 0.8008 X2–0.2862 X3 + 0.4622 X4–0.7755
X5 + 0.2428 X6. With the step, down procedure, three variable, i.e. maximum
temperature (X1), cumulative rainfall (X5) and wind speed (X6) were eliminated and
final equation fitted to the data is Y ¼ 37.4111 + 1.0622 X2 + 0.5299 X3–0.2868
X4. Stepwise regression analysis of the data in relation to weather variables revealed
that the R2 value varied from 0.53 to 0.86. The regression equation clearly
demonstrated that minimum temperature, morning RH and evening RH played a
major role in the development of French bean rust, in addition to other independent
variables.
Disease intensity is not influenced by varying levels of N, P and K. Farm
implements, insects, humans and animals help in local dissemination of the rust
fungus (Sumartini 1998).
7.2.4.3 Management
The disease can be managed by cultural, chemical, biological methods and host
resistance.
Cultural Practices
Moreno and Mora (1984) have suggested minimum tillage or no tillage to prevent
the spread of the disease and intercropping of bean with maize since severity and rate
of infection was more in monoculture. Mixed cropping reduced rust incidence level
by 51% and 25% with sole cropping and row intercropping, respectively (Fininsa
1996). Plant density and disease incidence are directly related (Flores Revilla et al.
1994). Mishra et al. (1998) evaluated seven dates of sowing under Orissa conditions
and Arka Komal variety was sown in between 12 October and 23 November; 9th
November was found as the optimum date of sowing because the rust incidence was
minimum. Sharma and Sohi (1989) also recommended some cultural practices like
long crop rotations, collection and destruction of plant debris, wider plant spacings
and removal of weeds to permit aeration and drying which check rapid build-up of
inoculum and spread of the disease under field conditions.
Host Resistance
More than 35 physiologic races of the rust fungus (U. appendiculatus) are present in
Australia, Brazil, Hawaii and the USA. Extensive cultivation of the new cultivars
may make them susceptible to new virulence. Golden Gate Wax and Brown
Kentucky Wonder 298 have been used in breeding for resistance to four races
(Dundas 1942). Cultivars/lines like Virginia 119, No.780 (Foster 1947), Hawaiian
Wonder (Frazier and Hendrix 1949), Seminole (Anonymous 1953), Redlands
Greenleaf and Redlands Pioneer (Anonymous 1966), Jackson Wonder and Strikton
(Makraw et al. 1973), Brazil K-12973 (Sokol et al. 1977), Catu and Carioca (Issa
1985) Serva Negra, Ag497 and Agrorrico (Azevedo and Kushalppa 1986), Gresham
(Anonymous 1989), PV 136, PV 88, PV 89 and PV 3 (Prakasam and Thumburaj
1991), white seeded Enganador, Chevere and CC 25-9B, Negro Inifap (Villar-
Sanchez and Lopez-Salinas 1993), Negro Cotaxtle 91 (Becerra Leor et al. 1994),
IBRN-6, IBRN-11, Hawaiian Wonder, Bush Bean S-9, Bush Blue Lake and Con-
tender (Ghimire et al. 1995), Ouro Negro (Faleiro et al. 1996), IIHR 220, stringless
bean (Mohan et al. 1997), Mimoso Rasteiro (Paula et al. 1998), SVM-1, Hans
(Sharma 1998) and IC 47643, PI 201489 and PI 247761 (Bhat et al. 1999) have
been found to be resistant in different parts of the world. Cv. Ouro Negro showed
immunity to 3 out of 4 races tested and PI 181395 was the best source of resistance to
rust, as it was immune to all the four races of U. appendiculatus (Faleiro et al. 1998).
Inheritance studies indicated that resistance to rust was controlled by a single,
dominant gene (Aghora et al. 2007). Line “Arka Anoop” developed by the breeding
programme is resistance to rust and has high pod yield and good pod quality.
Germplasm lines/cvs. like Alapa Trey, Sing Tamey and Local Collection were
found resistant to this disease under Solan, Himachal Pradesh conditions (Sharma
et al. 2014).
Biological Control
Verticillium lecanii has been suggested to be a potential biological agent for the
management of rust as it penetrates hyphae and invades urediniospores of the fungus
(Allen 1982). Under field conditions Verticillium lecanii reduced the disease inci-
dence and severity to 56% and 17% as compared to 98% and 85%, respectively, in
agrochemical package while seed yield was slightly less in biocontrol than agro-
chemical package which may be due to the use of fertilizer in latter package (Carrion
et al. 1999). Different isolates of Bacillus subtilis like APPL-1 (Baker et al. 1983,
1985) and FF-1, FF-5 and FF-6 (Mizubuti et al. 1995) have been selected as the
potential biocontrol agents. In culture medium B. subtilis isolates released a thermo-
stable substance(s) antagonistic to the rust fungus as pustule number was reduced by
>95% (Centurion et al. 1994). Isolate B 206 showed the greatest efficacy, when cells
of the antagonist were automized on to the plant.
Chemical Control
Different workers suggested various chemicals from time to time for keeping the
disease under check. Straib (1943) suggested the disinfection of stakes with
formaline (0.1%) before reuse. Systemic fungicides are more effective than the
non-systemic owing to the difficulty of obtaining under leaf coverage with the latter.
Efficacy of various systemic fungicides like oxycarboxin, carboxin, triforine,
bitertanol, triadimefon, tridemorph, hexaconazole and HWG 1608 has been reported
against this disease (Jaffer 1971; Rolim et al. 1981; Singh and Musymi 1979, 1984;
Prakasam and Thamburaj 1991; Gonzalez and Garcia 1996a, b; Sumartini 1998) and
possibly eradicated the already established infection of U. phaseoli in bean plants
after three sprays without any sign of phytotoxicity (Singh and Musymi 1979, 1984;
Gonzalez and Garcia 1996b).
Seed treatment with the thiram and subsequent sprays with mancozeb was found
best combination treatment for controlling rust (Mahanta and Dhal 2000). Three
sprays of hexaconazole (0.1%) at 15 days interval were found best in keeping the
disease under check (Bhat 2002). Contaf (0.1%) alone and in combination with
mancozeb (0.2%) were highly effective and reduced the disease incidence from
86.6% to 24.1% (Singh and Bhat 2002). Sprays of propiconazole were found to be
most effective in reducing the severity of rust to 10.93% as compared to control
(41.20%) and gave about 91.54% disease control (Shukla and Sharma 2009). Sprays
of Azoxystrobin (0.1%) were found significantly most effective in reducing the
disease severity to 4.64% and increased green pod yield by >97% (Sharma et al.
2018). This increased yield may be due to the phytotonic effect exhibited by
azoxystrobin sprays which delayed the crop senescence.
Systemic Resistance Inducer

Treatment of bean leaves with salicylic acid (SA) or 2, 6-dichloro-isonicotinic acid
(DCINA) induces resistance against the rust fungus U. fabae resulting in reduced
rust pustule density (Rauscher et al. 1999). Saccharin application in broad bean
(Vicia faba) induced resistance to the rust fungus Uromyces viciae-fabae (Boyle and
Walters 2005) and provided systemic protection to rust infection after 14 days of
application. Bion was also used to induce resistance in bean cultivars like Borlotto
Nano Lingua di Fuoco (BLF), Borlotto Taylor, Cannellino, Cannellino Montalbano,
Saxa and Top Crop, against U. appendiculatus. A single 0.3 mm BTH spray 7 days
before inoculation was sufficient to fully control the disease in all the examined
cultivars (Iriti and Faoro 2003).
References
Acosta-Gallegos JA et al (2001) Registration of “Negro Otoni” shiny black bean. Crop Sci 41:361
Aghora TS, Mohan N, Somkumar RG, Ganeshan G (2007) Breeding French bean (Phaseolus
vulgaris) for resistance to rust (Uromyces phaseoli Reben Wint). J Hortic Sci 2:104–107
Alberini JL, Mohan SK, de Menezes JR, da Silva WR, Oliari L, Meyer RC (1987) IAPAR 8 – Rio
Negro, a new French bean cultivar. Pesquisa-Agropecuaria Brasileira 22:995–998
Allen DJ (1982) Verticillium lecanii on the bean rust fungus, Uromyces appendiculatus. Trans Br
Mycol Soc 79:362–364
Anderson AL, Adams MW, Whitford G (1963) Charlevoix anthracnose resistant dark red kidney
bean. Mich State Univ Agric Stationn Res Rep:6
Anonymous (1953) Research work and workers. Seed World 72(11):80–81
Anonymous (1966) Division of Science Services, Biol. Branch, Plant Pathol. Section. Rep. Dep.
Agric. NSW 1964–65:75–78
Anonymous (1989) French bean (Phaseolus vulgaris). Plant Varities J 2:15–16
Araiyo GAA, Viera C, Chagas JM (1989) Performance of bean (Phaseolus vulgaris L.) cultivars in
the Zona da Mata area of Minas Gerais V. Revista Ceres 36:382–390
Azevedo LAS, Kushalppa AC (1986) Quantification of the resistance of Phaseolus vulgaris
cultivars to U. phaseoli typical. Fitopatol Bras 11:599–608
Baker CJ, Stavley JR, Thomas CA, Sasser M, Macfall JS (1983) Inhibitory effect of Bacillus
subtilis on Uromyces phaseoli var. typica and on development of rust pustules on bean leaves.
Baker CJ, Stavley JR, Mock N (1985) Biocontrol of bean rust by Bacillus subtilis under field
conditions. Plant Dis 69:770–772
Bardas GA, Lagopodi AL, Kadoglidou K, Tzavella KK (2009) Biological control of three
Colletotrichum lindemuthianum races using Pseudomonas chlororaphis PCL1391 and Pseudo-
monas fluorescens WCS365. Biol Control 49:139–145
Bashir S, Mustaq A, Sagar V, Najib M (2006) Identification of sources of resistance in French bean
to Phaeoisariopsis griseola causing angular leaf spot disease. Indian J Plant Prot 34:221–224
Becerra Leor EN, Lopezsalinas E, Acosta GJA (1994) Genetic resistance and chemical control of
bean rust in the humid tropics of Mexico. Revista Maxicana de Fitopatologia 12:35–42
Bhardwaj CL, Kalia NR, Singh LN (1992) Reaction of French bean germ-plasm against angular
leaf spot. Indian J Mycol Plant Pathol. 22:270–271
Bhardwaj CL, Nayital SC, Verma S, Kalia NR (1994) Effect of sowing date, variety and manage-
ment of angular leaf spot (Phaeoisariopsis griseola) on yield of French bean (Phaseolus
vulgaris). Indian J Agric Sci 64:336–338
Bhat MN (2002) Chemical control of rust and angular leaf spot of French bean in Sikkim. Plant Dis
Res 17:90–92
Bhat MN, Singh AK, Medhi RP (1999) Reduction of French bean (Phaseolus vulgaris) cultivars/
accession to rust pathogen Uromyces appendiculatus. Indian J Agric Sci 69:849–850
Chhetry GKN, Mishra RR, Sharma GD (1994) Effect of microclimatic factors on epidemiology of
leaf spot disease caused by Colletotrichum lindemuthianum and Phaeoisariopsis griseola of
French bean (Phaseolus vulgaris). Indian J Agric Sci 64(9):667–670
Borucka K, Marcinkowska JZ (2001) Occurrence of Colletotrichum lindemuthi-anum on Phaseolus
beans in Poland. Veg Crops Res Bull 54:147–151
Boudreau MA (1993) Effect of intercropping beans with maize on the severity of angular leaf spot
of beans in Kenya. Plant Pathol 42:16–25
Boyle C, Walters D (2005) Induction of systemic protection against rust infection in Broad bean by
saccharin: effects on plant growth and development. New Phytol 167:607–612
Bruggen VAHO, Whalen CH, Arneson PA (1986) Emergence, growth and development of dry
beans seedlings in respect to temperature, soil moisture and Rhizoctonia solani. Phytopathology
76:568–572
Buruchara RA, Gathuru EM, Mukunya DM (1988) Disease progress of angular leaf spot caused by
Isariopsis griseola Sacc. and its implications on resistance of some bean (Phaseolus vulgaris L.)
cultivars. Acta Hortic 218:321–328
Butler EJ (1918) Fungi and diseases in plants. Thacker Sprink and Co, Calcutta, p 547
Campos AJ, Fucikovsky ZL (1981) Study of some aspects of angular spot caused by Isariopsis
griseola Sacc. on bean. Fitopatologia 16:16–19
Carrion G, Romero A, Rico-Gray V (1999) Use of Verticillium lecanii as a biocontrol agent against
bean rust. Association Latinoamericana de Fitopatologia (ALF) Fitopatologia 34:214–219
Castro JL d, Ito MF, Dudienas C, Bulisani EA, D’Artegnan-de-Almeida L, De-Castro JL (1991)
Fungicide activity on two bean cultivars in Capao Bonito, Sau Paulo state, Brazil. Bragantia
50:309–321
Centurion MAPDAC, Kimati H, Pereira GT (1994) Machanism of action of antagonisits selected
for the biological control of bean rust (Uromyces phaseoli (Reben.) Wint.). Cientifica
(Jaboticobal) 22:163–175
Chakrabarty PK, Shyam KR (1988) Evaluation of systemic fungitoxicants against Colletorichum
lindemuthianum, the incitant of bean anthracnose. Indian J Plant Pathol 6:67–70
Chakrabarty PK, Shyam KR (1990) Reaction of some promising lines of French bean to different
isolates of Colletotrichum lindemuthianum in Himachal Pradesh. Indian J Plant Pathol 8:86–88
Chatterjee BN, Bhattacharya KK (1986) Principles and practices of grain legume production.
Oxford & IBH Publishing Co, New Delhi, p 492
Cook MT (1924) Common diseases of beans and peas. Agric Exp Station Circ 142:8
Correa-Victoria FJ, Saettler AW (1987) Angular leaf spot of red kidney beans in Michigan. Plant
Dis 71:915–918
Davison AD, Vaughan EK (1963) Longevity of uredospores of race 33 of Uromyces phaseoli var.
typica in storage. Phytopathology 53:736–737
de Oliviera SHF, de Oliveira SHF (2003) New fungicides and spraying schedules in the control of
anthracnose and angular leaf spot. Summa Phytopathol 29:45–48
Devi B, Gupta SK, Singh G (2019) Yield loss assessment in French bean due to rust (Uromyces
appendiculatus) in Himachal Pradesh. Plant Dis Res 34:41–44
Diaz-Plaza R, Teliz OD, Munoz DA (1991) Effect of diseases of bean grown under rainfed
conditions at Mixteca peblana. Revista-Mexicana-de-Fitopatol 9:21–30
Dowing KJ, Thomson JA (2000) Introduction of the serratia marcescens chi a gene into an
endophytic Pseudomonas fluorescens for the biocontrol of Phytopathogenic fungi. Canadian.
J Microbiol 46(4):363–369
Dumitras L, Sesan T (1990) Feature of antagonism of Trichoderma viride Pers ex Fr. to Rhizoctonia
solani Kuhn. Biologie Vegelata 32:87–92
Dundas B (1942) Breeding beans for resistance to powdery mildew and rust. Phytopathology
39:828
Eastman J (1944) Report of provincial plant pathologist. British Columbia Dept Agric Ann Rept
1943:R59–R-63
Elad Y, Chet I, Katan J (1980) Trichoderma harzianum biocontrol agent effective against Sclero-
tium rolfsii and Rhizoctonia solani. Phytopathology 70:119–121
Estevez-de-Jensen C, Percich JA, Graham PH, de Jensen CE (2002) Integrated management
strategies of bean root rot with Bacillus subtilis and Rhizobium in Minnesota. Field Crop Res
74(2–3):107–115
Faleiro FG, De Paula Jr TJ, De Barros EG, Frietos MAS, Moreira MA (1996) Common bean
resistance to Uromyces appendiculatus from zona da Mata region of the state of Minas Gerais,
Brazil. Fitopatol Bras 21:123–125
Faleiro FG, Vinhadelli WS, Ragagnin VA, Paula TJ Jr, Moreira MA, Barros EG (1998) Resistance
of the common bean to four races of Uromyces appendiculatus. Revista Ceres 263:11–18
Fenille RC, de Souza NL (1999) The role of the organic material amended and the soil moisture on
the pathogenicity of Rhizoctonia solani Kuhn AG-4 HGI in snap bean. Pesquisa Agropecuaria
Brasileira 34:1959–1967
Fininsa C (1996) Effect of intercropping bean with maize on common bacterial blight and rust
diseases. Int J Pest Manag 42:51–54
Flores Revilla C, Neito Angel D, De La Torre-Almaraz R (1994) Effect of varieties, foliar nutrients
and seedling rates on disease severity and yield in French bean (Phaseolus vulgaris). Revista
Mexicana de Fitopatologia 12:69–74
Fortugno C (1977) Angular leaf spot (Isariopsis griseola Sacc.) of bean. IDIA 317(320):26–33
Foster HH (1947) Reaction to rust under Mississippi conditions, of certain lines and vars. of pole
snap beans. Plant Dis Rep 31:378–383
Frazier WA, Hendrix JW (1949) Hawaiian Wonder, new rust resistant pole green bean. Circ Hawaii
Agric Exp Station 28:7
Garcia Veloz R, Marin MR, Veloz RR, Rodriguez Guerra R, Torres PI, Gonzalez CMM, Anaya
Lopez JL, Guevara Olvera L, Feregrino Perez AA, Loarca Pina G, Guevara Gonzalez RG (2010)
Antimicrobial activities of cascalote (Caesalpinia cacalaco) phenolics-containing extract
against fungus Colletotrichum lindemuthianum. Ind Crops Prod 31:134–138
Ghimire SR, Pradhanang PM, Lohar DP (1995) Use of fungicides and identification of resistant
varieties for the management of bean rust and anthracnose disease in common beans. Working
paper on Lumle Regional Agricultural Research Centre No. 95/76, 42p
Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research, 2nd edn. Wiley,
New York, p 680p
Gonalves-Vidigal MC, Sakiyama NS, Vidigal-Filho PS, Amaral-Junior AT, Poletine JP, Oliveira
VR (2001) Resistance of common bean cultivar AB 136 to races 31 and 69 of Colletotrichum
lindemuthianum: the Co-6 locus. Crop Breeding App Biotech 1:99–104
Gonzalez M, Garcia C (1996a) Evaluation of fungicides for the control of bean rust (Uromyces
appendiculatus). Agronomia Mesoamericana 7:86–89
Gonzalez M, Garcia C (1996b) Evaluation of losses due to the rust on bean (Phaseolus vulgaris L.)
in four sowing times in Cuba. Agronomia Mesoamericana 7:95–98
Grafton KF, Weiser GC, Little FLJ, Stavely JR (1985) Influence of resistance of two races of leaf
rust in dry edible bean. Crop Sci 25:537–539
Gupta SK, Thind TS (2018) Disease problems in vegetable production, 2nd edn. Scientific
Publishers, Jodhpur, p 586
Gupta SK, Dohroo NP, Shyam KR (1991) Antagonistic studies on seed-borne mycoflora of French
bean (Phaseolus vulgaris L.). Indian J Plant Pathol 9:62–63
Gupta SK, Mathew KA, Shyam KR, Joshi AK (1997) Screening of French bean germplasm against
root rot and web blight. Legume Res 20:57–60
Gupta SK, Mathew KA, Shyam KR, Sharma A (1999) Fungicidal management of root rot
(Rhizoctonia solani) of French bean. Plant Dis Res 14:20–24
Gupta SK, Mathew KA, Shyam KR (2000a) Seed transmission of Rhizoctonia solani in French
bean cultivars. Plant Dis Res 15:83–84
Gupta SK, Mathew KA, Shyam KR, Gupta DK (2000b) Role of fungicides in management of
anthracnose of French bean. In: Proceedings of Indian phytopathological society-golden jubilee
international conference on “Integrated Plant Disease Management for Sustainable Agricul-
ture”, vol II. Indian Phytopathological Society, IARI, New Delhi, pp 705–707
Gupta SK, Mathew KA, Shyam KR, Sharma AK (2000c) Evaluation of French bean germplasm
against angular leaf spot. Indian Phytopathol 53:488–489
Gupta SK, Mathew KA, Shyam KR (2000d) Management of angular leaf spot of French beam
through cultural practices. In: Proceedings of Indian phytopathological society-golden jubilee
international conference on “Integrated Plant Disease Management for Sustainable Agricul-
ture”, vol II. Indian Phytopathological Society, IARI, New Delhi, pp 701–704
Gupta SK, Mathew KA, Shyam KR (2005) Influence of inoculum density and environmental
factors on angular leaf spot of French bean. Plant Dis Res 20:25–28
Gupta SK, Jarial K, Kumar M (2008) Occurrence of French bean rust in Himachal Pradesh. J Mycol
Hagedorn DJ, Rand RE (1980) Wisconsin RRR 46 snap bean breeding line. Hortic Sci 15:529–530
Harter IL, Andrus CF, Zaumeyer WJ (1935) Studies on bean rust caused by Uromyces phaseoli var.
typica. J Agric Res 1:737–759
Hazarika DK, Das KK (1998) Biological management of root rot of French bean (Phaseolus
vulgaris L.) caused by Rhizoctonia solani. Plant Dis Res 13:101–105
Hidalgo R, Araya CM (1993) Optimum growth stage of common bean for chemical control of
anthracnose (Colletotrichum lindemuthianum) and angular leaf spot (Isariopsis griseola) in San
Carlos, Costa Rica. Agron Costarric 17:75–80
Hocking D (1967) A new virulent form of Phaeoisariopsis griseola causing circular leaf spot of
French bean. Plant Dis Rep 51:276–278
Hutchinson CM, Ram Ayyar CS (1915) The Indian rice beer ferment. Mem Dept Agric Indian Bac
Ser 1:137–168
Iriti M, Foro F (2003) Benzothiadiazole (BTH) induces cell death independent resistance in
Phaseolus vulgaris against Uromyces appendiculatus. J Phytopathol 15:171–180
Issa E (1985) Resistance to four diseases in cultivars of French bean, Phaseolus vulgaris L., bred in
Sao Paulo. Biologico 51:113–117
Issa E, Sinigaglia C, Oliveira DA (1982) Chemical control of angular leaf spot of bean (Phaseolus
vulgaris L.) due to Isariopsis griseola Sacc. Biologico 48(12):299–303
Jaffer AA (1971) Effect of fungicides on bean rust (Uromyces appendiculatus) (Pers.). Lev Misc
Rep Pestic Res Inst 765:8
Joshi D, Hooda KS, Bhatt JC (2008) Biocontrol efficacy of indigenous Trichoderma isolates against
root rot pathogens of French bean Uttrakhand hills. J Biol Control 21:119–126
Joshi D, Hooda KS, Bhatt JC, Mina BL, Gupta HS (2009) Suppressive effects of composts on soil-
borne and foliar diseases of French bean in the field in the western Indian Himalayas. Crop Prot
28:608–615
Khati P, Hooda KS (2006) Multiple disease resistance in Rajmash genotypes. Indian Phytopathol
59:521–522
Kikoka LP, Katunzi AL, Teri JM (1989) Evaluation of effect of cropping systems on bean diseases
and yield. In: Proceedings of the international pest management in tropical sub-tropical crop
systems, vol 89, pp 853–858
Kimati H, Galli F (1970) Glomerella cingulata (Stonem.) Spauld. et V. Schrenk. f. sp. phaseoli n.f.,
faseascegena de agente causal de anthracnose do feijoeiro. Annuals da Escola Superior de
Agricultura “Luisde Queiroz” 27:411–437
Kumar A, Sharma PN, Sharma OP, Tyagi PD (1997) Resistance to Colletotrichum lindemuthianum
in kidney bean accessions of diverse origin in Himachal Pradesh. Indian Phytopathol 50:59–64
Kumar A, Sharma PN, Sharma OP, Tyagi PD (1999) Epidemiology of bean anthracnose
Colletotrichum lindemuthianum under sub-humid mid-hills zone of Himachal Pradesh. Indian
Phytopathol 52(4):393–397
Lenne JM (1992) Colletotrichum diseases of legumes. In: Bailey JA, Jeger MA (eds)
Colletotrichum: biology, pathology and control. C.A.B. International, Wallingford, pp 134–166
Lopez SE, Acosta GJA, Becerra LEN, Frayre VG, Orozco SH, Beebe SE (1997) Registration of
Negro Tacana common bean. Crop Sci 37:1022
Macraw WM, Nasser SH, Sidky ST, Faris FS (1973) Producing a variety of beans, resistant to the
rust disease.1. Screening of local and imported varieties against the disease. Agric Res Rev
51:145–152
Mahanta IC, Dhal A (2000) Management of leaf spot and rust of rajmash (Phaseolus vulgaris)
through seed treatment and sprays of chemicals. Legume Res 23:271–272
Maringoni AC, Tofoli JG, Fregonese LH (1992) Fungicides action in vitro on Rhizoctonia solani
and on control of dry bean damping off. Summa Phytopathol 18:269–275
Maringoni AC, de Barros EM, de Barros EM (2002) Occurrence of isolates of Colletotrichum
lindemuthianum resistant to benzimidazole fungicides. Summa Phytopathol 28(2):197–200
Mathew KA, Gupta SK (1996) Studies on web blight of French bean caused by Rhizoctonia solani
and its management. Indian J Mycol Plant Pathol 26:171–176
Mathew KA, Gupta SK (1998) Biological control of root rot of French bean caused by Rhizoctonia
solani. J Mycol Plant Pathol 28:202–205
Mathew KA, Gupta SK, Shyam KR (1998) New strategies in fungicidal management of angular
leaf spot (Phaeoisariopsis griseola) of French bean. J Mycol Plant Pathol 28:123–133
Matz J (1921) Una enfermendad damina de la haleichuela. Porto Rico Insular St Cir 57:8
Mendes BMJ, Bergamin-Filho A (1989) Influence of temperature, wetness duration and leaf type on
the quantification of monocyclic parameters of bean rust. J Phytopathol 126:183–189
Menezes JR, Dianese JC (1988) Race characterization of Brazilian isolates of Colletotrichum
lindemuthianum and detection of resistance to anthracnose in Phaseolus vulgaris. Phytopathol-
ogy 78:650–655
Mishra HN, Mahapatra AK, Sahu S, Mahapatra AKB (1998) Performance of French bean under
different dates of sowing in eastern ghat highland zone of Orissa. Ann Agric Res 19(1):83–88
Mizubuti CSG, Maffia LA, Muchovej JJ, Romeiro RS, Batista UG (1995) Selection of isolates of
Bacillus subtilis with potential for the control of dry bean rust. Fitopatol Bras 20(4):540–544
Mohan N, Aghora T, Somkuwar RG (1995) Reaction of French bean germplasm to angular leaf
spot (Isariopsis griseola Sacc.). Legume Res 18:215–216
Mohan N, Aghora TS, Somkwar RG, Ganeshan G (1997) Sources of resistance to rust in French
bean (Phaseolus vulgaris). Indian J Agric Sci 67:85–86
Moreno RA (1977) Efecto de diferentes sistemas de cultivo sobre la severidad de la mancha angular
del frijol (Phaseolus vulgaris L.) caused for Isariopsis griseola Sacc. Acta Agron Palmira
7:164–169
Moreno RA, Mora LE (1984) Cropping pattern and soil management influence on plant disease
2. Bean rust epidemiology. Turrialba 34:41–45
Mukherjee S, Tripathi HS, Rathi YPS (2001) Integrated management of wilt complex in French
bean (Phaseolus vulgaris L.). J Mycol Plant Pathol 31:213–215
Mundel HH, Kiehn FA, Huang HC, Conner RL, Saindon G, Kemp G (2004) Morden 003 navy
bean. Can J Plant Sci 84:231–233
Muyolo NG, Lipps PE, Schmitthenner AF (1993) Reactions of dry bean, lima bean, and soyabean
cultivars to Rhizotonia root and hypocotyl rot and web blight. Plant Dis 77:234–238
Navarro AR, Puerta-E OD, Isaza L (1981) Combined effect of population density and chemical
control of leaf diseases and yield of bean. Fitopatol Colombiana 10:7–9
NHB (2017) National Horticulture Board. www.nhb.gov.in
Nitshe MJ, Cafati KC (1985) Identification of internal fungi present in bean seeds. Agricultura
Tecnica 45:227–234
Oliveira SHF, Barros BC, Castro JL (1992) Effect of fungicides on the control of bean foliar
diseases and seed quality. Summa Phytopathol 18:178–184
Padder BA, Sharma PN (2010) Assessment of yield loss in common bean due to anthracnose
(Colletotrichum lindemuthianum) under glass house conditions. Res J Agric Sci 1:184–188
Padder BA, Sharma PN, Kapil R, Pathania A, Sharma OP (2010) Evaluation of bioagents and
biopesticides against colletotrichum lindemuthianum and its integrated management in common
bean. Natl Sci Biol 2:72–76
Pastor-Corrales MA, Tu JC (1989) Estandarizacion de variedades differen-ciales y de designacion
de razas de Colletotrichum lindemuthianum (Abstr.). Phytopathology 81:694
Pastor-Corrales MA, Erazo OA, Estrada EI, Singh SP (1994) Inheritance of anthracnose resistance
in common bean accession G 2333. Plant Dis 78:959–962
Pathania A, Sharma PN, Sharma OP, Chahota RK, Ahmad B, Sharma P (2006) Evaluation of
resistance sources and inheritance of resistance in kidney bean to Indian virulences of
Colletotrichum lindemuthianum. Euphytica 149:97–103
Paul YS, Sharma RC (1990) Additions to the host range of Rhizoctonia solani. Plant Dis Res 5:234
Paula TJ Jr, Pinto CMF, da Silva MB, Nietsche S, de Carvalho GA, Falerio FG (1998) Resistance of
snap bean cultivars to anthracnose, angular leaf spot and rust. Revista Ceres 45:171–181
Persoon CH (1795) Observations mycologicae. In: Usteri P (ed) Annalen der Botanic, vol 3, pp
1–43
Pinto JMA, Souza EA, Oliveira DF (2010) Use of plant extracts in the control of common bean
anthracnose. Crop Prot 29:838–842
Prakasham V, Thamburaj S (1991) Efficacy of fungicides in control of rust disease of French bean
caused by Uromyces phaseoli (Reben.) Wimb. Indian J Mycol Plant Pathol 21:158–159
Prasad K, Weigle JL (1976) Association of seed coats factors with resistance to Rhizoctonia solani
in Phaseolus vulgaris. Phytopathology 66:342–345
Pyndji MM, Trutmann P (1992) Managing angular leaf spot of common bean in Africa by
supplementing farmer mixtures with resistant varieties. Plant Dis 76:1144–1147
Raffat FM (1992) Efficiency of certain fugicides in controlling Rhizoctonia damping off of bean.
Assuit J Agric Sci 23:37–47
Rajnauth G (1987) Web blight, an important disease of bean and pak-choi in Trinidad. Trop Agric
84:356–358
Rauscher M, Adam AL, Wirtz S, Guggenheim R, Mendgen K, Deising HB (1999) PR-1 protein
inhibits the differentiation of rust infection hyphae in leaves of acquired resistant broad bean.
Plant J 19:625–633
Ravi S, Doraiswamy S, Valluvaparidasan VD, Jeyalakshmi C (1999) Effect of biocontrol agents on
seed-borne Colletotrichum in French bean. Plant Dis Res 14(2):146–151
Rey G, Lozono JC (1961) Physiological studies on rust of bean (Phaseolus vulgaris) caused by
Uromyces appendiculatus. Acta Agron Palmira 11:147–186
Roberti R, Ghisellini L, Pisi A, Flori P, Filippini G (1993) Efficiency of two species of Trichoderma
as a biological control against Rhizoctonia solani Kuhn. Isolated from string bean root rot in
Italy. Adv Hortic Sci 7:19–25
Rodriques CH, Zambolim L, Martins HCP (1987) Effectiveness of fungicides in the control of
angular leaf spot (Isariopsis griseola) of bean (Phaseolus vulgaris) Fitopatol. Brasileira
12:40–45
Rolim PRR, Brignani Neto F, Roston AJ, Oliveira DA (1981) Chemical control of bean diseases.1.
Rust (Uromyces phaseoli (Pers.) Wint. Var. typica Arth.). Biologica 47:201–205
Saettler AW, Corea FJ (1988) Transmission of Phaeoisariopsis griseola by bean seed. J Seed
Technol 12:133–142
Sanchez AJH, Cardenas AM (1988) Etiology and damage of root rot disease in beans Phaseolus
vulgaris L. in the state of Durango. Revista Chapingo 12:43–49
Sanchez A, Echavez BR, Schroder EC (1994) Pseudomonas cepacia, a potential biofungicides for
root rot pathogens of beans. J Agric Univ Puerto Rico 78:55–57
Santos Filho HP, Ferraz S, Sediyama CS (1978) Effect of the time of inoculation of Isariopsis
griseola Sacc. on three bean cultivars. Fitopatol Brasileira 3:175–180
Sartorato A (1988) Angular leaf spot. Piracicaba Brazil, Asasociacao Brasileira para Pesquisa de
Potassa e do Fosfato, pp 491–501
Sartorato A, Zimmerman MJ, Rocha M, Yamada T (1988) Web blight. In: Cultura do Feijoero
Factores que afetam a Productividade, pp 503–520
Scribner FL (1888) Anthracnose of the bean Gloesporium lindemuthianum. U.S. D.A. Report
(1887), p 361
Shama S, Shama S (1989) Transmission of Rhizoctonia solani Kuhn. in seeds of bean (Phaseolus
vulgaris L.). Curr Sci 58:972–974
Shands WA, Schein RD (1962) Time-temperature interactions of Uromyces phaseoli during spore
germination and tube elongation. Phytopathology 52:27
Shaner G, Finney RE (1977) The effect of nitrogen fertilization on the expression of slow
mildewing resistance in Knox wheat. Phytopathology 67:1051–1056
Sharma SR (1977) Annual report, vol 1976. IIHR, Bangalore
Sharma SR (1989) Yield loss caudsed by rust in French bean. Plant Dis Res 4:92–94
Sharma AK (1998) Epidemiology and management of rust disease of French bean. Veg Sci
25:85–88
Sharma M, Gupta SK (2003) Ecofriendly methods for the management of root rot and web blight
(Rhizoctonia solani) of French bean. J Mycol Plant Pathol 33:345–361
Sharma SR, Sohi HS (1980) Assessment of losses in French bean caused by Rhizoctonia solani
Kuhn. Indian Phytopathol 33:366–369
Sharma SR, Sohi HS (1981) Effect of different fungicides against Rhizoctonia root rot of French
beans (Phaseolus vulgaris L.). Indian J Mycol Plant Pathol 11:216–220
Sharma SR, Sohi HS (1989) Diseases of French bean in India and their control. Indian Rev Life Sci
9:153–168
Sharma PN, Sugha SK (1995) Management of bean anthracnose through chemicals. Indian
Sharma RC, Barthe JP, Lafitte C, Touze A (1986) In vitro absorption of endopolygalacturonase
secreted by Colletotrichum lindemuthianum on cell walls of monogenically resistant and
susceptible cultivars to anthracnose. Can J Bot 64:2903–2908
Sharma PN, Sharma OP, Tyagi PD (1994) Status and distribution of bean anthracnose in Himachal
Pradesh. Himachal J Agric Res 20:91–96
Sharma PN, Kajal B, Rana JC, Ruby N, Sharma SK, Anju P (2012) Screening of common bean
germplasm against Colletotrichum lindemuthianum causing bean anthracnose. Indian
Sharma N, Gupta SK, Bharat NK, Sharma M (2014) Evaluation of French bean germplasm against
bean rust (Uromyces appendiculatus). Plant Dis Res 29(1):115–117
Sharma N, Savita S, Gupta SK, Sharma M (2018) Evaluation of fungicides against bean rust
(Uromyces appendiculatus). Plant Dis Res 33:174–179
Shear CL, Wood AK (1913) Studies of fungus parasites belonging to the genus Glomerella.
U.S.D.A. Bureau Plant Indian Bulletin 252
Shukla A, Sharma HR (2009) Fungicidal management of angular leaf spot and rust of bean
(Phaseolus vulgaris). J Plant Dis Sci 4:222–223
Sindhan GS (1983) Effect of temperature and relative humidity on the development of anthracnose
of French bean. Progr Hortic 15:132–135
Sindhan GS (1984) Evaluation of fungicides against occurrence of angular leaf spot of French bean
caused by Phaeoisariopsis griseola. Indian J Plant Pathol 2:39–42
Sindhan GS, Bose SK (1980) Effect of temperature and relative humidity on the development of
angular leaf spot of French bean. Progr Hortic 12:18–24
Sindhan GS, Bose SK (1981) Evaluation of fungicides against anthracnose of French bean caused
by Colletotrichum lindemuthianum. Indian Phytopath 34:325–329
Singh AK, Bhat MN (2002) Control of French bean rust through fungicides and a neem based
formulation. Indian Phytopathol 55:241–243
Singh G, Gupta SK (2019) Role of temperature, relative humidity and rainfall in the development of
French bean rust (Uromyces appendiculatus). Indian Phytopathol. https://doi.org/10.1007/
s42360-019-00133-w
Singh JP, Musiyimi ABK (1984) Chemical control of bean rust in Kenya. East African Agric
Forestry J 45:207–209
Singh JP, Musymi AK (1979) Control of rust and powdery mildews by a new systemic fungicide,
Bayleton. Pesticides 13:51–53
Singh AK, Saini SS (1980) Inheritance of resistance to angular leaf spot (Isariopsis griseola Sacc.)
in French bean (Phaseolus vulgaris L.). Euphytica 29:175–176
Singh BM, Sharma YR (1976) Screening of fungicides to control angular and floury leaf spots of
beans. Indian J Mycol Plant Pathol 6:148–151
Singh RY, Tripathi SP, Yadav GR (1995) Evaluation of different fungicides against various leaf
spot diseases of beans. Recent Hortic 2:111–112
Smolinska U, Kowalska B (2006) The effectivity of plant extracts and antagonistic microorganisms
on the growth inhibition of French bean pathogenic fungi. Veg Crops Res Bull 64:67–76
Sohi HS, Sharma RD (1974) Mode of survival of Isariopsis griseola Sacc. The causal agent of
angular leaf spot of beans. Indian J Hortic 31:110–113
Sokol PF, Gracia RM, Zimina TA, Pivovarov VF (1977) Some results of studies on the improve-
ment of horticultural plants under Cuban conditions. Inforem Cientifico 39:15–19
Srivastava LS, Gupta DK (1994) Management of angular leaf spot of ‘rajma’ through fungicides in
Sikkim. Indian Phytopathol 47:211–213
Srivastava LS, Gupta DK, Dhiman KR, Singh G (1995) Source of resistance in French bean
(Phaseolus vulgaris) to angular leaf spot (Phaeoisariopsis griseola) in Sikkim. Indian J Agric
Sci 65:305–307
Stiebeling HK, Clark F (1939) Planning for good nutrition. USDA Year Book, pp 321–349
Striab W (1943) Investigation on the biology and control of the bean rust (Uromyces phaseoli var.
typica (Pers.) Wint.). Gartenbavwirs 17:397–445
Sud AK, Sood VK (2002) Screening of indigenous and exotic collections of kidney bean against
bean anthracnose under dry temperate conditions of Himachal Pradesh. Crop Res Hisar
24:202–203
Sumartini (1998) Rust disease in French bean and its control. J Penelitian Pengembangan Pertanian
17:149–153
Sutton DC (1979) Systemic cross protection in bean against Colletotrichum lindemuthianum.
Australasian Plant Pathol 8:4
Tan CS, Tu JC (1995) Tillage effect of root rot severity, growth and yield of beans. Can J Plant Sci
75:183–186
Thakur RS, Sugha SK, Singh BM (1991) Evaluation of systemic fungicides for control of
Rhizoctonia solani under glass house conditions. Indian J Agric Sci 61:230–232
Trutmann P, Kayitare E (1991) Disease control and small multiplication plots improve seed quality
and small farm dry bean yields in Central Africa. J Appl Seed Prod 9:36–40
Trutmann P, Pyndji MM (1994) Partial replacement of local common bean mixtures by high
yielding angular leaf spot resistant varieties to conserve local genetic diversity while increasing
yield. Ann Appl Biol 125:45–52
Trutmann P, Paul KB, Cishabayo D (1992) Seed treatments increase yield of farmer varietal field
bean mixtures in the Central African highlands through multiple disease and bean fly control.
Crop Prot 11:458–464
Tu JC (1981) Anthracnose (Colletotrichum lindemuthianum) on white bean (Phaseolus vulgaris) in
Southern Ontario: spread of the disease from an infection focus. Plant Dis 5:477–480
Tu JC (1983) Epidemiology of anthracnose caused by Colletotrichum lindemuthianum on white
bean (Phaseolus vulgaris) in Southern Ontario: survival of the pathogen. Plant Dis 67:402–404
Tu JC (1992) Management of root rot diseases of peas, beans and tomatoes. Can J Plant Pathol
14:92–99
Tu JC, McNaughton ME (1980) Isolation and characterization of benomyl resistant biotypes of the
delta race of Colletotrichum lindemuthianum. Can J Plant Sci 60:585–592
Tu JC, Park SJ (1993) Root rot resistance in common bean. Can J Plant Sci 73:365–367
Tu JC, Varrtaja O (1981) The effect of the hyperparasite (Gliocladium virens) on Rhizoctonia solani
and Rhizoctonia root rot of the white beans. Can J Bot 59:22–27
Tu JC, Zheng J (1993) Compatability of biocontrol agents with DCT. Mededelingen van de
Faculteit Landbouwwetenschappen Universiteit Gent 58:1359–1364
Tu JC, Tan CS, Park SJ (1992) Effect of soil moisture in root soil on plant growth and root rot
severity of resistant and susceptible bean cultivars. Mededelingen van de Facuteit
Landbouwwetenschappen Universteit Gent 57:381–386
Upmanyu S, Gupta SK (2005) Influence of environmental factors on the progress of root rot and
web blight (Rhizoctonia solani) of French bean. Indian Phytopathol 58:79–83
Upmanyu S, Gupta SK, Shyam KR (2002) Innovative approaches for the management of root rot
and web blight (Rhizoctonia solani) of French bean. J Mycol Plant Pathol 32:317–331
Upmanyu S, Gupta SK, Sharma MK (2004) Evaluation of French bean (Phaseolus vulgaris)
germplasm against web blight (Rhizoctonia solani). Indian J Agric Sci 74:448–450
Vander Plank JE (1963) Plant disease – epidemics and control. Academic Press, New York, p 349
Vierra RF, Paula TJ Jr, Berger PG (1998) Effect of fungicides and cartap insecticides on the control
of bean foliar diseases during the winter-spring season. Summa Phytopathol 24:17–22
Villar-Sanchez B, Lopez-Salinas E (1993) Negro Inifap, a new var. of French bean for Chiapas state
and similar tropical regions. Rev Fitotec Mex 16:208–209
Voland RP, Epstein AH (1994) Development of suppressiveness to diseases caused by Rhizoctonia
solani in soils amended with composted and non-composted manure. Plant Dis 78:461–466
Wagacha JM, Muthomi JW, Mutitu EW, Mwaura FB (2007) Control of bean rust using antibiotics
produced by Bacillus and Streptomyces species – translocation and persistence in Snap Beans. J
Appl Sci Environ Manag 11:165–168
Wolk M, Sarkar S (1994) Antagonism in vivo of Bacillus spp. against Rhizoctonia solani and
Pythium spp. Anzeiger fur Schadlingskunde pflanzenschutz Umweltschutz 67:1–5
Wolk M, Sarkar S (1995) In vivo antagonism of fluorescent pseudomonads against Rhizoctonia
solani and Pythium aphanidermatum on cucumbers and beans. Anzeiger fur Schadlingskunde
pflanzenschutz Umweltschutz 68:3–8
Xin FG, Gouren Z, Baodong L, Jing HH, Jine Z (2000) Microscopic and ultramicroscopic
investigation into the infection process of snap bean rust (Uromyces appendiculatus). Acta
Phytopathologica Sinica 30:53–59
Ziedan EH, Mahamoud SYM (2002) Calcium and sulfur soil treatment to improve biological
control with Trichoderma harzianum for root rot disease control. Assiut J Agric Sci 33:149–160
Whitefly-Transmitted Plant Viruses
and Their Management 8
P. S. Soumia, G. Guru Pirasanna Pandi, Ram Krishna,
Waquar Akhter Ansari, Durgesh Kumar Jaiswal, Jay Prakash Verma,
and Major Singh
Abstract
Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are major agricultural pests

that cause economic damage worldwide. These pests commonly referred to a
group of tiny, soft-bodied, sap-sucking winged insects generally inhabiting on the
underside of the leaf. Moreover, they are enormously polyphagous showing
intercrop movement, high reproduction, resistances to insecticides and virus
transmission. Since decades, genetic complexity of whiteflies is debatable.
Despite the presence of several whitefly species, biotype and genetic species
concepts also exist. Like most arthropods, whiteflies too harbour endosymbionts
(both primary and secondary), essential for its survival and development. These
endosymbionts are specific to species, host and geographic location, which
enable easy differentiation among populations of the same species. Apart from
causing losses through direct feeding, they also act as vector for various econom-
ically important plant viruses like Begomovirus, Geminivirus, etc., and transmit
viral particles via persistent and semi-persistent mode of transmission. Manage-
ment of whitefly populations, and, in particular, management of the viral plant
diseases it transmits, is difficult. At present, the use of insecticides is the main
approach employed to manage whiteflies. However, due to both environmental
concern and resistance issue, this practice is greatly restricted. Hence, integrated
P. S. Soumia (*) · M. Singh

ICAR-Directorate of Onion and Garlic Research, Pune, Maharashtra, India
G. Guru Pirasanna Pandi
ICAR-National Rice Research Institute, Cuttack, Odisha, India
R. Krishna · D. K. Jaiswal · J. P. Verma
Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi, UP,
India
W. A. Ansari
ICAR- India Institute of Vegetable Research, Varanasi, UP, India

https://doi.org/10.1007/978-981-15-6275-4_8
176 P. S. Soumia et al.
pest management programme with all available tactics would help in reducing the
pest population.
Keywords
Begomovirus · Biotype · Endosymbionts · Geminivirus · Vector management ·

Whiteflies
8.1 Introduction
Crop diversification with vegetables, flowers, medicinal plants and fruits provides a
viable option to enhance farmers’ income. Various biotic and abiotic stresses may
hamper agricultural production. Among biotic stresses, insect pests and diseases are
the major constraints resulting about 10–30% yield loss in various crops (Rai et al.
2014). Sucking pests like whiteflies cause incalculable loss globally as they pose
serious threat to many host plants. Crops grown under protected conditions like
greenhouse as well as in open fields are very prone to whitefly attack causing damage
through direct feeding and virus transmission. Previously, sporadic infestation of this
pest and the diseases they transmit was managed through a cocktail of insecticides;
however, uncontrollable outbreaks at recent times have become serious cause for
concern. Once established under favourable microclimate, pest density rises to
outbreak levels where its control becomes difficult. Intervention to control pest
should also be made when population reaches the economic threshold limit (ETL),
while subsequent delay with initial control measures may prove expensive to
farmers. In field crops like cotton, greenhouse crops like tomatoes and cucumbers
and ornamental species like poinsettia and gerbera, whiteflies are of major concern.
Many weed species act as alternate hosts to whiteflies and often serve as sources of
infestations.
Whiteflies/snow flies are tiny, sap-sucking, soft-bodied winged insects com-
monly found in clusters on the undersurface of the leaves (Liu et al. 2007; Zhang
et al. 2007; Wan et al. 2009). They are so named as their entire body including wings
is covered with white waxy powder. Adults of these whiteflies resemble small
moths, while the nymphs look more like a scale insect with a flattened oval shape.
Both nymphs and adult stages of whitefly have piercing and sucking mouthparts and
usually penetrate the phloem and suck the sap by inserting their stylets into the leaf.
During this feeding process, they acquire plant viruses. Adult whiteflies may dis-
perse and transmit the virus to new plants at subsequent feeding. They also secrete
honeydew, a sticky substance on which saprophytic sooty mould develops and
thereby hinders the photosynthetic efficiency of the plant. Irrespective of the instars,
whiteflies at low densities are usually not damaging, and adults do not cause
significant damage unless they transmit plant viruses. Excessive feeding affects
plant growth by causing distortion, discoloration, yellowing or silvering of leaves.
The role of whitefly is more important as a vector of various plant viruses than its
direct damage as a sucking pest. The disease attains significance because the virus is
8 Whitefly-Transmitted Plant Viruses and Their Management 177
capable of attacking the crop in all the stages of its growth period (Brown 1994;
Rajinimala et al. 2005). However, the combination of direct feeding loss and vector
behaviour has promoted this pest to one of the most damaging pests in agricultural
production (Perring et al. 1993).
During the past decades, whiteflies have already taken a heavy toll and risen as
important sucking pests of most vegetables and horticultural and ornamental crops
around the world. The escalating whitefly population has resulted in increased usage
of insecticides, which is not only harmful to the environment but also resulted in
resistance problems. Also, various whitefly species and biotypes morphologically
look similar, yet have subtle physiological differences. Due to these differences,
whiteflies exhibit differential behavioural response towards management strategies.
Hence, proper identification of the whitefly (i.e. species, biotype) is critical for
successful control measures.
8.2 Whitefly Taxonomy and Genetic Structure
Whiteflies are genetically complex and are economically most devastating pests.
Substantial effort is made in investigating the taxonomy and systematics of whitefly
for decades; still its species status is being a subject of debate. Despite their name,
whiteflies are not true flies; these are small hemipterans which belong to
Aleyrodidae, the only family in the superfamily Aleyrodoidea.
8.2.1 Systematic Position
Kingdom: Animalia
Phylum: Arthropoda
Class: Insecta
Order: Hemiptera
Suborder: Sternorrhyncha
Superfamily: Aleyrodoidea
Family: Aleyrodidae
Several species of whiteflies exist, of which Bemisia tabaci (tobacco whitefly)

and Trialeurodes vaporariorum (greenhouse whitefly) are few belligerent pests that
cause significant damage to a wide range of economically important crop plants.
Apart from several species, biotype and genetic species concepts also exist. Biotype
concept in whitefly became evident with B. tabaci variant, a totally different
population from the native population of southern United States (De Barro et al.
2011). To identify these biotypes, molecular tools like various DNA markers such as
mitochondrial cytochrome c oxidase subunit I (MtcoxI) or the ribosomal internal
transcribed spacer (ITS) regions were used. Microsatellite markers are also effective
for population genetic studies in insects (Dalmon et al. 2008; Boopathi et al. 2014).
Recent studies with microsatellites have revealed six genetic populations with little
or no gene flow between B. tabaci from the Asian and Pacific region. Further, out of
the six genetic populations, four subsequently split into two subpopulations. How-
ever, B. tabaci had 24 morphologically indistinguishable species which barely
interbreed and form different phylogenetic clades, hence called as cryptic species
complex exhibiting high variability in their biology and genetics (Dinsdale et al.
2010; De Barro et al. 2011).
Based on genetic differences in B. tabaci, 33 extant biotypes have been reported
(Brown 2010; Gill and Brown 2010; Hadjistylli et al. 2010). Subsequent research
suggested roles of Wolbachia and mating interference in B. tabaci revealed 11 well-
defined genetic groups with at least 36 putative species (Dinsdale et al. 2010; De
Barro et al. 2011; Boykin et al. 2012). These putative species are morphologically
indistinguishable and genetically distinct and differ in their virus transmission
capability, host range, fecundity and insecticide resistance (Dinsdale et al. 2010;
Wang et al. 2010). Middle East Asia Minor 1 (MEAM1, known as B biotype) and
Mediterranean (MED, known as Q biotype) (Hu et al. 2011; Skaljac et al. 2013) are
two widespread putative species that caused extensive damage to numerous crops
worldwide. Later on, three putative species (MEAM1, MED and JpL (Lonicera
japonica)) were identified. MEAM1 and MED were first detected in 1998 and 2004
(Lee et al. 2000, 2005), respectively, whereas JpL was first recorded in 2014 (Lee
et al. 2014). B biotype was first reported from Kolar district of Karnataka, India
(Rekha et al. 2005). Whole genome-wide variants between Asia II 1 (indigenous to
Indian subcontinent and South-East Asia) and MEAM1 (originated from Middle
East that has spread globally in recent decades) contribute to resolving species
delimitation of whitefly. MtcoxI sequence analysis helped in identifying cryptic
species showing 3.5% pairwise divergence within B. tabaci species. About 42 dis-
tinct species have been reported: Africa, Asia I, Asia I-India, Asia II 1–12, Asia III,
Asia IV, Asia V, Australia, Australia/Indonesia, China 1–5, Indian Ocean, Ru,
Middle East Asia Minor I-II (MEAM), Mediterranean (MED), MEAM K, New
World 1–2, Japan 1–2, Uganda, Italy 1 and Sub-Saharan Africa 1–5 (Firdaus et al.
2013; De Barro et al. 2011; Boykin et al. 2007; Hu et al. 2018; Roopa et al. 2015).
Lately, in the identification of cryptic species within this species complex, 4%
genetic divergence was found more realistic than 3.5% (Lee et al. 2013).
Like most arthropods, whiteflies too harbour endosymbionts (bacterium called
Candidatus Portiera aleyrodidarum) confined to bacteriocyte cells (Thao et al.
2004; Sloan and Moran 2012). This bacterium is essential for host survival and
development and has a long co-evolutionary history with all members of the
subfamily Aleyrodidae (Thao et al. 2004; Moran and Telang 1998; Baumann
2005). Apart from primary endosymbiont, secondary endosymbionts, namely,
Arsenophonus, Cardinium, Hamiltonella, Hemipteriphilus, Fritschea, Rickettsia
and Wolbachia, have been reported from B. tabaci populations around the world.
8.3 Whitefly: An Overview
Whiteflies consist of more than 1500 species in approximately 126 genera (Martin,
2004), but relatively few transmit plant viruses (not even 1%). Whiteflies in the
Bemisia and Trialeurodes genera are vectors of various plant viruses. Reports
suggest that the following five species of whitefly are identified as transmitting
virus to plant: Bemisia tabaci, Bemisia afer (Priesner & Hosny), Trialeurodes
abutilonea Haldeman (banded wing whitefly), Trialeurodes vaporariorum
Westwood (greenhouse whitefly) and Parabemisia myricae Kuwana (bayberry
whitefly) (Ng and Falk 2006; Gamarra et al. 2010; Navas-Castillo et al. 2011).
Whitefly vector diversity is much lower than other vectors, yet considered as the
second most important type of vector due to its capacity to transmit many plant
viruses. Notably it transmits 90% of the viruses belonging to the genus Begomovirus
which currently comprises around 200 members. Further, whitefly-transmitted
viruses include ipomoviruses and criniviruses of the family Potyviridae and
Closteroviridae, respectively.
8.4 Whitefly Distribution
Whitefly is a serious pest in agricultural, horticultural and ornamental crops; apart

from direct losses by its feeding, the major concern lies with its role as a virus vector.
Depending on the molecular phylogenetic data and genetic group of reciprocity,
about 35 cryptic species complex are recognized worldwide in B. tabaci (De Barro
et al. 2011). B. tabaci species complex has been reported in 83 countries globally in
all six continents, namely, Asia, Africa, Australia, Europe, South America and
Central America. In Indian, infestation of whitefly was first reported on cotton
(Misra and Lambda 1929). Earlier B. tabaci was considered as a single species,
but recent reports confirmed it as a complex species comprising Asia I, Asia I-India,
Asia II 1, Asia II 5, Asia II 7, Asia II 8, Asia II 11, Asia II 13, MEAM K, China 3,
MEAM1 types (Kanakala and Ghanim 2019). B. tabaci species complex has been
reported in almost all Indian states starting from Jammu and Kashmir to Tamil Nadu
and Arunachal Pradesh to Gujarat.
8.5 Whitefly Host Range
B. tabaci is polyphagous and has a broad host range which includes some of the
common plants like cassava (Lal 1981), cinnamon (Koya et al. 1983), tomato
(Bhardwaj and Kushwaha 1984; Qiu et al. 2007), eggplant (Balaji and Veeravel
1995), okra (Bhagabati and Goswami 1992), sunflower (Men and Kandalkar 1997;
Qiu et al. 2007), black pepper (Ranjith et al. 1992), pulses (Patel and Srivastava
1998), cucumber, groundnut, cauliflower, cabbage and tobacco (Ellango et al. 2015)
and several other host plants (Lisha et al. 2003).
8.6 Whitefly Life Cycle
Life cycle of all whiteflies is similar, staring from egg to adult phase through four
nymphal instars. Both male and female adults are similar, except that the female is
usually larger. Eggs are oval shaped and laid on the underside of the leaf in
horseshoe or circular patterns. Subsequently, upon hatching from the eggs,
crawlers/first instar nymphs (about 0.3 mm long) wander over the leaf surface.
After a week’s time, crawlers gradually settle down and remain sedentary. Later
on, they pass through second, third and fourth nymphal instars. Both legs and
antennae are lost after the first moult, and the subsequent nymphs remain fixed to
the leaf surface. Generally, whitefly nymphs are flat and oval in shape and yellowish
or black in colour and often resemble small-scale insects. Second and third instar
nymphs are pale green. Fourth instar nymphal colour is acquired based on the host
plant, upon which they feed. For example, yellowish individuals are associated with
herbaceous plants while black on woody plants. Fourth instar nymphs feed initially
and thereafter ceases feeding upon maturity (i.e., during gradual transformation into
adult internally). From the fourth instar nymphs, winged adults emerge and hence
often incorrectly called as pupa. After eclosion, adult whiteflies are often pale green
or yellow in colour and thereafter secrete a white waxy coating. Irrespective of the
instars, all stages of whitefly suck the plant sap and excrete the excess liquid as
honeydew. Whitefly populations have several generations in a year, where warm
weather generally flourishes the population with its peak at spring and autumn. The
entire life cycle may be completed in 18 days at temperatures of 28 C, depending on
species. All growth stages can often be found on leaves at any one time (Fig. 8.1).
8.7 Whitefly and Virus Association and Disease Transmission
Geminiviruses are a large family of plant viruses with circular, single-stranded DNA
genome (H). The genome organization and molecular properties have classified
Geminiviruses into seven genera, namely Becurtovirus, Begomovirus, Curtovirus,
Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus according to Interna-
tional Committee on Taxonomy of Viruses. In tropical and semi-tropical region,
B. tabaci majorly acts as a vector of Begomovirus and transmits virus to both
monocots and dicots (Navas-Castillo et al. 2011; Brown et al. 2010). As far as
whiteflies are concerned, it transmits viruses by two modes, namely, persistent and
semi-persistent; their acquisition and retention time has been discussed in Fig. 8.2.
Likewise, the number of viruses transmitted by different species of whitefly was
given in Tables 8.1 and 8.2 (a, b).
1st instar nymph

(crawler)
Egg
1 2
White fly
life cycle
3
6
2nd instar nymph
Adult fly
5
4
4th instar nymph 3rd instar nymph
Fig. 8.1 Life stages of Bemisia tabaci. 1. Egg, 2. First instar nymph (Crawler), 3. Second Instar
nymph, 4. Third instar nymph, 5. Fourth instar nymph and 6. Adult fly (Source: Naveen 2016)
Whitefly virus transmission

mode
Persistent Semi-persistent
Acquistion time - hours Acquistion time - minutes
Retention time - days to entire life Retention time - hours to days
Circulative
Propagative
Virus cannot replicate in the
Virus can replicate in the hemolymph
hemolymph
Fig. 8.2 Transmission mode of viruses by whitefly

Table 8.1 Summary of virus transmitted by whitefly species (Navas-Castillo et al. 2011)
No. of approved
Virus (genus; family) Transmission mode Whitefly species speciesa
Begomovirus; Circulative, B. tabaci 192
Geminiviridae persistent T. ricini 1
Ipomovirus; Potyviridae Semi-persistent B. tabaci 4
Crinivirus; Semi-persistent B. tabaci 4
Closteroviridae B. afer 1
T. abutilonea 4
T. vaporariorum 4
Carlavirus; Semi-persistent B. tabaci 3b
Betaflexiviridae
Torradovirus; Secoviridae Unknown B. tabaci 2c
T. vaporariorum 1
a
According to King et al. (2011) except as noted
b
Includes Cucumber vein-clearing virus (Menzel et al. 2011)
c
Includes Tomato necrotic dwarf virus (Wintermantel and Hladky 2013)
8.8 Whitefly Management
Management of whitefly populations and in particular the viral disease it transmits is

difficult. Polyphagous nature, high reproductive rate, short generation time,
biotypes, rapid evolution of resistance to insecticides and the relatively protected
location of the individuals on the underside of the leaves contribute to its survival
and dominance in the agroecosystem. Invariably different management strategies
have been employed vector management worldwide. Hence, durable and cost-
effective pest-control strategies that include good agricultural practices like routine
monitoring fields, breaking the pest continuum, conserving natural enemies and
using pesticides only when necessary are required. Whiteflies can be controlled
effectively when crops are grown under protected environment like greenhouses or
polyhouses. If farmers practice open cultivation, they will have to adopt some
preventive practices to avoid complete crop loss:
• Management of virus and vector reservoirs

• Host plant resistance
• Use of mulching
• Virus-free seed/planting material
• Trap crop
• Application of insecticides
8
Table 8.2 Whiteflies transmitting viral diseases with global significance

2a) B. tabaci transmitting viral diseases
Monetary loss
(US$) and yield
Crop Disease Other host Affected area Affected region loss References
Tomato Tomato Eggplants, potatoes, 7 million ha Middle East Asia; Early stage – 100% CABI (2019)
yellow leaf tobacco, beans and peppers Mediterranean Basin; North 30 Days after
curl disease America, Australia planting – 80%
Cassava Cassava Soybean, 2.6 million ha Africa 1.9–2.7 billion; Legg et al. (2006)
mosaic Jatropha, castor bean, Indian subcontinent Early stage – 90%
disease Ceara rubber 30 Days after
planting – 80%
Bean Bean golden Phaseolus lunatus, 4 million ha North America, Early stage – 75% EPPO (2019)
yellow P. acutifolius and South America
mosaic virus P. coccineus,
Vigna luteola
Macroptilium lathyroides,
Malvastrum
coromandelianum
Cucurbit Cucurbit Alfalfa, lettuce, 1 million ha Africa, Asia, Europe, North 40–60% Abrahamian and Abou-
Whitefly-Transmitted Plant Viruses and Their Management
yellow snap bean greenhouse/ America Jawdah (2014) and

stunting polyhouse CABI (2019)
disorder
virus
Cucurbit Cucurbit Lettuce, spinach, 2 million ha Asia, Africa, North 60% Abrahamian and Abou-
chlorotic sugar beet America Jawdah (2014)
yellows
virus
Sweet Sweet potato Beets, sowbane, devil’s 4–5 million Africa, South America 90% Data from FAO and
potato mild mottle snare, tomato, tobacco, ha regions calculated from
virus petunia and zinnia this study
183
(continued)
Table 8.2 (continued)
184
2b) Trialeurodes spp. transmitting viral diseases (Jones 2003; CABI, 2019)
Monetary loss (US$)
Crop Disease Other host Affected region and yield loss
Tomato Tomato Callistephus chinensis, Cynara cardunculus, C. scolymus, Lactuca sativa, Asia, Europe, Greenhouse – 80–
infectious Lycopersicon esculentum, Nicotiana glauca, Petunia hybrida, Physalis North America 100%
chlorosis ixocarpa, Picris echioides, Ranunculus spp. Field condition – 50%
virus
Tomato Tomato Datura stramonium, Europe, Greenhouse – 80–
chlorosis Lycopersicon esculentum, North America 100%
virus Solanum nigrum Field condition – 50%
Potato Potato Catharanthus roseus, Lycopersicon spp., Polygonum spp., Rumex South America Up to 50%
yellow vein obtusifolius, Solanum nigrum, S. tuberosum, Tagetes spp.
virus
Beetroot Beet Beta vulgaris, Lactuca sativa, Cichorium endivia, Cucumis melo, C. sativus, Australia, Early stage – 50%
pseudo- Capsella bursa-pastoris, Taraxacum officinalis, Conium maculatum Europe,
yellow North America
virus
Sweet Sweet Ipomoea batatas Asia, Africa, 50% or more
potato potato North America
chlorotic
stunt virus
P. S. Soumia et al.
8.8.1 Management of Virus and Vector Reservoirs
Control of weeds and alternate hosts near to the main crop could reduce the
off-season survival of vector and virus load effectively (Adkins et al. 2011).
Virus-infected fields are often the most important source of inoculums. For example,
virus-infected tomato fields are often the most important source of the tomato yellow
leaf curl virus (TYLCV) and its vector (Polston and Lapidot 2007). Hence, removal
of virus-infected plants can contain the spread of the disease to healthy plants. Not
every time visible symptoms appear. Symptomless infection of pepper can also
provide a reservoir of TYLCV for tomato (Morilla et al. 2005; Polston et al.
2006). Hence, to reduce the initial TYLCV inoculum levels, pest continuum needs
to be disrupted through creating host/crop-free period of tomato and pepper (Polston
et al. 2009). This will reduce the spread of virus as without the susceptible host plant
it cannot spread and also it breaks the phonological synchrony.
8.8.2 Host Plant Resistance
Potential source of resistance have been identified in wild germplasm that could reduce
the virus spread. Screening is done for both the vector (white fly) as well as the virus in
wild germplasm; if the screened material was found resistant against any one, it shall
be considered as a good donor to develop resistant genoptypes (Table 8.3).
8.8.3 Nutrient Management
Like in most crops, nutrient management is an essential component in pest manage-

ment. Excessive use of nitrogenous fertilizer promotes luxuriant plant growth,
thereby increasing the susceptibility to whitefly infestation. Also phosphorus and
magnesium levels of the plants need to be monitored, as deficiencies in these
nutrients are believed to contribute to whitefly infestations.
8.8.4 Use of Mulching
Under open-field situation, plastic/polyethylene soil covers (mulch) are a popular

strategy for protection against whiteflies and the viruses they transmit (Table 8.4).
Apart from reducing the pest inoculum, it also inhibits germination and growth of
weeds.
8.8.5 Virus-Free Seed/Planting Material
To avoid the infestation of any pest and disease at the field level, healthy and virus-
free planting material like seeds or vegetatively propagated planting material is best.
Vegetatively propagated crops like cassava, banana and sweet potato are particularly
Table 8.3 Summary of virus resistant sources

Crop Virus Resistance source References
Tomato TYLCV Solanum pimpinellifolium, Lapidot et al.
S. peruvianum (PI 126926, PI (2014)
126930, PI 390681), S. chilense
(LA1969, LA2779, LA1932) and
S. habrochaites (B6013), S. arcanum
(LA0441)
Tomato Tomato infectious Wild Solanum species (acylsugars Lapidot et al.
chlorosis virus derived gene) (2014)
(TICV)
Watermelon Squash vein Wild citron - PI 500354 (Citrullus Kousik et al.
yellowing virus lanatus var. citroides) (2009)
(SqVYV) PI 386024 (C. colocynthis)
PI 459074 and PI 392291
(C. lanatus var. lanatus)
Watermelon Zucchini yellow Virus-tolerant rootstocks Wang et al.
mosaic virus and (2002)
PRSV-W
Watermelon Cucurbit yellow PI 313970 and TGR-1551 Lapidot et al.
stunting disorder (2014)
virus (CYSDV)
Bean TYLCV Breeding line - GG12 Monci et al.
(2005)
Cassava Cassava mosaic African landraces (CMD2 gene) Akano et al.
virus (CMD) (2002)
Table 8.4 Types of mulching used for controlling whiteflies

Coloured (yellow)
Particulars mulching Silver (aluminium) coated plastic mulching
Mode of Attractant Deterrent
action whiteflies attract to the Strongly reflects light, which acts as a deterrent
mulch instead of to the host to the invading whiteflies
Crop period Early crop Early crop
Successful Tomato field against Tomato mottle virus in Florida; Cucurbit leaf
field TYLCV in Israel crumple virus (CuLCrV) in Zucchini squash
application (Cucurbita pepo L.)
vulnerable to virus infection. Hence, tissue culture technique is successfully

employed to produce virus-free seed/planting material for vegetatively propagated
crops (Clark et al. 2012). In cassava, tissue culture and virus indexing with
thermotherapy has been routinely used for many years to ensure virus-free germ-
plasm exchange between continents.
8.8.6 Trap Crops/Barrier Crops
Trap crops are those crops which are grown along with the main crop to attract the
pest towards them, so that the main crop suffers no or little damage. Similarly, as the
name suggests, barrier crops are crops grown along the borders of the main field as
barrier to the movement of the pest from nearby fields. Both trap crops and barrier
crops have shown to be effective in reducing the whitefly populations, consequently
reducing the level of virus infection (Table 8.5).
8.8.7 Sticky Traps
Generally, yellow sticky traps or yellow pan traps are used to attract the whiteflies. It
helps in both monitoring and mass trapping of the pest. These traps (25/acre) are
hanged close to the crop canopy or at 30 cm from the ground.
8.8.8 Biological Control
In general, biological control is more expensive than chemical control and does not
eliminate the pest completely from the agroecosystem. Biological control is often
successfully used to suppress whitefly populations in greenhouses in Europe but is
less widespread in the United States. Natural enemies include entomopathogens,
predators and parasitoids. The only disease-causing organisms known to attack
whiteflies are fungi. Currently, commercially available entomopathogenic fungi
are Mycotal® (Verticillium lecanii), BotaniGard® (Beauveria bassiana) and
Table 8.5 List of trap/barrier crops used against whiteflies/viruses

Trap crop
Main crop Trap crop Vector/virus to be trapped
Tomato Squash Bemisia tabaci (TYLCV)
Tomato Cucumber B. tabaci (TYLCV)
Cotton Cantaloupes B. tabaci
Cotton Sunflower B. tabaci
Cassava Soybean Aleurotrachelus socialis, Trialeurodes variabilis
Barrier crop/intercropping
Tomato Sorghum B. tabaci
Tomato Maize B. tabaci (TYLCV)
Cassava Maize, cowpea CMD (Cassava mosaic disease)
Cotton Maize B. tabaci
Cow pea Pearl millet Virus transmission
Cucumber Maize CVYV
Squash Maize Squash leaf curl virus
Zucchini Okra, sun hemp B. tabaci
PreFeRal® (Paecilomyces fumosoroseus). However, various predators and

parasitoids are also found effectively reducing the whitefly population. Habitat
management through border cropping with perennial plants provides year-round
refuge for these predators. General predators like the coccinellid (both adult and
grub) beetles feed on whiteflies. Another predatory beetle, Delphastus catalinae,
consumes whitefly eggs and nymphs and attacks all species of whitefly. It avoids
eating whitefly nymphs that have parasitoids developing within them, which means
that it can be released together with a parasitoid without interfering with parasitism.
Apart from predators, a nymphal parasitoid, Encarsia formosa, is smaller than the
whiteflies it attacks and oviposits on whitefly nymphs; subsequent parasitoid larva
develops within the host body by consuming it from the inside over a period of
1–2 weeks. This parasitoid could parasitize three whitefly species, namely, banded
winged whitefly (Trialeurodes abutiloneus), greenhouse whitefly (T. vaporariorum)
and sweet potato whitefly (B. tabaci), but provides the best control against green-
house whitefly, especially at cooler temperatures. Likewise, Eretmocerus eremicus
is another parasitoid that is commercially available, provides better control against
B. tabaci than Encarsia inaron and performs better at higher temperatures.
8.8.9 Chemical Control
Whiteflies are mainly controlled through multiple applications of insecticides with

different modes of action. This method delays the development of insecticide
resistance and gives better results at the field level and limits the spread of
whitefly-transmitted viruses. The list of insecticides used in India against whiteflies
with respect to crop has been given in detail (Table 8.6). In India, only 5 different
modes of action insecticides are available for whitefly management, whereas the
United States had registered 11 different group of synthetic chemical along with 6
different groups of mixer and biopesticides (Table 8.7). Comprehensive control of
whiteflies with conventional insecticides is difficult to achieve. Therefore, novel
insecticides with relative target pest specificity are recommended for the effective
management of whiteflies, which prove to be less harmful to natural enemies and the
environment. Accordingly, they are also more suitable for integrative combination
with other methods.
8.8.9.1 Insect Growth Regulators (IGRs)

Insect growth regulators like buprofezin and novaluron are chitin synthesis inhibitors
effective in managing the pest. Chitin is the integral part of the insect exocuticle, due
to action of chitin synthesis inhibitor; chitin is not produced as the result procuticle
of the whitefly nymph loses its elasticity and the insect is unable to moult. While
pyriproxyfen is a juvenile hormone (JH) mimic that affects the hormonal balance in
insects, suppresses embryogenesis, metamorphosis and adult formation.
Table 8.6 List of insecticides recommended against whitefly in India (CIBRC 2020)
Mode of action Application rate
a.i Formulation
Insecticide name Crop Site of action Code (g/ha) (g/ml)
Acetamiprid 20% Cotton Nicotinic acetylcholine 4A 10– 50–100
SP receptor (nAChR) agonists 20
Diafenthiuron Cotton Inhibitors of mitochondrial 12A 300 600
50% WP ATP synthase
Flonicamid 50% Cotton Chordotonal 29 75 150
WG organModulators
Monocrotophos Cotton Acetylcholinesterase 1B 200 1333
15% SG (AChE) inhibitors
Profenofos 50% Cotton Acetylcholinesterase 1B 500 1000
EC (AChE) inhibitors
Spiromesifen Tomato Inhibitors of acetyl-CoA 23 150 625
22.9% SC carboxylase
Spiromesifen Cotton Inhibitors of acetyl-CoA 23 144 600
22.9% SC carboxylase
Thiamethoxam Cotton Nicotinic acetylcholine 4A 3 10
30% FS receptor (nAChR) agonists
Thiamethoxam Cotton Nicotinic acetylcholine 4A 300 430
70% WS receptor (nAChR) agonists
Thiamethoxam Tomato Nicotinic acetylcholine 4A + 3A 44 200
12.6% + Lambda- receptor (nAChR) agonists
cyhalothrin 9.5% + Sodium channel
ZC modulators
8.8.9.2 Neonicotinoid Insecticides

Neonicotinoids are nicotine mimics which bind to the nicotinic acetylcholine recep-
tor (nAChR) of both the central and peripheral nervous systems, resulting in excita-
tion and paralysis followed by insect death. Imidacloprid was the first commercial
neonicotinoid successfully used for controlling agricultural pests.
8.8.9.3 Diafenthiuron
Diafenthiuron is a thiourea derivative which directly affects insect respiration via
inhibition of oxidative phosphorylation and disruption of mitochondrial ATP
synthesis.
8.8.9.4 Pyridine Insecticides (Pymetrozine)

Pymetrozine is an azomethine pyridine insecticide which affects the nerves con-
trolling the salivary pump and thereby causes immediate and irreversible cessation
of feeding due to obstruction in stylet penetration, followed by starvation and insect
death.
Table 8.7 List of insecticides recommended against whitefly in the United States (Vegetable
Production Handbook for Florida, 2020 and Lapidot et al. 2014)
Mode of action
Insecticide name Group Site of action Code
Oxamyl Carbamates Acetylcholinesterase 1A
(AChE) inhibitors
Methamidophos, Organophosphates Acetylcholinesterase 1B
Acephate (AChE) inhibitors
Endosulfan Cyclodiene GABA-gated 2A
Organochlorines chloride channel
blockers
Beta-cyfluthrin, Bifenthrin, Pyrethroids Sodium channel 3A
Esfenvalerate, Gamma-cyhalothrin, Pyrethrins modulators
Lambda-cyhalothrin, Pyrethrins +
Piperonyl butoxide,
Zeta-cypermethrin
Acetamiprid, Clothianidin, Neonicotinoids Nicotinic 4A
Dinotefuran, Imidacloprid, acetylcholine
Thiamethoxam receptor (nAChR)
competitive
modulators
Sulfoxaflor Sulfoximines Nicotinic 4C
acetylcholine
receptor (nAChR)
competitive
modulators
Flupyradifurone Butenolides Nicotinic 4D
acetylcholine
receptor (nAChR)
competitive
modulators
Pyriproxyfen Pyriproxyfen Juvenile hormone 7C
mimics
Pymetrozine Pymetrozine Chordotonal organ 9B
TRPV channel
modulators
Afidopyropen Pyropenes Chordotonal organ 9D
TRPV channel
modulators
Novaluron Benzoylureas Inhibitors of chitin 15
biosynthesis
affecting
CHS1
Buprofezin Buprofezin Inhibitors of chitin 16
biosynthesis, type 1
Fenpyroximate METI acaricides Mitochondrial 21A
and insecticides complex I electron
transport inhibitors
(continued)

Mode of action
Insecticide name Group Site of action Code
Spiromesifen, Tetronic and Inhibitors of acetyl- 23
Spirotetramat tetramic acid CoA carboxylase
derivatives
Chlorantraniliprole Diamides Ryanodine receptor 28
modulators
Flonicamid Flonicamid Chordotonal organ 29
Modulators
Lambda-cyhalothrin + Mixes of more than one active ingredient 3A + 28
chlorantraniliprole
Bifenthrin + imidacloprid Mixes of more than one active ingredient 3A + 4A
Lambda-cyhalothrin + Mixes of more than one active ingredient 3A + 4A
Thiamethoxam
Bifenthrin + Avermectin B1 Mixes of more than one active ingredient 3A + 6
Thiamethoxam + chlorantraniliprole Mixes of more than one active ingredient 4A+ 28
Buprofezin + flubendiamide Mixes of more than one active ingredient 16 + 28
8.8.9.5 Ryanodine Receptor Insecticides (the Diamides)

Ryanodine receptors are a class of ligand-gated calcium channels which controls the
release of calcium from intracellular stores. Cyazypyr™ is the commercially avail-
able ryanodine formulation specific for sucking pests such as whiteflies and aphids.
8.8.10 Genetically Engineered Resistance
Genetic engineering is a potent tool that broadens and enriches the resistance gene
pool against virus diseases. Viral coat protein gene expression in transgenic plants
confers resistance against virus. This type of induced resistance effect is categorized
as ‘coat-protein-mediated’ protection, a part of pathogen-derived-protection (PDR)
strategies. Further for plant transformations, constructs showing mutated or
truncated virus genes or virus RNA sequences are used, which interfered with
virus infection or silenced the expression of viral genes. Most transgenics expressing
truncated virus genes are model crops and not the field or horticultural crop itself.
8.8.10.1 Replication (Rep)-Associated Proteins

Transgenic tomato plants expressing truncated geminivirus Rep genes exhibited
resistance against Tomato yellow leaf curl Sardinia virus (TYLCSV). The Rep
genes particularly interfered with related viral infection and not with those viral
strains which showed low identities at amino acid level.
8.8.10.2 Movement Proteins (mp)

Transgenic tomato plants with mutated Bean dwarf mosaic virus (BDMV) mp gene
conferred resistance against Tomato mottle virus (ToMoV). This gene provides a
wide range of resistances; however, its over-expression induces toxic effects making
them impractical.
8.8.10.3 Gene Silencing

Target gene that needs to be suppressed/silenced can be achieved by blocking their
expression either through transcriptional gene silencing by DNA methylation or
post-transcriptional gene silencing (PTGS) by degradation of mRNA. PTGS can be
triggered by the expression of dsRNAs homologous to virus sequences. PTGS was
used successfully against the begomoviruses Mung bean yellow mosaic virus
(MYMV), Tomato Leaf Curl Virus (ToLCV), etc.
8.8.10.4 Antisense RNA

In vivo base pairing of RNA molecules with sequence complementation to the viral
RNA can prevent RNA translation or induce PTGS. This strategy has been success-
fully exploited to target and selectively suppress the expression of geminivirus genes
of tomato golden mosaic virus (TGMV), ToLCV and others; however, this approach
failed when tested against other geminiviruses.
8.9 Conclusion
Apart from causing direct yield loss through infestation, whiteflies also act as vector
for various plant viruses inflicting 60–100% yield loss in different crops. Further,
whiteflies can easily adapt to changing climatic conditions, especially in subtropical
and tropical agroecosystems and in temperate regions under greenhouse conditions.
Managing the vector itself can reduce the disease incidence and further spread of the
same to new regions. Hence, vector management is of prime importance especially
for controlling whitefly-transmitted viral diseases. Careful integration of various
IPM components like host plant resistance, physical/mechanical methods, biocontrol
and need-based chemical control with selective novel insecticides would discourage
pest population explosion and also minimize the pesticide load at levels that pose
risk to human health and the environment. Currently, whitefly management solely
relies upon the predominant use of insecticides. Adopting IPM package will alleviate
the numerous concerns that accompany the use of chemicals, including environmen-
tal pollution and resistance development. Moreover, uses of novel insecticides are
relatively target specific and therefore pose minimal hazard to natural enemies and
the environment. Further, these new molecules should be compatible with other
management strategies to enhance the efficiency of the existing IPM package.
References
Abrahamian P, Abou-Jawdah Y (2014) Whitefly-transmitted criniviruses of cucurbits: current
status and future prospects. Virus Dis 25:26–38
Adkins S, Webster CG, Kousik CS, Webb SE, Roberts PD, Stansly PA, Turechek WW (2011)
Ecology and management of whitefly-transmitted viruses of vegetable crops in Florida. Virus
Res 159:110–114
Akano AO, Dixon AGO, Mba C, Barrera E, Fregene M (2002) Genetic mapping of a dominant gene
conferring resistance to cassava mosaic disease. Theor Appl Genet 105:521–525
Balaji K, Veeravel R (1995) Effect of constant temperature on the duration of different develop-
mental stages of whitefly, Bemisia tabaci (Genn.) on eggplant. Madras Agric J 82:62–63
Baumann P (2005) Biology of bacteriocyte-associated endosymbionts of plant sapsucking insects.
Annu Rev Microbiol 59:155–189.
Bhagabati KN, Goswami BK (1992) Incidence of yellow vein mosaic disease of okra (Abelmoschus
esculentus) in relation to whitefly (Bemisia tabaci) population under different sowing dates.
Indian J Virol 8:37–39
Bhardwaj SC, Kushwaha KS (1984) Whitefly, Bemisia tabaci (Genn.) infesting tomato in
Rajasthan. Bull Entomol 25:76–97
Boopathi T, Mohankumar S, Karuppuchamy P, Kalyanasundaram M, Ravi M, Preetha B et al
(2014) Genetic Evidence for Diversity of Spiralling Whitefly, Aleurodicus dispersus
(Hemiptera: Aleyrodidae) Populations in India. Fla Entomol 97:1115–1122
Boykin LM, Shatters RG Jr, Rosell RC, McKenzie CL, Bagnall RA, De Barro P et al (2007) Global
relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of
mitochondrial COI DNA sequences. Mol Phylogenet Evol 44:1306–1319
Boykin LM, Armstrong KF, Kubatko L, De Barro P (2012) Species delimitation and global
biosecurity. Evol Bioinforma 8:S8532
Brown JK (1994) The status of Bemisia tabaci (Genn.) as a pest and vector in world
agroecosystems. FAO Plant Prot Bull 42:3–32
Brown JK (2010) Phylogenetic biology of the Bemisia tabaci sibling species group. In: Stansly PA,
Naranjo SE (eds) Bionomics and management of a global pest. Springer, Amsterdam, pp 31–67
CABI (2019) Tomato yellow leaf curl virus (leaf curl) data sheet. (Accessed on 25th January 2020)
CIBRC (2020). http://ppqs.gov.in/divisions/pesticides-monitoring-ocumentation /pesticide- moni-
toring-and-documentation-unit
Clark CA, Davis JA, Abad JA, Cuellar WJ, Fuentes S, Kreuze JF, Valkonen JP (2012) Sweetpotato
viruses: 15 years of progress on understanding and managing complex diseases. Plant Dis
96:168–185
Dalmon A, Halkett F, Granier M, Delatte H, Peterschmitt M (2008) Genetic structure of the invasive
pest Bemisia tabaci: evidence of limited but persistent genetic differentiation in glasshouse
populations. Heredity (Edinb) 100:316–325
De Barro PJ, Liu SS, Boykin LM, Dinsdale AB (2011) Bemisia tabaci: a statement of species status.
Annu Rev Entomol 56:1–19
Dinsdale AB, Cook L, Riginos C, Buckley YM, De Barro PJ (2010) Refined global analysis of
Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) mitochondrial cyto-
chrome oxidase 1 to identify species level genetic boundaries. Ann Entomol Soc Am
103:196–208
Ellango R, Singh ST, Rana VS, Gayatri Priya N, Raina H, Chaubey R, Rajagopal R (2015)
Distribution of Bemisia tabaci genetic groups in India. Environ Entomol 44:1258–1264
EPPO Reports (2019) Paris. https://www.gd.eppo.int/taxon/BEMITA. Accessed 12 Apr 2020
Firdaus S, Vosman B, Hidayati N, Supena EDJ, Visser RG, van Heusden AW (2013) The Bemisia
tabaci species complex: additions from different parts of the world. Insect Sci 20:723–733
Gamarra HA, Fuentes S, Morales FJ, Glover R, Malumphy C, Barker I (2010) Bemisia afer sensu
lato, a vector of Sweet potato chlorotic stunt virus. Plant Dis 94:510–514
Gill R, Brown JK (2010) Systematics of Bemisia and Bemisia relatives: can molecular techniques
solve the Bemisia tabaci complex conundruma – taxonomist’s view point. In: Stansly PA,
Naranjo SE (eds) Bionomics and management of a global pest. Springer, Amsterdam, pp 5–29
Hadjistylli M, Brown JK, Roderick GK (2010) Tools and recent progress in studying gene flow and
population genetics of the Bemisia tabaci sibling species group. In: Stansly PA, Naranjo SE
(eds) Bionomics and management of a global pest. Springer, Amsterdam, pp 69–103
Hu J, De Barro P, Zhao H, Wang J, Nardi F, Liu SS (2011) An extensive field survey combined with
a phylogenetic analysis reveals rapid and widespread invasion of two alien whiteflies in China.
PLoS One 6(1):e16061
Hu J, Zhang X, Jiang Z, Zhang F, Liu Y, Li Z et al (2018) New putative cryptic species detection
and genetic network analysis of Bemisia tabaci (Hempitera: Aleyrodidae) in China based on
mitochondrial COI sequences. Mitochondrial DNA Part A 29:474–484
Jones DR (2003) Plant viruses transmitted by whiteflies. European J Plant Pathol 109:195–219
Kanakala S, Ghanim M (2019) Global genetic diversity and geographical distribution of Bemisia
tabaci and its bacterial endosymbionts. PLoS One 14(3):e0213946
King AM, Lefkowitz QE, Adams MJ, Carstens EB (2011) Virus taxonomy: Ninth report of the
International Committee on taxonomy of viruses. Academic Press, San Diego
Kousik CS, Adkins S, Turechek WW, Roberts PD (2009) Sources of resistance in US plant
introductions to watermelon vine decline caused by Squash vein yellowing virus. HortScience
44:256–262
Koya MA, Gautam SS, Banerjee SK (1983) Bemisia tabaci (Genn.). A new pest of cinnamon.
Indian J Entomol 45:198
Lal SS (1981) An ecological study of the whitefly Bemisia tabaci (Genn.) population on cassava,
Manihot esculenta. Pestology 5:11–17
Lapidot M, Legg JP, Wintermantel WM, Polston JE (2014) Management of whitefly-transmitted
viruses in open-field production systems. In: Advances in virus research, vol 90. Academic
Press, Cambrige, MA, pp 147–206
Lee ML, Ahn SB, Cho WS (2000) Morphological characteristics of Bemisia tabaci (Gennadius)
(Homoptera: Aleyrodidae) and discrimination of their biotypes in Korea by DNA markers.
Korean J App Entomol 39(1):5–12
Lee MH, Kang SY, Lee SY, Lee HS, Choi JY, Lee GS et al (2005) Occurrence of the B- and
Q-biotypes of Bemisia tabaci in Korea. Korean J App Entomol 44:169–175
Lee W, Park J, Lee GS, Lee S, Akimoto SI (2013) Taxonomic status of the Bemisia tabaci complex
(Hemiptera: Aleyrodidae) and reassessment of the number of its constituent species. PLoS One
8:e63817. pmid:23675507
Lee W, Lee SM, Kim CS, Choi HS, Akimoto SI, Lee KY et al (2014) Three species of the Bemisia
tabaci (Hemiptera: Aleyrodidae) complex in the Republic of Korea; detection by an extensive
field survey combined with a phylogenetic analysis. Fla Entomol 97(1):155–161
Legg JP, Owor B, Sseruwagi P, Ndunguru J (2006) Cassava mosaic virus disease in East and
Central Africa: epidemiology and management of a regional pandemic. Adv Virus Res
67:355–418
Lisha VS, Antony B, Palaniswami MS, Henneberry TJ (2003) Bemisia tabaci (Genn.) biotypes in
India. J Econ Entomol 96:322–327
Liu W, Hou ML, Wen JH, Li JW (2007) Influence of potassium fertilizer on life table parameters of
laboratory populations of Bemisia tabaci. Chinese Bull Entomol 44(5):722–726
Men UB, Kandalkar HG (1997) Pest complex of sunflower, Helianthus annus in Maharastra. PKV
Res J 21:61–63
Menzel W, Abang MM, Winter S (2011) Characterization of Cucumber vein-clearing virus, a
whitefly (Bemisia tabaci)-transmitted carlavirus. Arch Virol 156:2309–2311
Misra CS, Lambda SK (1929) The cotton whitefly (Bemisia gossypiperda). Bulletin Agricultural
Research Institute Pusa 196, IARI, Pusa, Bihar
Monci F, García-Andrés S, Maldonado JA, Moriones E (2005) Resistance to monopartite
begomoviruses associated with the bean leaf crumple disease in Phaseolus vulgaris controlled
by a single dominant gene. Phytopathology 95:819–826
Moran NA, Telang A (1998) Bacteriocyte-associated symbionts of insects. Bioscience:295–304
Morilla G, Janssen D, García-Andrés S, Moriones E, Cuadrado IM, Bejarano ER (2005) Pepper
(Capsicum annuum) is a dead-end host for Tomato yellow leaf curl virus. Phytopathology
95:1089–1097
Navas-Castillo J, Fiallo-Olivé E, Sánchez-Campos S (2011) Emerging virus diseases transmitted by
whiteflies. Annu Rev Phytopathol 49:219–248
Naveen NC (2016) Studies on the monitoring of insecticide resistance and its mechanisms in the
intraspecific populations of whitefly, Bemisia tabaci (Gennadius) towards resolving the taxo-
nomic complexities (Hemiptera: Aleyrodidae). Unpublished Doctoral dissertation, Banaras
Hindu University, Varanasi, India
Ng JCK, Falk BW (2006) Virus-vector interactions mediating nonpersistent and semipersistent
transmission of plant viruses. Annu Rev Phytopathol 44:183–212
Patel MB, Srivastava KP (1998) Host preference of whitefly Bemisia tabaci (Genn.) for oviposition
and development on different grain legumes. Bull Entomol 30:118–120
Perring TM, Cooper AD, Rodriguez RJ, Farrar CA, Bellows TS Jr (1993) Identification of a
whitefly species by genomic and behavioral studies. Science 259:74–77
Polston JE, Lapidot M (2007) Management of Tomato yellow leaf curl virus: US and Israel
perspectives. In: Tomato yellow leaf curl virus disease. Springer, Dordrecht, pp 251–262
Polston JE, Cohen L, Sherwood TA, Ben-Joseph R, Lapidot M (2006) Capsicum species: symp-
tomless hosts and reservoirs of Tomato yellow leaf curl virus. Phytopathology 96:447–452
Polston JE, Schuster DJ, Taylor JE. (2009) Identification of weed reservoirs of Tomato yellow leaf
curl virus in Florida. Tomato Research Report for, 43
Qiu BL, Coats SA, Ren SX, Idris AM, Caixia XU, Brown JK (2007) Phylogenetic relationships of
native and introduced Bemisia tabaci (Homoptera: Aleyrodidae) from China and India based on
mtCO1 sequencing and host plant comparisons. Prog Nat Sci-Mater 17:645–654
Rai AB, Halder J, Kodandaram MH (2014) Emerging insect pest problems in vegetable crops and
their management in India: an appraisal. Pest Manag Hortic Ecosyst 20:113–122
Rajinimala N, Rabindran R, Ramiah M, Kamlakhan A (2005) Virus vector relationship of bitter
gourd yellow mosaic virus and whitefly Bemisia tabaci. Acta Phytopathol Acad Sci Hung
40:23–30
Ranjith AM, Pillay VS, Sasikumaran S, Mammooty KP (1992) New record of whitefly (Bemisia
tabaci) on black pepper. Indian J Agric Sci 62:166–168
Rekha AR, Maruthi MN, Muniyappa V, Colvin J (2005) Occurrence of three genotypic clusters of
Bemisia tabaci (Gennadius) and the rapid spread of the B-biotype in South India. Entomol Exp
Appl 117:221–233
Roopa HK, Asokan R, Rebijith KB, Hande RH, Mahmood R, Kumar NK et al (2015) Prevalence of
a new genetic group, MEAM-K, of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in
Karnataka, India, as evident from mtCOI sequences. Fla Entomol 98:1062–1071
Skaljac M, Zanic K, Hrncic S, Radonjic S, Perovic T, Ghanim M (2013) Diversity and localization
of bacterial symbionts in three whitefly species (Hemiptera: Aleyrodidae) from the east coast of
the Adriatic Sea. Bull Entomol Res 103:48–59
Sloan DB, Moran NA (2012) Endosymbiotic bacteria as a source of carotenoids in whiteflies. Biol
Lett 8:986–989
Thao ML, Baumann L, Baumann P (2004) Organization of the mitochondrial genomes of
whiteflies, aphids, and psyllids (Hemiptera, Sternorrhyncha). BMC Evol Biol 4(1):25
Wan FH, Zhang GF, Liu SS, Luo C, Chu D et al (2009) Invasive mechanism and management
strategy of Bemisia tabaci (Gennadius) biotype B: Progress Report of 973 Program on Invasive
Alien Species in China. Sci China C Life Sci 52:88–95
Wang P, Ruan YM, Liu SS (2010) Crossing experiments and behavioral observations reveal
reproductive incompatibility among three putative species of the whitefly Bemisia tabaci. Insect
Sci 17(6):508–516
Wang Y, Gaba V, Yang J, Palukaitis P, Gal-On A (2002) Characterization of synergy between
Cucumber mosaic virus and potyviruses in cucurbit hosts. Phytopathology 92:51–58
Wintermantel WM, Hladky LL (2013) Genome characterization of Tomato necrotic dwarf virus, a
Torradovirus from southern California. Phytopathology 103:S160
Zhang GF, Lü ZC, Wan FH (2007) Detection of Bemisia tabaci (Homoptera: Aleyrodidae) remains
in predator guts using a sequence-characterized amplified region marker. Entomol Exp Appl
123:81–90
Recent Advances in Management
of Bacterial Diseases of Crops 9
M. R. Ravikumar, H. S. Mahesha, J. U. Vinay, and K. Dinesh
Abstract
The number of diseases caused by bacterial plant pathogens was less as compared
to fungi, but the loss caused by these diseases is huge. In the recent past, some of
the bacterial diseases which had minor importance earlier were major constraints
in crop production due to changing climate, which led to lesser productivity. For
the management of bacterial diseases, farmers solely rely on chemicals,
i.e. antibiotics and antibacterial chemicals. Many of the antimicrobial agents
currently available for agricultural use are highly toxic, non-biodegradable and
responsible for causing chronic contamination of the ecosystem. In addition, an
increasing number of phytopathogens develop resistance against antibiotics and
even residual problems in food products. To address this problems, there is a need
of novel technologies in plant protection, which includes nanotechnology,
chitosan as defence elicitor, CRISPR/CAS, transgenic crops, bacteriophages
and endophytes.
Keywords
Bacterial diseases · Management · Nanotechnology · Elicitors · Transgenics ·
Antagonists
M. R. Ravikumar (*)
College of Agriculture, Hanumanamatti, UAS, Dharwad, Karnataka, India
H. S. Mahesha
Crop Improvement Division, ICAR-IGFRI, Jhansi, Uttar Pradesh, India
J. U. Vinay · K. Dinesh
Department of Plant Pathology, UAS, Dharwad, Karnataka, India

https://doi.org/10.1007/978-981-15-6275-4_9
198 M. R. Ravikumar et al.
9.1 Introduction
India had a population of 1.21 billion in 2011 and projected population of 1.5 billion
during 2030. The population had been growing at a rate of 1.76% per annum in the
decade of 2001 to 2011. The projected estimate of the total food grain demand is
311 Mt. comprising 122 Mt. of rice, 115 Mt. of wheat, 47 Mt. of coarse grains and
27 Mt. of pulses by year 2030 (Kumar 2016). Every year there will be an increasing
demand for food due to the growing population of the world. But the natural
resources that are available for agriculture are limited: these include water, agricul-
tural land, arable soil, biodiversity, availability of non-renewable energy, human
labour and fertilizers (Smil 2001). It is possible to cut the gap between demand and
supply of food grain by adopting good crop protection practices. Crop protection in
general and management of plant diseases in particular, play an important role in
meeting the future demand with respect to both quality and quantity of food (Strange
and Scott 2005). This goal became more challenging because of crop yield losses to
various bacterial and fungal diseases, which accounts to about 15% (Oerke and
Dehne 2004).
In case of different plant pathogenic groups, fungi and fungi-like organisms stand
first in number as well as monitory loss, followed by bacteria and viruses. Bacteria
are very small, simple, unicellular microorganisms. Although considered structurally
simple, bacteria are extremely diverse from a metabolic standpoint and are found
almost everywhere on earth and their biological properties and predominant repro-
duction by binary fission relate them in prokaryotes. Plant-associated bacteria may
be beneficial or detrimental. Although many bacteria are strictly saprophytes and
they are very beneficial to man because of their necessarily required activities to
human beings includes digestion in animals, nitrogen fixing ability in roots of certain
legume crops, the decomposition of plant remains and animal carcasses, and sewage
disposal systems.
However, several bacteria are responsible for causing severe fatal diseases in
humans, animals and plants. Besides these, some bacterial species which generally
live in and around the crop plants in which they incite various diseases of economic
significance are known as plant pathogenic bacteria. In general, a rod-shaped
bacterium mostly infects various plants (Mansfield et al. 2012). Bacterial diseases
are more prevalent in the subtropical and tropical regions of the world (Ashbolt
2004). The first bacterial disease ever discovered was anthrax (caused by Bacillus
anthracis) in cattle and sheep in the year 1876. This discovery was immediately
followed by the discovery of fire blight of pear and apple (causal agent is Erwinia
amylovora) by T. J. Burrill from the University of Illinois (1877–1885). More than
200 species of phytopathogenic bacteria have been identified so far and almost all of
them are parasites within the plant either in soil or on the surface of plants. The
survey conducted by Mansfield et al. (2012) reveals the top 10 bacterial pathogens
based on their economic/scientific importance including, in rank order, (1) Pseudo-
monas syringae pathovars, (2) Ralstonia solanacearum, (3) Agrobacterium
tumefaciens, (4) Xanthomonas oryzae pv. oryzae, (5) Xanthomonas campestris
pathovars, (6) Xanthomonas axonopodis pathovars, (7) Erwinia amylovora,
9 Recent Advances in Management of Bacterial Diseases of Crops 199
(8) Xylella fastidiosa, (9) Dickeya (dadantii and solani) and (10) Pectobacterium
carotovorum (and Pectobacterium atrosepticum).
The important bacterial diseases in the southern part of the Indian subcontinent
include bacterial blight of pomegranate (Xanthomonas axonopodis pv. punicae),
bacterial wilt of ginger, tomato, chilli and eggplant (Ralstonia solanacearum), black
rot of cabbage (X. campestris pv. campestris), tip over of banana (Erwinia
carotovora subsp. carotovora), bacterial leaf spot of betel vine (X. campestris
pv. betlicola), bacterial leaf spot of grapes (Xanthomonas sp.), citrus canker
(X. axonopodis pv. citri) and bacterial spot of tomato (X. campestris
pv. vesicatoria). To address the above problems for the efficient management, new
approaches need to be followed.
9.2 Recent Approaches for Management of Bacterial Diseases

of Plants
9.2.1 Biocontrol and Endophytes for Management of Bacterial

Plant Disease
Soil microorganisms coexist in association with plant roots and also interfere with
plant physiological functions and other associated microbial community in the soil.
It is estimated that bacteria occupy 7% to 15% of the total root surface area. Of these,
some bacteria positively affect plants and have been designated as plant growth-
promoting rhizobacteria (PGPR) (Kloepper 1978). In vivo biocontrol agent selection
is not a simple task due to the diversity of agents and interactions with the host plant,
and therefore, efficient search methods are required. In recent years, biocontrol of
phytopathogenic organisms has been considered as one of the major and potential
control strategies. The use of biocontrol strategies offers several advantages over the
chemical control, since it is economical, self-perpetuating and usually free from
residual side effects. However, in reality, it will not immediately nor totally replace
chemicals, but the use of biocontrol agents can significantly enhance quality of life,
the environment and agricultural productivity.
Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae is one of
the destructive diseases in rice. Phenazine antibiotic produced by some fluorescent
pseudomonads has excellent antibacterial activity against X. oryzae pv. oryzae.
Different Bacillus spp. applied to rice plants as seed treatment before sowing; a
root dip prior to transplantation and two foliar sprays prior to inoculation could
suppress 59% of bacterial leaf blight (Vasudevan et al. 2002). Bacterial wilt caused
by Ralstonia solanacearum is one of the most important bacterial diseases of plants
of commercial value in the tropics, subtropics and regions with warm temperature
around the world. Biocontrol of R. solanacearum is mostly based on antagonism
(antibiosis) activity. Solanaceous crops other than potato and other vegetatively
propagated crops were protected biologically from bacterial wilt by dipping the
root system of seedlings before transplanting. Pseudomonas putida strain has been
developed for management of tobacco and tomato bacterial wilt. Other method to
deliver bacterial antagonist is by seed treatment, either by seed dressing, seed coating
or seed pelleting. Treatment of tomato seed with water suspension of P. putida strain
Pf-20 suppressed bacterial wilt into some degree (Asrul et al. 2004). Crown gall,
caused by Agrobacterium tumefaciens, is distributed worldwide and is responsible
for nursery and field losses among the large variety of plants especially stone fruit
trees. The method of control is by inoculation of planting material with
non-pathogenic A. radiobacter strain K84 immediately before sowing or planting.
For over 15 years, crown gall on many different host plants has been successfully
controlled by K84 in many countries. The inhibition of the pathogen by Agrocin 84
(a bacteriocin produced by K84), is mainly due to competition for site and certain
nutrient. biological site competition and competition of a certain nutrient that
common these bacteria.
Endophytes occur almost everywhere in various parts of the world with
diversified climatic conditions, and their association with algae, bryophytes,
pteridophytes, gymnosperms and angiosperms has been studied in detail. Informa-
tion compiled from more than three decades of research on endophytic fungi could
reveal that 347 host plants belonging to 119 plant families have been screened out of
17,527 angiosperms and 67 gymnosperm species reported from India (Karthikeyan
et al. 2005). Pre-treatment of cotton seedlings with B. bassiana also resulted in
reduced severity of bacterial blight disease caused by Xanthomonas axonopodis
pv. malvacearum (Griffin et al. 2006; Ownley et al. 2008). Machavariani et al.
(2014) reported on endophytic isolates from the medicinal plants Aloe arborescens,
Mentha arvensis, Lysimachia nummularia, Fragaria vesca and Arctium lappa. The
endophytic isolates were identified as Nocardiopsis, Streptomyces and
Micromonospora. The assay was done by the well diffusion method. The isolates
showed activity against different strains of bacteria such as S. aureus strain FDA
209P, S. aureus 209P/UF-2, Micrococcus luteus strain 9341, B. subtilis strain 6633
and E. coli strain 25922. Some examples of endophytes against phytopathogenic
bacteria are presented in tabular form.
9.2.2 Bacteriophages
Bacteriophages (phages) are obligate intracellular parasites that multiply inside

bacteria by using some or all of the host biosynthetic machinery. Phages specific
to X. oryzae pv. oryzae are found in the water of rice field, irrigation canal and rivers.
The population density of bacteriophage is correlated with the number of its bacterial
host. However, the problem in using of bacteriophage in the biocontrol of this
pathogen was its inactivation by UV light, variable bacterial sensitivity and the
rapid development of resistance to the phage (Mayer 2007). Svircev et al. (2019)
reported development of antibiotic resistance, popularity of organic fruit production
and the consumers’ desire for pesticide-free food has reinvigorated interest in
biological control of Erwinia amylovora. Application of bacteriophages as
biologicals exploits the ability of lytic phages to kill the pathogen. The results of
past field trials were variable from failed trials to 50–65% efficacy. Tomato bacterial
wilt caused by Ralstonia solanacearum is difficult to control. The phage that had
widest spectra against several isolates of R. solanacearum was then chosen for the
next experiment. A bacteriophage from a region in Central Java showed the wide
host range compared with the others. Clear phages were produced in the lawn of
R. solanacearum in CPG medium (Arwiyanto et al. 2019).
9.2.3 Nanotechnology
Nanotechnology is leading for the development of new concepts and agricultural

products with immense potential to manage the aforementioned problems. Nano-
technology has substantially advanced in medicine and pharmacology but has
received comparatively less interest among agricultural applications. The application
of nanotechnology in agriculture is currently being explored in plant hormone
delivery, seed germination, water management, transfer of target genes,
nanobarcoding, nanosensors and controlled release of agrichemicals. As agricultural
nanotechnology develops, the potential to provide a new generation of pesticides
and other active compounds for plant disease management will greatly increase.
Prabhu et al. (2015) have reported the antibacterial activity of iron oxide
nanoparticles against Xanthomonas sp. and Proteus vulgaris at 50 mg/ml.
Chikkanaswamy (2018) proved the antibacterial activities of green synthesized
mango-based copper nanoparticles (CuNPs) against X. axonopodis pv. citri (citrus
canker) under glasshouse condition. Vinay et al. (2018) proved the antibacterial
activity of green synthesized Pseudomonas fluorescens-based ZnNPs against
Xanthomonas oryzae pv. oryzae and X. axonopodis pv. punicae at 500–1250 ppm.
Poovizhi and Krishnaveni (2015) had reported the antibacterial activity of zinc oxide
nanoparticles against Xanthomonas axonopodis pv. citri. Ocsoy et al. (2013) studied
the effect of DNA-directed silver (Ag) nanoparticles against bacterial spot caused by
Xanthomonas perforans in tomato; most of the X. perforans strains are copper
resistant. These Ag-dsDNA-GO composites had effectively decreased the cell via-
bility of X. perforans in culture and on plants. At a very low concentration (16 ppm)
of Ag-dsDNA-GO, composites had shown excellent antibacterial activity in culture
with significant advantages in improved stability, stronger adsorption properties and
enhanced antibacterial capability. Application of AG-dsDNA-GO at 100 ppm on
tomato seedlings in an experiment conducted in greenhouse significantly reduced the
severity of bacterial spot disease; there was no phytotoxicity observed. Silver
nanoparticles synthesized from cotton stem showed antibacterial activity against
X. axonopodis pv. malvacearum and X. campestris pv. campestris. Furthermore,
AgNPs exhibited strong antioxidant activity and no phytotoxicity on cowpea.
Overall, the findings suggest that cotton stem extract could be efficiently used in
the synthesis of AgNPs and showed antimicrobial activity against plant pathogenic
microbes (Vanti et al., 2019). Vinay and Narguad (2019) reported an excellent
antibacterial activity of green synthesized water-soluble chitosan-based iron
nanoparticles (Ch-FeNPs) against Xanthomonas oryzae pv. oryzae and
X. axonopodis pv. punicae at 500–1250 ppm. They noticed bacterial growth inhibi-
tion due to treatment of iron nanoparticles.
Bryaskova et al. (2011) studied the effect of three different bacteria (Staphylo-
coccus aureus, E. coli and P. aeruginosa) in order to study the antibacterial potential
of synthesized silver nanoparticles. Concentration, physiology, metabolism, intra-
cellular selective permeability of membranes and the type of microbial cell are the
different factors for the basis of antimicrobial activity of the nanoparticles. The
significant antibacterial activity was observed in ZnO NPs against Staphylococcus
aureus, Streptococcus pyogenes, Bacillus cereus, Pseudomonas aeruginosa, Pro-
teus mirabilis and Escherichia coli. The synthesized ZnO NPs have shown
antibacterial efficacy against both Gram-positive and Gram-negative pathogens
(Gupta et al., 2018). Synergistic effects of ZnO NPs and streptomycin showed
increased efficacy as indicated by the increased zone of clearance in comparison to
their individual effects (either ZnO NPs or streptomycin). Nanoparticles synthesized
from titanium dioxide (TiO2) induced photocatalysis, resulting in antimicrobial
effects against the bacterial spot pathogen Xanthomonas perforans (Paret et al.
2013). Interestingly, doping the TiO2 NPs with Ag and Zn significantly increased
the photocatalytic activity against X. perforans. Treatment of X. perforans-infected
tomato plants with TiO2/Zn NPs at approximately 500–800 ppm significantly
reduced bacterial spot severity compared with untreated and copper controls (Paret
et al. 2013). ZnO and Ag NPs also exhibited promising antimicrobial activity against
E. amylovora with minimum inhibitory concentrations. Some NPs can exert antimi-
crobial activity through the release of ions, such as Ag+, Zn2+ and Cu2+, which are
toxic to bacteria. The release of Ag+ greatly contributed for such activity of Ag NPs
(Lok et al. 2007).
9.2.4 CRISPR/Cas9- Novel Tool for Management of Plant Bacterial

Diseases
CRISPR (Clustered Regular Interspaced Palindromic Repeats)/Cas9 (CRISPR-

associated protein) is a recent breakthrough in gene editing technology. It can be
utilized to exploit defensive mechanism in plants against pathogen attack with
recognition and degradation of the invading pathogenic genes by bacterial immune
system. CRISPR/Cas9-mediated genetically engineered resistance can be inherited
to further generation of crops after segregation of Cas9/sgRNA transgene in F1
generation. CRISPR/Cas9 proves itself as a fascinating tool to revolutionize plant
breeding for developing various disease-resistant cultivars (Ghimire 2017). Phyto-
pathogenic bacteria are difficult to control, mainly because of undetected latent
infections and also the lack of suitable agrochemicals. Generally speaking, bacterio-
logical plant control is based on prevention and exclusion of the pathogen by using
genetic resistance, agronomic practices and biocontrol agents (Kerr 2016). CRISPR/
Cas9 mutagenesis of OsSWEET13 has been performed in rice to achieve resistance
to bacterial leaf blight (BLB) disease caused by γ-proteobacterium Xanthomonas
oryzae pv. oryzae (Zhou et al. 2015). OsSWEET13 is a susceptibility (S) gene
Table 9.1 Effective endophytes against bacterial plant pathogens

Crop Fungal endophytes Targeted pest/pathogen
Sugarcane Epicoccum nigrum Xanthomonas albilineans
Eggplant Pseudomonas fluorescens Ralstonia solanacearum
encoding a sucrose transporter involved in plant-pathogen interaction. X. oryzae

produces an effector protein, PthXo2, which induces OsSWEET13 expression in the
host and the consequent condition of susceptibility. In a previous work concerning
OsSWEET14 promoter mutagenesis adopting a TALEN approach, the disruption of
this gene rendered the X. oryzae effector unable to bind OsSWEET14 and ultimately
resulted in disease resistance (Li et al. 2012). Zhou et al. (2015) obtained a null
mutation in OsSWEET13 in order to better explore PthXo2-dependent disease
susceptibility, and resultant mutants were resistant to bacterial blight.
Two recent research works have reported the employment of CRISPR/Cas9 with
the aim of producing citrus plants resistant to citrus bacterial canker. Citrus canker is
caused by Xanthomonas citri subsp. citri (Xcc). Jia et al. (2016) generated canker
resistant mutants by editing the PthA4 effector binding elements in the promoter
region of the Lateral Organ Boundaries 1 (CsLOB1) gene in Duncan grapefruit.
Mutated lines showed a decrease in typical canker symptoms 4 days post inoculation
with Xcc, and no further phenotypic alterations were detectable. Furthermore, no
potential off-target mutations in other LOB family genes were found by PCR
sequencing. Peng et al. (2017) confirmed the link between CsLOB1 promoter
activity and citrus canker disease susceptibility in Wanjincheng orange (Citrus
sinensis Osbeck). The complete deletion of the EBEPthA4 sequence from both
CsLOB1 alleles induced resistance enhancement to citrus canker (Table 9.1, 9.2
and 9.3).
9.2.5 Antimicrobial Peptides: Emerging Candidates for Plant

Protection
Antimicrobial peptides (AMPs) are the short polymers of amino acids with peptide
bond having 50 amino acids or short polymers of amino acids having
broad-spectrum antimicrobial activity against bacteria/fungi/viruses/nematodes.
Antimicrobial peptides (AMPs) are also called as host defence peptides (HDPs)
and cell-penetrating peptides (CPPs). These are isolated from many organisms,
namely, insects, amphibians, humans, microorganisms and plants. AMPs are part
of the nonspecific host defence system and are active against different types of
microorganisms including phytopathogens.
Microscopic analysis revealed wide-scale damage to the microorganism’s mem-
brane, in addition to inhibition of pathogen growth. In planta potent antibacterial
activity was demonstrated. Treatment with the lipopeptides of Arabidopsis leaves
infected with Pseudomonas syringae efficiently and rapidly reduced the number of
bacteria with no toxicity on the plant tissues (Makovitzki et al. 2007). The ultrashort
204
Table 9.2 CRISPR/Cas9 applications for bacterial disease resistance

Plant Target
species Targeted pathogen gene Gene function Strategy References
Oryza Bacterial blight SWEET13 Sucrose transporter gene Agrobacterium-mediated transformation of Li et al. (2012)
sativa (Xanthomonas oryzae embryogenic callus with Cas9/gRNA and Zhou et al.
pv. oryzae) expression plasmid (2015)
vectors and TALEN
Citrus Citrus canker LOB1 Susceptibility (S) gene promoting Agrobacterium-mediated Jia et al. (2016)
paradisi (Xanthomonas citri pathogen growth and pustule transformation of epicotyl with Cas9/gRNA
subsp. citri) formation expression plasmid vectors
Citrus Citrus canker LOB1 Susceptibility (S) Agrobacterium-mediated transformation of Peng et al.
sinensis (Xanthomonas citri gene promoting pathogen growth epicotyl with Cas9/gRNA expression (2017)
Osbeck subsp. citri) and pustule plasmid vectors
formation
Malus Fire blight (Erwinia DIPM-1 Susceptibility factor involved in PEG-mediated protoplast transformation Malnoy et al.
domestica amylovora) DIPM- fire blight disease with CRISPR (2016)
2 DIPM-4 ribonucleoproteins
M. R. Ravikumar et al.
Table 9.3 The effective concentration of native chitosan or its derivatives against bacterial plant
pathogens (Rabea and Steurbaut 2010; Badawy et al. 2014)
Effective concentration
Plant pathogenic bacteria Chitosan/its derivative (ppm)
Agrobacterium tumefaciens N-(o,o-dichlorobenzyl) 500
chitosan
Agrobacterium tumefaciens Quaternary N-(benzyl) 500
chitosan
Agrobacterium tumefaciens N-(benzyl) chitosan 800
Xanthomonas campestris Chitosan 500
Erwinia carotovora Chitosan 200
Erwinia carotovora N-(o,o-dichlorobenzyl) 480
chitosan
Erwinia carotovora Quaternary N-(benzyl) 600
chitosan
Erwinia carotovora N-(benzyl) chitosan 700
Erwinia carotovora subsp. carotovora Chitosan 5000
Clavibacter michiganensis subsp. Chitosan 1000
michiganensis
Erwinia carotovora N-(α-methylcinnamyl) 1025
chitosan
(Xing et al. 2015)
lipopeptides could serve as native-like antimicrobial agents economically feasible

for use in plant protection. The synthetic LFchimera showed potential antibacterial
activities against phytopathogenic bacteria such as Ralstonia solanacearum,
Xanthomonas campestris, Erwinia amylovora, Pseudomonas syringae and
Pectobacterium carotovorum. LFchimera was effective against bacterial strain in a
dose-dependent manner (Chahardoli et al. 2017) and can improve the potential of an
antimicrobial peptide in plant disease management. The synthetic antimicrobial
peptide, i.e. Shiva-1, was isolated from the silk moth and introduced in transgenic
tobacco (Jaynes et al. 1993). Lactoferrin is another iron-binding glycoprotein known
to have antibacterial properties. The expression of a human lactoferrin gene in
tobacco delayed the onset of symptoms caused by R. solanacearum from 5 to
25 days. This resistance appears to be due to the truncation of lactoferrin, resulting
in a smaller peptide with strong antibacterial activity (Mitra and Zhang 1994).
9.2.6 Transgenic Approaches
Plant pathogens can cause significant reduction in crop yield. Due to infection of
many invasive pathogens, there is a possible threat to wipe out plant species.
Hence, plant pathologists and biotechnologists trying their best to develop
pathogen-resistant plants against some diseases caused by bacteria of economic
importance (Wani et al. 2010). Many molecular approaches have been proposed to
enhance plant resistance to bacterial pathogens like P. syringae; these strategies
include the use of antibacterial proteins from different insect vectors and their
transformation in plants for development of disease resistance (Huang et al. 1997)
and inactivation of virulence factors resulted the immunity of plants against the
relevant bacterial species (Anzai et al. 1989). The resistance nonbacterial genes can
also be introduced by transgenic approaches for broad-spectrum resistance against
the devastating pathogens.
Recently, the utilization of RNAi has emerged as an important tool to counter the
bacterial genome at transcriptional and post-transcriptional level. Small interference
RNA has been proved effective against the crown gall disease in Arabidopsis,
Nicotiana and Lycopersicum species caused by Agrobacterium tumefaciens by
transformation of inverted repeats of this pathogen genes ipt and iaaM to encode
precursors of biosynthesis for two important phytochromes auxin and cytokinins
(Escobar et al. 2001). Phenolic compounds (a group of secondary metabolites) are
widely distributed in plants and have shown to possess antimicrobial properties. The
anti-Xylella activity of 12 phenolic compounds, representing phenolic acid, couma-
rin, stilbene and flavonoid, was evaluated using an in vitro agar dilution assay.
Overall, these phenolic compounds were effective in inhibiting X. fastidiosa growth,
as indicated by low minimum inhibitory concentrations (MICs). In addition, pheno-
lic compounds with different structural features exhibited different anti-Xylella
capacities. Particularly, catechol, caffeic acid and resveratrol showed strong anti-
Xylella activities. Differential response to phenolic compounds was observed among
X. fastidiosa strains isolated from grape and almond. Elucidation of secondary
metabolite-based host resistance to X. fastidiosa will have a broad implication in
combating X. fastidiosa-caused plant diseases. It will facilitate future production of
plants with improved disease resistance properties through genetic engineering or
traditional breeding approaches and will significantly improve crop yield (Maddox
et al. 2010).
9.2.7 Organic Elicitor: Chitosan in Disease Management
Chitosan is a deacetylated derivative of chitin that is naturally present in the insect

body wall, in fungal cell wall and in crustacean shells from which it can be easily
extracted. Chitosan has been reported to possess antimicrobial activity. Chitosan also
behaves as a resistance elicitor inducing both local and systemic plant defence
responses when applied to plants directly (Orzali et al. 2017). Chitosan has been
emerging as one of the most promising polymers for the efficient delivery of
agrochemicals and micronutrients in nanoparticles (Kashyap et al. 2015). Chitosan
and its derivatives inhibited the growth of a wide range of bacterial plant pathogens
(Liu et al. 2001 (Fei Liu) Wisniewska-Wrona et al. 2007). Based on the available
evidences, bacteria appear to be generally less sensitive to the antimicrobial action of
chitosan than fungi. Gram-negative bacteria are more susceptible to chitosan (Park
et al. 2004; Du et al. 2009). They suggested that hydrophilicity in Gram-negative
bacteria is significantly higher than that in Gram-positive bacteria, which makes
them more sensitive to chitosan (Chung et al. 2004). Moreover, the Gram-negative
cell envelope contains an additional outer membrane composed by phospholipids

and lipopolysaccharides, which face the external environment. The highly charged
nature of lipopolysaccharides will confer an overall charge of negative to the cell
wall of the Gram-negative bacteria. Therefore, Gram-negative bacteria with high
electronegative charge will interact more effectively with the polycationic chitosan
compared with Gram-positive bacteria. Chitosan was evaluated against several
bacterial pathogens and effective concentration for suppression of growth of bacteria
is presented in tabular form hereunder.
The in vitro antibacterial effect of chitosan and its ability in protection of
watermelon seedlings from Acidovorax citrulli were evaluated. The disease index
of watermelon seedlings planted in soil and the death rate of seedlings planted in
perlite were significantly reduced by chitosan at 0.40 mg/mL compared with the
control (pathogen) (Li et al. 2013). Chitosan solution at 0.10 mg/mL inhibited the
growth of Xanthomonas pathogenic bacteria from different geographical origins.
The surviving cell numbers in the chitosan solution decreased more than 3.86
log10CFU/mL compared with the control after 6 h of incubation regardless of the
bacterial strain (Li et al. 2008). Pre-treatment of tomato seedlings with 10 μg/mL
chitosan before Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) inocula-
tion significantly decreased bacterial damages in cotyledons compared with control.
Not only does chitosan inhibit bacterial cell growth but also it affects the already
established biofilms (Mansilla et al. 2013). The polysaccharide chitosans represent a
renewable source of natural biodegradable polymers and meet with the emergence of
more and more food safe problems. A wider comprehensive knowledge of the
mechanism of action of chitosan in pathogens and plants will increase the chance
of its successful application to control disease spread in plants.
References
Anzai H, Yoneyama K, Yamaguchi I (1989) Transgenic tobacco resistant to a bacterial disease by
the detoxification of a pathogenic toxin. Mol Gen Genet 219:492–494
Arwiyanto T, Aryani WT, Gusti E, Restina D (2019) Control of tomato bacterial wilt (Ralstonia
solanacearum) by grafting and bacteriophage application. 4th international symposium on
biological control of bacterial plant diseases. J Pl Pathol Biocontrol, July 9-11, Viterbo, Italy
Ashbolt NJ (2004) Microbial contamination of drinking water and disease outcomes in developing
regions. Toxicology 198:229–238
Asrul TA (2004) The Influence of Tomato Seeds Treatment with Pseudomonas putida Pf-20 against
Bacterial Wilt Disease. Agrosains, 17
Badawy ME, Rabea EI, Taktak NE (2014) Antimicrobial and inhibitory enzyme activity of
N-(benzyl) and quaternary N-(benzyl) chitosan derivatives on plant pathogens. Carbohydr
Polym 111:670–682
Bryaskova R, Pencheva D, Nikolov S, Kantardjiev T (2011) Synthesis and comparative study on
the antimicrobial activity of hybrid materials based on silver nanoparticles (AgNps) stabilized
by polyvinylpyrrolidone (PVP). J Chem Biol 4:185
Chahardoli M, Fazeli A, Ghabooli M (2017) Antimicrobial activity of LFchimera synthetic peptide
against plant pathogenic bacteria. Arch Phytopathol Plant Protec 50:1008–1018
Chikkannaswamy (2018) Synthesis, characterization and evaluation of copper and sulphur nano-
particle against major plant pathogens, Ph. D. Thesis, Uni. Agri, Sci., Dharwad
Chung YC, Su YP, Chen CC, Jia G, Wang HL, Wu JG, Lin JG (2004) Relationship between
antibacterial activity of chitosan and surface characteristics of cell wall. Acta Pharma
25:932–936
Du WL, Niu SS, Xu YL, Xu ZR, Fan CL (2009) Antibacterial activity of chitosan tripolyphosphate
nanoparticles loaded with various metal ions. Carbohydr Polym 75:385–389
Escobar MA, Civerolo EL, Summerfelt KR, Dandekar AM (2001) RNAi-mediated oncogene
silencing confers resistance to crown gall tumorigenesis. Proc Natl Acad Sci 98:13437–13442
Fei LX, Lin GY, Zhi YD, Li Z, De YK (2001) Antibacterial action of chitosan and
carboxymethylated chitosan. J Appl Polym Sci 79:1324–1335
Ghimire B (2017) Use of Crispr/Cas9 for development of disease resistant cultivars in plant
breeding. Int J Appl Sci Biotechnol 5:403–409
Griffin MR, Ownley BH, Klingeman WE, Pereira RM (2006) Evidence of induced systemic
resistance with Beauveria bassiana against Xanthomonas in cotton. Phytopathology 96
Gupta M, Tomar RS, Kaushik S, Mishra RK, Sharma D (2018) Effective antimicrobial activity of
green ZnO nano particles of Catharanthus roseus. Front Microbial 9:2030
Huang Y, Nordeen RO, Di M, Owens LD, McBeath JH (1997) Expression of an engineered
cecropin gene cassette in transgenic tobacco plants confers disease resistance to Pseudomonas
syringae pv. tabaci. Phytopathology 87:494–499
Jaynes JM, Nagpala P, Destéfano-Beltrán L, Huang JH, Kim J, Denny T, Cetiner S (1993)
Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced
resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Sci 89:43–53
Jia H, Orbovic V, Jones JB, Wang N (2016) Modification of the PthA4 effector binding elements in
Type I Cs LOB 1 promoter using Cas9/sg RNA to produce transgenic Duncan grapefruit
alleviating XccΔpthA4: dCs LOB 1.3 infection. Plant Biotechnol J 14:1291–1301
Karthikeyan M, Ramanujam B, Krishnan R, Subramanian M, Velusamy J, Rajendran S,
Rethinasamy V (2005) Endophytic Pseudomonas fluorescens Endo 2 and Endo 35 induce
resistance in black gram (Vigna mungo L. Hepper) to the pathogen Macrophomina phaseolina.
J Plant Interact 1:135–143
Kashyap PL, Xiang X, Heiden P (2015) Chitosan nanoparticle based delivery systems for sustain-
able agriculture. Int J Biol Macromol 77:36–51
Kerr A (2016) Biological control of crown gall. Australas Plant Pathol 45:15–18
Kloepper JW (1978) Plant growth-promoting rhizobacteria on radishes. In Proc. of the 4th Internet.
Conf. on Plant Pathogenic Bacter, Station de Pathologie Vegetaleet Phytobacteriologie, INRA,
Angers, France, 1978. Vol. 2, pp. 879–882
Kumar P (2016) Demand vs supply of food in India-futuristic projection. Proc Indian Natl Sci Acad
82:1579–1586
Li B, Wang X, Chen R, Huangfu W, Xie G (2008) Antibacterial activity of chitosan solution against
Xanthomonas pathogenic bacteria isolated from Euphorbia pulcherrima. Carbohydr Polym
72:287–292
Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012) High-efficiency TALEN-based gene editing
produces disease-resistant rice. Nat Biotechnol 30:390
Li T, Huang S, Zhou J, Yang B (2013) Designer TAL effectors induce disease susceptibility and
resistance to Xanthomonas oryzae pv. oryzae in rice. Mol Plant 6:781–789
Lok CN, Ho CM, Chen R, He QY, Yu WY, Sun H, Che CM (2007) Silver nanoparticles: partial
oxidation and antibacterial activities. J Biol Inorg Chem 12:527–534
Machavariani NG, Ivankova TD, Sineva ON, Terekhova LP (2014) Isolation of endophytic
actinomycetes from medicinal plants of the Moscow region, Russia. World Appl Sci J
30:1599–1604
Maddox CE, Laur M, Tian L (2010) Antibacterial activity of phenolic compounds against the
phytopathogen Xylella fastidiosa. Curr Microbiol 60:53
Makovitzki A, Viterbo A, Brotman Y, Chet I, Shai Y (2007) Inhibition of fungal and bacterial plant
pathogens in vitro and in planta with ultrashort cationic lipopeptides. Appl Environ Microbiol
73:6629–6636
Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS et al (2016) DNA free genetically edited
grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7:1904
Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Toth IAN (2012) Top
10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629
Mansilla AY, Albertengo L, Rodríguez MS, Debbaudt A, Zúñiga A, Casalongué CA (2013)
Evidence on antimicrobial properties and mode of action of a chitosan obtained from crustacean
exoskeletons on Pseudomonas syringae pv. tomato DC3000. Appl Microbiol Biotechnol
97:6957–6966
Mayer G (2007) Bacteriology-chapter seven bacteriophage
Mitra A, Zhang Z (1994) Expression of a human lactoferrin cDNA in tobacco cells produces
antibacterial protein(s). Plant Physiol 106:977–981
Ocsoy I, Paret ML, Ocsoy MA, Kunwar S, Chen T, You M, Tan W (2013) Nanotechnology in plant
disease management: DNA-directed silver nanoparticles on graphene oxide as an antibacterial
against Xanthomonas perforans. ACS Nano 7:8972–8980
Oerke EC, Dehne HW (2004) Safeguarding production—losses in major crops and the role of crop
protection. Crop Protect 23:275–285
Orzali L, Corsi B, Forni C, Riccioni L (2017) Chitosan in agriculture: a new challenge for managing
plant disease. Biol Activ Appl Mar Polysacch:17–36
Ownley BH, Griffin MR, Klingeman WE, Gwinn KD, Moulton JK, Pereira RM (2008) Beauveria
bassiana: endophytic colonization and plant disease control. J Invert Pathol 98:267–270
Paret ML, Vallad GE, Averett DR, Jones JB, Olson SM (2013) Photocatalysis: effect of light-
activated nanoscale formulations of TiO2 on Xanthomonas perforans and control of bacterial
spot of tomato. Phytopathology 103:228–236
Park SI, Daeschel MA, Zhao Y (2004) Functional properties of antimicrobial lysozyme-chitosan
composite films. J Food Sci 69:215–221
Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Zou X (2017) Engineering canker-resistant plants
through CRISPR/Cas9-targeted editing of the susceptibility gene Cs LOB 1 promoter in citrus.
Plant Biotechnol J 15:1509–1519
Prabhu YT, Rao KV, Kumari BS, Kumar VSS, Pavani T (2015) Synthesis of Fe3O4 nanoparticles
and its antibacterial application. Int Nano Lett 5:85–92
Poovizhi J, Krishnaveni B (2015) Synthesis, characterization and antimicrobial activity of zinc
oxide nanoparticles synthesized from Calotropis procera. Int J Pharm Sci Drug Res 7:425–431
RabEa EI, StEuRbaut W (2010) Chemically modified chitosans as antimicrobial agents against
some plant pathogenic bacteria and fungi. Plant Protect Sci 46:149–158
Smil V (2001) Feeding the world: a challenge for the twenty-first century. MIT press, Cambridge,
MA
43
Svircev AM, Anany H, Wang Q, Castle AJ (2019) Successful control of fire blight: can
bacteriophages do the job? In: Fourth international symposium on biological control of bacterial
plant diseases, p 34
Vanti GL, Nargund VB, Vanarchi R, Kurjogi M, Mulla SI, Tubaki S, Patil RR (2019) Synthesis of
Gossypium hirsutum-derived silver nanoparticles and their antibacterial efficacy against plant
pathogens. Appl Organomet Chem 33:e4630
Vasudevan P, Kavitha S, Priyadarisini VB, Babujee L, Gnanamanickam SS (2002) Biological
control of rice diseases. In: Gnanamanickam (ed) Biological control of crop diseases. Marcel
Dekker Inc, New York, pp 11–32
Vinay JU, Nargund VB (2019) Green synthesis of water soluble chitosan based iron nanoparticles
and their antibacterial activity. J Farm Sci 32:466–470
Vinay JU, Nargund VB, Jahagirdhar S, Patil RR, Hegde RV (2018) Green synthesis of zinc
nanoparticles using Pseudomonas fluorescens extract and their antibacterial activity against
Xanthomonas spp. Int J Curr Microbiol App Sci 7:1280–1291
Wani SH, Sanghera GS, Singh NB (2010) Biotechnology and plant disease control-role of RNA
interference. Am J Plant Sci 1:55
Wiśniewska-Wrona M, Niekraszewic A, Ciechańska D, Pospieszny H, Orlikowski LB (2007)
Biological properties of chitosan degradation products. Polish Chitin Soc Monogr 12:149–156
Xing K, Zhu X, Peng X, Qin S (2015) Chitosan antimicrobial and eliciting properties for pest
control in agriculture: a review. Agron Sustain Dev 35:569–588
Zhou J, Peng Z, Long J, Sosso D, Liu B, Eom JS, White FF (2015) Gene targeting by the TAL
effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J 82:632–643
Resistance Breeding and Exploitation
of Wild Relatives for New Resistance 10
Sources
N. K. Singh, Anjali Joshi, Smrutishree Sahoo, and Birendra Prasad
Abstract
Increasing yield from same piece of land and resources has now become more
imperative since the share of resources is decreasing continuously due to increas-
ing population. Change in environmental parameters are confronting with plants
by changing dynamics as well as emergence of novel parasites. Resistance
breeding though has been the traditional objective of plant breeding programme.
With changing scenario, effective and diverse-resistant sources, particularly from
wild relatives and from other sources, seem to be essential for durability of the
resistance. Furthermore, precise tools are required for identification and transfer
of genes for developing resistant genotypes. This chapter includes description of
necessity of resistance breeding, types of resistance, breeding tools used in
development of resistant genotypes and wild relatives that can be used as
potential sources of resistant genes.
Keywords
Resistance sources · Wild relatives · Breeding tools · Specific race · Nonspecific
race · Biotic stress
10.1 Introduction
Every living organism takes food in one or other form to sustain, grow and
reproduce. Availability has not been an issue when production avenues were con-
siderably high than the consumption capacity of all the dependent creatures. With the
changing scenario due to anthropogenic activities, industrialization, infrastructural
N. K. Singh (*) · A. Joshi · S. Sahoo · B. Prasad

Department of Genetics and Plant Breeding, G. B. Pant University of Agriculture & Technology,
Pantnagar, Uttarakhand, India

https://doi.org/10.1007/978-981-15-6275-4_10
212 N. K. Singh et al.
development and decline in efficiency of production avenues, the emphasis has

shifted to the use of alterative and innovative approaches to minimize the gap
between production and consumption. Agriculture sector has been witness to
major changes not only in intensive use of resources but also in evolutionary changes
of diverse cropping system with dramatic shift in method and time of crop cultiva-
tion. Thus, agriculture has grown continuously and dynamically to support the
requirement of food, feed, medicines and raw materials for industries. Furthermore,
growth in agriculture is highly anticipated till population growth is stabilized. In fact
agriculture is considered to be a gamble of monsoon in majority of the developing
countries, because monsoon rainfalls influence the availability of water and success-
ful crop cultivation. Plants growth and development are affected by climatic
conditions as favourable biotic and abiotic factors ensure positive interactions
leading to optimum growth and performance of the crop. However, change in
climatic conditions exert stresses not only on plants but also on all the living
organisms and also disturb the dynamic balance between crop and different biotic
and abiotic factors. Altered environmental parameters are not only interfering with
genetic potential of crop leading to altered or abnormal growth and development but
also it affects population dynamics as well as emergence of novel race of
microorganisms that may or may not interact with different stages of plants. Plants
being sessile are exposed continuously to good or adverse environmental conditions
of varying intensity, including attack by pathogens, wounding by insects, exposure
to ultraviolet radiation, low temperature and decrease in water and nutrient avail-
ability. It is quite remarkable that in spite of ever-changing environmental
parameters, plants are able to adapt, continue to grow, develop and importantly
remain productive. To accomplish this feat, plants rely heavily on their ability to
coordinate the perception of environmental stimuli with alterations in developmental
and physiological programmes required for adaptation and survival. Plant response
to external signals is a very complex and highly coordinated process that involves
the quick perception of stress stimuli, the activation of many signalling networks and
changes in the expression of hundreds of genes and ultimately altered metabolites
that probably plant needs. In addition, it is imperative that these genes are only
transcribed in response to the right signal, at the right time, in the right place and for
the appropriate amount of time. Since the available knowledge and system is unable
to perceive and understand the language of plants, it is very difficult to know whether
plants feel pain or discomfort and also it is difficult to pin-point exactly when a plant
is in abnormal state. It is accepted that a plant is healthy or normal, when it can carry
out its physiological functions to the best of its genetic potential.
In case we noticed that the ability of the cells of a plant or plant part to carry out
one or more of essential functions is interfered with by either a pathogenic organism
or an adverse environmental factor, the activities of the cells are disrupted, altered or
inhibited, the cells malfunction or die, and the plant becomes diseased. Initially, the
distress is localized and by and large is invisible. Their action becomes more
prominent and affects many plant parts, and consequential changes are visible
morphologically. In fact, these visible changes are the symptoms of the disease.
The visible or otherwise measurable adverse changes in a plant, produced in reaction
10 Resistance Breeding and Exploitation of Wild Relatives for New Resistance. . . 213
to infection by an organism or to an unfavourable environmental factor, are a

measure of the amount of disease in the plant. Disease in plants, then, can be defined
as the series of invisible and visible responses of plant cells and tissues to a
pathogenic organism or environmental factors that result in adverse changes in the
form, function or integrity of the plant and may lead to partial impairment or death of
plant parts or of the entire plant.
Prevost proved as early as in 1807 that diseases are caused by microorganisms.
The physiological functions that are likely to be interrupted due to pathogens are
dependent on the cells and tissues that are invaded by the pathogenic organism. For
example, infection of roots may cause roots to rot and make them unable to absorb
water and nutrients from the soil; infection of xylem vessels, as happens in vascular
wilts and in some cankers, interferes with the translocation of water and minerals to
the crown of the plant; infection of the foliage, as happens in leaf spots, blights, rusts,
mildews, mosaics and so on, interferes with photosynthesis; infection of phloem
cells in the veins of leaves and in the bark of stems and shoots, as happens in cankers
and in diseases caused by viruses, mollicutes and protozoa, interferes with the
downward translocation of photosynthetic products; and infection of flowers and
fruits interferes with reproduction. Although infected cells in most diseases are
weakened or die, in some diseases, namely in case of crown gall, infected cells are
induced to divide much faster (hyperplasia) or to enlarge a great deal more (hyper-
trophy) than normal cells and to produce abnormal amorphous overgrowths
(tumours) or abnormal organs. Microorganisms that can cause disease are generally
referred to as pathogens, usually they cause disease in plants by disturbing the
metabolism of plant cells through enzymes, toxins, growth regulators, and other
substances they secrete and by absorbing foodstuffs from the host cells for their own
use. Some pathogens may also cause disease by growing and multiplying in the
xylem or phloem vessels of plants, thereby blocking the upward transportation of
water or the downward movement of sugars, respectively, through these tissues.
Environmental factors cause disease in plants when abiotic factors, such as tempera-
ture, moisture, mineral nutrients and pollutants, occur at levels above or below a
certain range tolerated by the plants. Devastating diseases caused by pathogens and
pests have threatened crop production, human health and the stability of global
economies. Management of pathogenic organism is the potential option to minimize
distortion and unusual changes in plants leading to normal production potential.
Replacing plant genes by more efficient allelic form to incorporate resistance
potential is still a more prospective option as it does not add any additional input
in growing crops, and also this option is environmentally safe. The availability of
gene (s) conferring higher level of resistance is essentially required either in
cultivated form or use of wild relatives for exploring and domesticating wild alleles,
and genes conferring resistance to pathogenic organisms is another novel avenue for
successful resistance in breeding programme.
10.2 Resistance Breeding
Plant breeding has been the most successful approach for developing new crop
varieties since domestication occurred, making possible major advances in feeding
the world and societal development. Crops are susceptible to a large set of pathogens
including fungi, bacteria and viruses, which cause significant economic losses; the
enhancement of plant resistance played an important role in adjusting crop produc-
tion to meet the requirement of global population growth. Approaches to disease
control that depend on resistant varieties and agrochemicals are usually highly
effective whenever they are deployed. Dynamic evolutionary properties of plant
pathogens, however, limit the cultivation of new varieties in localized or in larger
areas once a virulent race that counter the existing resistant gene is evolved. As such,
disease control approach based on the existing resistant gene has become ineffective.
Incorporation of novel genes and deployment of new genotypes in the areas are
therefore required as a control measure of plant diseases. In fact resistant gene-based
disease control strategies are like continuous war against pathogens in which
genotypes equipped with different weapons that are ‘resistant genes’ are deployed
strategically in time and geographical frames to manage plant diseases.
Following the ancient domestication of crop species, plant breeding occurred
only informally for thousands of years. During that time, farmers might have chosen
to save seed from the healthiest or highest-yielding plants from one generation to the
next, but they lacked the scientific knowledge of inheritance to permit deliberate
breeding for traits or understand the causes and effects of the widely used method of
mass selection. Domestication syndrome leads to development of modern crop with
high production potential and adapted to high input environments, on the one hand,
but, on the other hand, consequential domestication bottleneck witnessed the loss of
many adaptive alleles leading often susceptibility of crop to diseases, insects and
abiotic stresses. To find resistance genes, it is often necessary to go back to their wild
ancestors and close relatives. In fact this seems to be a problematic option due to the
inevitable setback in optimized yields and other agronomical parameters attained
when crossing to wild relatives, on one hand, however, on the other hand, it seems to
have great prospect for novel resistant gene (s) and resistant breeding point of view.
Pathogens are major constraints for field and horticultural crop production,
impairing both yield and quality. Breeding for disease resistance is the most efficient,
economical and environmental-friendly way of control. It is, therefore, a crucial
component of sustainable agriculture that can be performed by using several
approaches from classical breeding to genetic engineering. However, detailed under-
standing of pathogen biology, host–pathogen interaction and the efficient resistance
mechanisms at the cellular and molecular levels are required to improve the effi-
ciency of the breeding process.
The act of changing the genetic make-up of plants has been done by humans in
distant past to fulfil their immediate needs and services. The only thing that has
changed from then to now is the level of understanding we have on the subject.
However, human experimentation with plant breeding has developed many of our
modern crops even if the breeder didn’t have much knowledge on the subject. From
the very beginning, plant breeding had been a common activity. In fact, Gregor
Mendel’s work on how genes behave in terms of phenotypic appearance and how
they could be passed to offspring was the first major event to spark an interest in the
science behind plant breeding. Until the 1900s, it had been ignored. However, once
three scientists having trouble with breeding came across it, Mendel’s findings had
been publicized. It is in fact very tedious to trace the exact date that humans began
breeding plants. Yet, it is well known that R.J. Camerarius of Germany is credited
for first reporting sexual reproduction in plants in 1694. Since then, tools have been
made, new plant breeds have been developed and the general knowledge humans
have on this subject has grown greatly and become more and more scientific and
specific.
10.3 History of Resistance Breeding
Theophrastus, in the third century BC, noted that cultivated varieties differed in their
ability to avoid disease. That diseases are produced by a pathogen was conclusively
shown by Benedict Prevost, he showed that wheat bunt was produced by a fungus.
During the middle of nineteenth century, various workers noted that crop varieties
differed for disease resistance. In 1904, Blakeslee described mating type differentia-
tion in Rhizophus. In 1905, Biffen demonstrated that resistance to yellow rust in
wheat is governed by a recessive gene showing classical Mendelian segregation ratio
in F2. Breeding for disease resistance is believed to have started with the work of
Orton in 1900, who selected lines of cotton resistant to Fusarium wilt by growing
cotton on wilt sick soil. He also used hybridization to develop wilt (Fusarium
oxysporum) resistant varieties of water melon.
In 1894, Erikson showed that pathogen, although morphologically similar, dif-
fered from each other in their ability to attack different related host species. Later, in
1911, Barrus showed that different isolates of a microorganism differed in their
ability to attack different varieties of the same host species. This finding has formed
scientific basis for physiological races and or pathotypes. Later on, Johnson and
Newton (1940) established in case of black rust of wheat that the ability of a
pathogen to infect a host strain, i.e. pathogenicity, is genetically determined. Thus,
both the ability of a host to resist invasion by a pathogens as well as the ability of a
pathogen to infect a host strain, i.e. pathogenicity, is genetically determined. The
breeding for resistance to diseases and insect pests gained further momentum when
Flor (1955, 1956) proposed the gene-for-gene hypothesis which states that ‘for each
gene conditioning rust reaction in the host, there is a specific gene conditioning
pathogenicity in the parasite’. In other words, each genetic locus conditioning
resistance or susceptibility in the host has a corresponding locus in the pathogen
controlling avirulence or virulence. The gene-for-gene concept provides a useful
working model for studying host parasite systems, even when genetic information is
not fully available. Thus, gene-for-gene hypothesis added knowledge on host patho-
gen interaction and helped in planning effective resistance breeding programme. The
gene-for-gene hypothesis has thus (a) prompted the identification of new major
genes for disease resistance, (b) enabled the development of varied and usually
effective strategies for the use of major gene resistance in space and/or over time to
manage diseases, (c) provided a clear understanding of the host–parasite
interactions, the nature of gene action and co-evolution of host–parasite systems
and (d) enabled planning of breeding programme for the development of disease-
resistant varieties.
Available evidences indicate that the pathogens are more dynamic for generating
new variation in pathogenicity by a variety of reproduction methods and mutation.
The evolution of different races of the same pathogen is a continuous feature. Thus,
the resistance breeding objective should not only be to develop varieties resistant to
the prevalent pathotypes of the pathogens but also be vigilant with access of diverse
alleles for resistance to face the challenges once emerged due to evolution of the new
virulent pathotypes in future. Thus, resistance breeding requires continuous inter-
vention by various classical and molecular tools along with diverse pool of resis-
tance sources for breeding genotypes with appreciably high level of resistance along
with high yield potential.
10.4 Complexities in Breeding for Resistant Varieties
The breeding for disease resistance is supposed to be more complex and complicated
than the breeding for other traits. The complication is because of the dynamic nature
of pathogens leading to evolution of pathogenic races or biotypes that are new and
can overcome the crop resistance potential since the gene deployed in the crop is
specific to existing pathogenic race. This in fact poses an additional hurdle to
breeding programmes, once a new genotype for resistance to a specific pathogen
race may show susceptibility to other races. Therefore, when a pathogenic race is
mutated and a new race emerges, plant breeders have to initiate a new breeding effort
to search an effective allelic form of a gene and to deploy the same in the crop to
develop a resistant cultivar. In reality, this process is an endless battle against the
pathogen. Another problem is the shift of prevalent existing pathogenic races in a
region, since they may also reduce the life span of a resistant cultivar that after a few
growing seasons become susceptible. It must also be recognized that the number of
insects that causes yield reduction is large, including those that attack the crops
during the growing season, feeding on leaves, pods, fruits and roots. An additional
class of insects that causes losses by feeding on the harvested crop, like borer,
weevils among others exists and causes significant losses both in terms of quantity
and quality. Historical evidences show that biotic stresses occur, in high or low
intensity, in all agricultural areas around the world. In some areas, the stresses caused
by pests and weeds may not be relevant in a specific year, but they bounce back in
another years or seasons. Migration of insect-pests is another burden on breeding for
resistance cultivars. For example, Fall army worm, an exotic insect, entered in India
and was reported for the first time in May 2018 from a maize field located in southern
region of the country. Within a year of its appearance in India, Fall army worm
spread to most of the maize-growing regions causing severe damage. Additionally,
climate change is bringing new pests and weeds to relevance in crop production,
especially in the tropical regions. Climate changes are also affecting insect-pests to
remain active and becoming burden for sequential crop in the field bypassing the
seasonal boundary. Overall, global warming has caused and probably may cause
even larger incidence by insect-pests, diseases and weeds on farmers’ fields globally
in general, but impact seems to be more devastating on the fields of medium and
resource-poor farmers. Some biotic stresses that have been considered secondary in
the past may emerge as biotic stress of major relevance with climate change.
Breeding efforts for developing insect-pests resistant cultivars have not been as
effective as for disease resistance because of non-availability of potential and
effective gene sources in primary gene pool of the crop species. However, some
resistant cultivars have been developed over the years against many insect-pests.
Success for breeding insect-pests resistance has been remarkable by exploring and
transferring gene(s) showing resistance across the species and genera boundary with
the help of biotechnology. The contribution of plant breeding throughout history in
helping agriculture to produce food, feed, fibre and fuel is very well documented in
the scientific literature (Vencovsky and Ramalho 2006; Duvick et al. 2004). How-
ever, what will happen in the coming decades with the new challenging scenario will
demand from breeders new and more efficient strategies to manage biotic stresses to
sustain agricultural production and productivity of different crops for producing
adequate amount of food, feed, fibre, industrial raw materials, etc. Thus,
complexities in breeding for biotic stresses are more which seem to increase with
changing climatic conditions as well as cropping patterns. Search for novel genes
seems to be the foremost priority followed by efficient and precise tools to identify
and incorporate resistant genes in crops from the sources beyond the primary gene
pools.
10.5 Nature of Resistance
Plant breeding tools can be used to develop varieties that show resistance against
specific race (race-specific, qualitative, vertical resistance) or race-nonspecific resis-
tance (quantitative, race-nonspecific, horizontal resistance and field resistance) or
integration of both kind of resistance.
10.5.1 Qualitative Resistance
It is also known as a major gene resistance and is based on one or few genes with
major effect and provides race-specific, high level resistance (vertical resistance).
Qualitative resistance, often associated with rapid cell death called hypersensitive
response (HR) around the contact point of pathogen, is generally quickly overcome
when deployed in the field, though there are exceptions. In fact, race-specific
resistance is conditioned by the interaction of specific genes in the host with those in
the pathogen. The genetic principles underlying host–pathogen interaction were
elegantly established by Flor (1955) while working on rust (Melampsora lini) of
flax and elaborated that resistance or susceptibility of a cultivar is dependent on
gene/allele for resistance or susceptibility in the host and the presence of
corresponding gene/allele for virulence or avirulence in the race of pathogen. A
similar system has been shown to exist for most of the cereal crops and their rust
pathogen. The ability of the pathogen to change its racial identity into another new
virulent form necessitates an on-going search for new sources and types of resistance
that can be utilized in breeding for disease resistance. Qualitative resistance is
generally effective against biotrophic pathogens (pathogens that derive their nutri-
tion from living host cells). Observed start of an epidemic is delayed and effective
amount of initial inoculums reduces once qualitative resistance is deployed in a
variety. This type of resistance mechanism has been deployed against many
pathogens in many crops namely coffee (Coffea arabica L.) – Hemileia vastatrix
Berk. & Br., maize (Zea mays L.) – Puccinia sorghi Schw., oats (Avena sativa L.) –
P. coronata Cda., wheat (Triticum aestivum (L.) Thell.) – P. graminis f. sp. tritici
Pers., wheat – P. recondita, wheat – P. striiformis Westend, barley (Hordeum
vulgare) – Erysiphe graminis D.C. f. sp. hordei, flax (Linum usitatissimun L.) –
Melampsora lini (Ehrenb.) Desmaz. These resistance genes often cluster together in
certain chromosome arms, sometimes so tightly that they can be considered as
complex loci, and true allelic series also occur. In the flax-flax rust pathosystem,
34 R-genes have been identified in seven regions: K(2), L(14), M(7), N(3), P(6), D
(1) and Q(1). Regions N and P are linked, as well as regions N and K. The N region
consists of at least two closely linked loci. The M region, together with seven
resistance alleles, also consists of some closely linked loci. The L region, with
14 resistance alleles, behaves as a locus with an allelic series, but intra-allelic
recombination has been reported (Islam and Shepherd 1991). In barley, most of
the resistance genes to powdery mildew are located on one arm of chromosome
5 and one arm of chromosome 4 (Jorgensen 1990). On the short arm of chromosome
10 in maize, at least 16 resistance genes to P. sorghi are found on the complex locus
Rp1 and the loci Rp5 and Rp6 within three centimorgans of each other (Saxena and
Hooker 1968). The three downy mildew [Peronospora effusa(Grev.) Tul.] resistance
genes known in spinach are tightly linked.
10.5.2 Quantitative Resistance
As the name indicates, quantitative resistance has gradient of phenotypic class that is
determined by many genes, each contributing small, but together become important
to confer significant amount of disease and pest tolerance. Multiple genes typically
form the genetic basis and generally provide a level of resistance against many races
(non-race specific, horizontal). Such genetic foundation supports the durability of
quantitative resistance and ultimately the durability of a variety on farmers’ field.
Quantitative resistance (QR) is more often associated with resistance to necrotrophic
pathogens (pathogens that derive nutrition from dead cells). The utility of horizontal
resistance is more prospective in long epidemic in which disease increases with
small beginnings to relatively very great heights. Quantitative resistance seems to be
more effective and durable when large area is covered with crop varieties showing
race-nonspecific resistance (Parlevliet 2002). Barley leaf rust (P. hordei) resistance
showed polygenic inheritance and all cultivars in Western Europe, including the
very susceptible cultivars, carry at least some QR. Most cultivars carry considerable
levels of QR, thus preventing the barley leaf rust from becoming a major pathogen in
Western Europe (Parlevliet 1979). In rice, cross between two very susceptible
cultivars, some lines were obtained that were considerably more susceptible than
either parent, while a few other progeny lines were moderately resistant to bacterial
blight, Xanthomonas campestris pv. oryzae. The progenies performance beyond the
parental values meant that both highly susceptible cultivars carried many genes with
small effects for QR that differed from each other (Koch and Parlevliet 1991). Thus,
even the so-called very susceptible genotypes may harbour some QR, confirming the
experience with barley leaf rust. Similar observations were reported from quantita-
tive trait loci (QTL) analyses done in the pathosystems maize/Cercospora zeae-
maydis by Tehon & Daniels, pea (Pisum sativum L.)/Ascochyta pisi Lib. and tomato
(Lycopersicon esculentum Mill.) /Ralstonia solanacearum (Smith 1896 and
Yabuuchi et al. 1996). In crosses between a susceptible and a QR parent, QTLs
for QR were found that originated not only from the QR parent but also from the
susceptible parent when a cross was analysed where crossing between a susceptible
and a QR parent was used to generate the experimental materials (Young 1996).
Varieties with clear-cut visible resistance due to genes with larger effects are
required for release against major prevalent pathogens of the areas. Such varieties are
generally recommended for cultivation due to high score of resistance. Under this
situation, the effect of QR is not visible in spite of presence of genes with small
effects on resistant phenotype. After a number of years of cultivation, resistant scores
of the varieties goes down, as the major gene resistance is not effective any more.
However, after the resistance “breaks down”, QR becomes visible, if present. All
cultivars selected for their major gene resistance appear to carry moderate to fair
levels of QR hidden behind that major gene (Anonymous 1958–1998). This hidden
QR is sometimes indicated as residual resistance which is due to the presence of
some level of QR. Potatoes have a range of viruses which may affect them. Apart
from major resistance genes, QR also exists against those viruses. This QR is often
expressed through a reduced frequency of infected plants (incidence). The Dutch
recommended list of potato cultivars discerns between major gene resistance and
QR. All potato cultivars listed from the period 1958 to present carry low to high
levels of QR to each of the four viruses assessed: Potato virus X, Potato virus Y and
Potato virus A and Leaf roll virus. Therefore, QR is present almost everywhere.
Cultivars without any QR are very rare. For this type of resistance, breeders do not
need to look for primitive genotypes from centres of diversity nor to related wild
species. The resistance is found in adapted cultivars, a fortunate situation as it makes
breeding easier. McIntosh (1996) concluded that the ideal sources of resistance are
those present in closely related, commercial genotypes, and any effort to transfer
resistance from related species and genera should be considered long term.
10.6 Breeding Methods for Disease Resistance
Breeding methods used to modify genetic make-up of plants by integrating gene

(s) that confer resistance to biotic stresses can be broadly classified into two
categories:
10.6.1 Classical Breeding Approach

10.6.2 Novel approaches
10.6.2.1 Molecular Breeding approach
10.6.2.2 Transgenic breeding approach
10.6.1 Classical Breeding Approach
Classical breeding approaches used for developing resistant varieties include the
same approaches as used for developing high yielding varieties. In practice, resis-
tance to existing races of prevalent pathogens is considered directly or indirectly an
integral part in yield improvement programmes. In resistance breeding programme,
progenies or populations are required to be screened for reaction to the targeted plant
pathogens under hot spots natural conditions or under artificially inoculated
conditions to identify genotypes with high level of resistance score. In fact reliable
screening against the plant pathogens is essentially required to validate the parental
lines planned to be used in the beginning of a resistance breeding programme or to
screen and testify the level of resistance in segregating/stabilizing populations
derived from a hybridization programme. In both the cases, sick plots/hot spots
sites are very useful and helpful in reliable screening.
The simplest breeding approach is to search out the resistant lines, genetic stocks,
advanced breeding lines, landraces by screening using enough inoculums and
disease pressure either in the areas where abundant pathogenic races are available
or under ambient controlled condition or in the disease screening nursery. Multi-
location and multi-environment testing will be more reliable in exploring lines with
true resistance potential. In fact, standard screening techniques should be used for
preparation of inoculums, transfer of inoculums at right parts of the plant at right
stage under appropriate environmental conditions followed by scoring of response in
terms of data on appropriate scale. Based on the data of multi-location and multi-year
trials, resistant genotypes are selected.
There are many sources that can be explored for resistant gene in the breeding
programme. Primary gene pool sources of resistant genes are land races, farmers’
varieties, commercial varieties, natural or induced mutants, exotic and indigenous
germplasm, and elite lines. The primary gene pools are the best source of resistant
gene, since they are ready to use materials that a breeder can use freely. The
secondary gene pool sources of resistance include those lines that are considered to
be wild relatives or progenitors of cultivated crops. These sources are rather more
important in terms of allelic divergence and may provide a rather strong source of
resistant gene. However, crossability with cultivated plants and transfer of tightly
linked undesirable genes are major hurdles in domestication of wild alleles for
disease resistance. Tertiary gene pool sources include those species which are
quite distantly related, and crossing between primary and tertiary gene pool sources
are normally not possible and various improved and advanced tools are required to
make genomic influx. Secondary and tertiary gene pools have great potential to
support the biotic stress breeding programme with potential resistant genes.
Classical breeding methods based on major gene or gene with major effect
towards disease resistance is simple and straightforward. In case the major resistance
is available in primary gene pool, backcross breeding approach will be more
pertinent to transfer a single desired character to an otherwise superior genotype
(the recurrent parent) without altering the genetic make-up of recurrent parent.
Success from this approach depends largely on (i) the availability of a potential
genotype or variety that has good adaptability and yield potential to serve the
purpose of recipient or recurrent parent, (ii) the identification of the transferred
character in segregating populations and (iii) no existence of linkage disequilibrium
between any undesirable trait with the desirable trait to be transferred from a
genotype that possessed the resistant gene and serve the purpose of donor
(non-recurrent) parent. This approach has been very useful in transferring simply
inherited traits, especially genes that have clear-cut evidence of resistance/suscepti-
bility. The backcross breeding methods can be used successfully in self-pollinated
crops for development of pure line varieties. The method has same level of signifi-
cance and importance in cross pollinated crops where the objective is to develop a
hybrid cultivar resistant to a particular disease conditioned by a gene with major
effect. In case of self-pollinated crops, product is developed at the end of backcross
breeding programme, however in case of cross pollinated or hybrid breeding
programme, parental lines resistance to disease is developed at the end of the
programme. These improved parental lines are crossed to develop disease resistance
hybrid cultivar. Backcross breeding approach can also be used for pyramiding
diverse major genes for resistance to a pathogen/insect in one and the same variety.
It is believed that the greater the number of major genes for resistance in a variety,
greater would be its longevity. Further deployment of effective resistance gene in
time and space can be another effective strategy for utilization of resistance sources
in management of diseases and minimization of crop losses due to biotic stresses.
Backcross breeding can also be used successfully in development of multiline
variety having many resistance genes in component lines. In fact a multiline variety
is a population of plants that is agronomically uniform but heterogeneous for genes
that condition reaction to pathogens. This concept was first given by Jensen (1952)
for use in oats. Similar approach was suggested by Borlaug (1959) in wheat for
controlling stem rust. Multiline variety has genotypic diversity with respect to
vertical resistance genes. Each component line of a multiline must have strong
resistance gene to ensure the reduction of initial inoculums and spread of diseases
for longer duration. Except disease-resistant genes, component of multiline must

have phenotypic uniformity with respect to agronomic characters, namely plant
height, plant morphology, maturity and seed shape, colour and size. Pedigree and
bulk methods of selection in segregating population generated by biparental or
multi-parental crossing are other approaches that can be used for development of
genotypes with high resistance score. Again the success of the programme depends
on the availability of resistant sources used in crossing programme followed by
handling of segregating population, comprehensive screening and identification of
potential genotypes. These methods are heavily dependent on gene sources from
primary gene pool.
In case resistance gene is not available in germplasm, induced mutation followed
by mutation breeding approach can be adopted to screen the variability induced due
to mutagen and subsequently to identify the individual plants showing resistance. In
the subsequent generations, progeny rows are tested under adequate disease pressure
to further validate and identify a stable mutant line. In case of mutation breeding,
initial genetic material should be a well-established variety in case of self-pollinated
crops or well-proven parental lines of a hybrid cultivar. Natural or spontaneous
mutant can also be screened for identification of source of resistance to a particular
pathogen. Mutagenized lines can serve the purpose of variety if other required
parameters are at par with standard cultivars or alternately can be used as source
of resistance gene (donor) in backcross breeding programme. Frequencies of desir-
able mutant (disease resistant) or specific mutant are extremely low, therefore large
population size should be grown for increasing probability of desirable mutant.
To induce the desired genetic variability, tools and techniques for induction of
variability in vitro in somatic cells is another effective way, in case resistance gene is
not available or available resistance source is not as effective as required.
Somaclonal variation is the variation seen in plants that have been produced by
plant tissue culture. Somaclonal variation is not restricted to, but is particularly
common in plants regenerated from callus. The variations can be genotypic or
phenotypic, which in the latter case can be either genetic or epigenetic in origin.
Typical genetic alterations are changes in chromosome numbers (polyploidy and
aneuploidy), chromosome structure (translocations, deletions, insertions and
duplications) and DNA sequence (base mutations). The phenomenon of high
variability in individuals from plant cell cultures or adventitious shoots is called
somaclonal variation. Therefore, it can be defined as the variation that occurs
because of genetic mutation caused by in vitro conditions or by chimeral separation.
Somaclonal variation is usually undesirable. In some cases, somaclonal variation can
lead to new cultivars (e.g., disease resistance, new leaf pattern) that may have
desirable ornamental characteristics or increased pest resistance. Disease-resistant
somaclonal variants can be obtained by (1) Screening of plants regenerated from
cultured cells or their progeny are subjected to disease test, and resistant plants are
selected. (2) Cultured plant tissues are selected for resistance to the toxin or culture
filtrate produced by the pathogen, and plants are regenerated from the selected cells.
Cell selection strategy is most likely to be successful in cases where the toxin is
involved in disease development. Resistance was first reported in sugarcane for eye
spot disease caused by Helminthosporium sacchari.
The choice of any one method of selection depends on the breeder, stage of the
breeding program, stage of germplasm development, stage of knowledge of the
populations and objectives of the breeding program. In cross pollinated crops,
quantitatively inherited disease resistance can be enhanced by recurrent selection
(RS) – any breeding system designed to increase the frequency of desired alleles for
particular quantitatively inherited characters by repeated cycles of selection (Sleper
and Poehlman 2006). Number of selection cycles may be repeated as long as
superior genotypes with higher score of resistance are generated. The improved
population can be used as a variety per se; alternatively, it can be used as source for
development of inbred lines that can be used as parents of a synthetic or hybrid
cultivar. With simplest form of intra-population improvement which target only one
population, the method of recurrent selection can be extended to several divergent
populations. Individuals in a population can be evaluated on the basis of their
phenotype or on the basis of the performance of their half-sib or full-sib progenies.
In both intra- and inter-population improvement approaches, the ultimate aim is to
improve the frequency of genes conferring resistance against a quantitatively
inherited disease. Improved populations are in fact elite group of plants that can be
used as such in bulk as an open pollinated variety for cultivation in those areas where
crop is damaged by pathogens. Furthermore, such cultivars do not require seed
replacement every year and the farmers’ harvest can be used for sowing next year.
Alternatively, improved population can support hybrid development programme in
areas where seed production and supply network is well established.
10.6.2 Novel Approaches in Biotic Stress Resistance Breeding
Why Novel Approaches?

Advancement in genetic research at molecular level leads to the development of
many handy but useful tools that can be integrated with classical plant breeding to
achieve the specific objective of gene transfer, gene pyramiding, gene addition, gene
knockout, alteration in nucleotide sequence of a gene and ease accessibility of any
gene across the crossing barrier boundary for improvement in general and develop-
ment of resistance cultivars against biotic stresses in particular. Novel molecular-
based tools can be used successfully in resistance breeding programme, as it is
advantageous over classical resistance breeding methods as depicted below:
• Can exploit horizontal variability, thus widen option for genetic manipulation.
• Precise transfer of specific functional fragment of DNA.
• Precise up and down regulation of gene.
• Reduces time required in gene transfer.
• Novelty can be introduced.
• Effective screening is easy.
• Gene can be precisely modified partly or wholly.
• Many resistant genes for a disease or for different diseases can be pyramided.
10.6.2.1 Molecular Breeding

Molecular breeding (MB) or molecular plant breeding (MPB) in broad sense
includes use of genetic manipulation performed at DNA molecular levels to improve
plants characteristics and covers genetic engineering or gene manipulation, molecu-
lar marker-assisted selection and genomic selection. However, often MB or MPB
implies molecular marker-assisted breeding (MAB) and it is defined as the applica-
tion of molecular biotechnologies, specifically molecular markers, in combination
with linkage maps and genomics, to alter and improve plant characteristics on the
basis of genotypic assays. This term is used to describe several modern breeding
strategies, including marker-assisted selection (MAS), marker-assisted backcrossing
(MABC), marker-assisted recurrent selection (MARS), and genome-wide selection
(GWS) or genomic selection (GS) (Ribaut et al. 2010).
MAS is the process in which a marker is used for indirect selection of a genetic
determinant of biotic stresses. This method involves selection of plants carrying
genomic regions that are involved in the expression of traits of interest through the
application of molecular markers. The development and availability of an array of
molecular markers and dense molecular genetic maps in crop plants have made
application of MAS possible for traits governed by major genes as well as those
governed by many genes and expression are quantitative in nature (Choudhary et al.
2008). The success of MAS depends on several factors, including the number of
target genes or genomic regions to be transferred and the distance between the
flanking markers and the target genomic site. With availability of different marker
systems and high throughput genotyping and phenotyping along with improved
statistical approaches, MAS is gaining considerable importance as it can improve the
efficiency of plant breeding through precise transfer of genomic regions of interest
and acceleration of the recovery of favourable alleles of the recurrent parent genome
(Wijerathna 2015).
With the application of MAS, individual plants can be selected based on their
genotype, during the selection procedure. For most traits, homozygous and hetero-
zygous plants cannot be distinguished by conventional phenotypic screening. MAS
can be used to assist selection of parents, increasing the effectiveness of backcross
breeding and improving sex-limited traits (Zhou et al. 2007). MAS can be used to
investigate heterosis for hybrid crop production (Reif et al. 2003), and there is the
potential for use of DNA marker data along with phenotypic data to select hybrids
(Jordan et al. 2003). There are various advantages of using MAS in rice breeding.
For example, it may be simpler than phenotypic screening; therefore, it can reduce
time, effort and resources. Selection of quality traits in rice generally requires
expensive screening procedures that are made feasible through MAS. Additionally,
MAS can be conducted at the seedling stage, and undesirable plant genotypes can
quickly be eliminated (Khan et al. 2015). The advantages associated with the use of
markers includes speed, consistency, efficiency, biosafety and the ability to skew the
odds in our favour, even while dealing with complex traits.
Gene pyramiding is the transfer or pyramiding more than one resistance/tolerance
genes/QTLs into a single genotype (Collard and Mackill 2008). Pyramiding of
resistance genes into a single line for each disease is a novel strategy in resistance
breeding to prevent the breakdown of resistance against specific disease or stress.

Pyramiding of genes/QTLs that confer resistance to biotic stresses and tolerance
against various types of abiotic stresses is now feasible because of advancements in
molecular markers (Das and Rao 2015). MAS has been found to work efficiently for
transferring genes from pyramided lines into new plants and into improved varieties
(Magar et al. 2014). Breeders have used marker-assisted selection to “pyramid”
resistance conferred by several separate resistance genes/QTLs with the help of
closely linked markers against diseases such as bacterial blight, rice blast and insect
as gall midge in rice, leaf rust resistance and powdery mildew resistance in wheat,
and insect pest resistance in cotton, as well as many other traits (Das and Rao 2015;
Pradhan et al. 2015; Suh et al. 2015; Shamsudin et al. 2016). To get the desired
population with required gene combinations without unwanted genes, backcrossing
with the recurrent parent is required. The use of molecular markers, which were
unlinked to the assembled genes/QTLs for back ground selection, enhances the
proportion of recovery of the recipient genome. The gene pyramiding scheme can
be distinguished into two parts, development of a pedigree, which is designed to
accumulate all target genes in a single genotype known as the root genotype, and a
fixation step, which is intended to fix the target genes into a homozygous state to
derive the ideal genotype from one single genotype. The brown plant hopper (BPH),
Nilaparvata lugens, has been one of the most devastating pests to rice crops in
Vietnam and Asia. There is successful report of the use of SSR and STS markers in
pyramiding two BPH resistance genes Bph14 and Bph15 into three elite japonica
varieties Shengdao 15, Shengdao 16 and Xudao 3, using marker-assisted backcross
breeding programmes (Xu 2013). Rice cultivation across tropical and semi-tropical
regions of the world is affected by bacterial blight (BB) disease caused by
Xanthomonas oryzae pv. oryzae (Xoo). A total of 38 R genes of BB have been
identified in rice (Khan et al. 2014). Resistant cultivars with one or two major
resistant genes are unsustainable in the field, and the only way to delay such a
breakdown of BB resistance is to pyramid many resistance genes using MAS
(Rafique et al. 2010). Rice sheath blight disease caused by Rhizoctonia solani
Kuhn reduces trivial yield in rice-growing areas around the globe (Yellareddygari
et al. 2014; Yadav et al. 2015). Genetic studies have shown that SB resistance can be
controlled by polygenic QTLs. It is possible to pyramid SB resistance QTLs into rice
varieties using MAS.
Marker-assisted selection (MAS)/Marker-assisted backcrossing (MAB) has been
used successfully in many crop species for introgressing resistance genes. It is an
important tool for pyramiding different genes for a disease or vice versa. In India,
many varieties/hybrid have been developed using MAB/MAS (Table 10.1).
10.6.2.2 Transgenic Breeding for Biotic Stress Tolerance

Genetic variations are unanimously accepted as the basic necessity for improvement
of any trait using classical breeding approach and so is the case with molecular or
transgenic breeding programme. Resistance genes present in taxonomically different
plant species or in wild relatives have no meaning since breeder or molecular breeder
of other crops cannot use such variability which is referred to as horizontal
Table 10.1 MAS derived varieties in India for different characters

Crop Varieties Characters
Pearl millet HHB 67 improved Downey mildew
Rice Pusa 1612 (Pusa Sugandh 6) Blast (Piz5 and Pi54)
Pusa 1592 Bacterial blight (xa13 and Xa21)
Pusa Basmati 1609 Blast (Piz5 and Pi54)
Improved Pusa Basmati 1 Bacterial blight (xa13 + Xa21)
Improved Samba Mahsuri Bacterial blight (xa5 + xa13 + Xa21)
Wheat PBW-343 improved Lr37/Yr17 and Lr76/Yr70
HD 2329 Lr24 and Lr28
WH147 Lr24 + Lr28,Lr24 + Lr37,Lr28 + Lr37
HD2687 Lr24 + Lr28 + Yr15
variability. Transfer of genes between plant species has played an important role in
crop improvement for many decades. Genes expected to confer disease resistance are
isolated, cloned and transferred into many crop species. Useful traits, viz., resistance
to diseases, insects and pest, have been transferred to crop varieties from nonculti-
vated plants. Transfer of useful alleleic variants or novel gene (s) across the species
and genera trans-boundary requires identification, introduction, validation followed
by transfer of the gene into desired plant species. Once a gene known to confer
resistance is identified from any sources or even chemically synthesised and is
finally cloned, its transfer become easy and in principle it can be transferred to any
plant species using different plant transformation techniques. The overall process of
genetic transformation involves introduction, integration and expression of foreign
gene in the recipient host plant. Plants that carry additional stably integrated and
expressed transferred gene (transgenes) from other genetic sources are referred to as
transgenic plants. The capacity to introduce and express diverse foreign genes in
plants was first described in tobacco by Agrobacterium mediated and vectorless
approach (Horsch et al. 1984) Development and deployment of strong major genes
over time and space may check epidemics in alternate years and geographic regions
so that the virulent pathotype against any one of them doesn’t evolve and doesn’t
survive even if it did evolve. Based on the principle of crop rotation to control certain
soil-borne pathogens, transgenic crops with different resistant gene can be of great
help in management of pathogens. Refugia approach can also be integrated while
recommending cultivation of transgenic crop based on single major gene to delay the
emergence and also manage the population of virulent strains. The term ‘refugia’ is
classically defined as an area in which a population of organisms can survive over a
period of unfavourable conditions. Exploiting refugia as a means of delaying the
evolution of resistance has been explored in biological systems, in particular in the
management of agricultural pests.
The transgenic breeding approach can use many strategies to incorporate genes
responsible for disease resistance. Diverse genes reported to confer different func-
tion related directly or indirectly to disease resistance can be used for development of
GM crops. Targets that can be used for modification in host for enhancing heritable
tolerance against different pathogens can be based on:
Pathogen
Pathogenecity factor
Induced
Constitutive defence 1(b) Detection of Pathogen mimicry (3)
pathogen (2a)
defence (1a)
Defence regulation (2b)
Nucleus
Host cell
Fig. 10.1 A simplified model of defence illustrating successful transgenic strategies. Strategy
1 concerns direct interference with pathogenicity or inhibition of pathogen physiology. Thus, 1a
involves constitutive expression of antimicrobial factors and 1b involves pathogen-induced expres-
sion of one or more genes in the transgenic plant. Strategy 2 concerns the regulation of the natural
induced host defences. 2a concerns altering recognition of the pathogen (e.g. R-genes) and 2b
concerns downstream regulatory pathways (e.g. SAR) and includes transcription factors. Strategy
3 is pathogen mimicry: the manipulation of the plant to prime recognition of a specific pathogen
through pathogen-derived gene sequences. (Adapted from Collinge et al. 2008)
1. Expression of antifungal compounds—PR proteins, phytoalexins, defensins

2. Enzymes that destroy or neutralize components of pathogen arsenals
polygalactouronase inhibitors, oxalic acid (toxin) oxidase
3. Alterations of structural components-peroxidase for lignin production, callose
synthase
4. Resistance genes
5. Components of signalling pathways
A simplified model of defence in host illustrating successful transgenic strategies

that can be adopted for deployment of genes conferring resistance to pathogens has
been proposed (Fig. 10.1).
Transgenic breeding approach has been used for introgression of resistant gene
(s) in many plant species. The diverse sources have been used to isolate the genes
that confer resistant property directly or indirectly. Both physical and biological
approaches of genetic transformation have been used to transfer gene into recipient
plants. Though there are many successful examples of introgressing resistance gene
(s) against various plant pathogens, it has been more successful in cases of diseases
caused by viruses (Table 10.2). Use of transgenic variety in control of papaya ring
spot virus is an excellent example of successful use of transgenic breeding approach.
Table 10.2 Genetically engineered biotic stress-resistant varieties

Crop/Even/ Gene
Trade name introduced Gene source Product Function
Common bean (Phaseolus vulgaris L.)
EMBRAPA ac1 (sense Bean Golden Sense and antisense Inhibits the
5.1 and Mosaic Virus RNA of viral replication synthesis of the
antisense) (BGMV) protein (Rep); no viral replication
functional viral protein of the
replication protein is Bean Golden
produced Mosaic Virus
(BGMV), thereby
conferring
resistance to the
BGMV
Papaya (Carica papaya)
Rainbow, prsv_cp Papaya Coat protein (CP) of the Confers
SunUp ringspot virus papaya ringspot virus resistance to
(PRSV) (PRSV) papaya ringspot
virus (PRSV)
through
“pathogen-
derived
resistance”
mechanism
63–1 prsv_cp Papaya Coat protein (CP) of the Confers
ringspot virus papaya ringspot virus resistance to
virus (PRSV)
through
“pathogen-
derived
resistance”
mechanism
Huanong Replicase Papaya Confers resistance to Papaya ringspot
no.1 domain of ringspot virus papaya ringspot virus virus (PRSV)
the papaya (PRSV) (PRSV) through gene
ringspot silencing mechanism
virus
(PRSV)
X17–2 prsv_cp Papaya Coat protein (CP) of the Confers
ringspot virus papaya ringspot virus resistance to
virus (PRSV)
through
“pathogen-
derived
resistance”
mechanism
(continued)

Crop/Even/ Gene
Plum (Prunus domestica)
C-5 ppv_cp Plum pox virus Coat protein of plum Confers
(PPV) pox virus (PPV) resistance to plum
pox virus (PPV)
through
“pathogen-
derived
resistance”
mechanism
Potato (Solanum tuberosum L.
Hi-Lite cry3A Bacillus cry3A delta endotoxin Confers
NewLeaf™ thuringiensis resistance to
Y potato subs. coleopteran
tenebrionis insects by
selectively
damaging their
midgut lining
pvy_cp Potato Virus Y Coat protein of the Confers
(PVY) potato virus Y (PVY) resistance to
potato virus Y
(PVY) through
“pathogen-
derived
resistance”
mechanism
New Leaf™ cry3A Bacillus cry3A delta endotoxin Confers
Y Russet thuringiensis resistance to
Burbank subs. coleopteran
potato tenebrionis insects by
selectively
damaging their
midgut lining
pvy_cp Potato Virus Y Coat protein of the Confers
(PVY) potato virus Y (PVY) resistance to
potato virus Y
(PVY) through
“pathogen-
derived
resistance”
mechanism
(continued)

Crop/Even/ Gene
New Leaf™ cry3A Bacillus cry3A delta endotoxin Confers
Plus Russet thuringiensis resistance to
Burbank subs. coleopteran
potato tenebrionis insects by
selectively
damaging their
midgut lining
cp4 epsps Agrobacterium Herbicide tolerant form Decreases
(aroA: tumefaciens of binding affinity
CP4) strain CP4 5-enolpyruvulshikimate- for glyphosate,
3-phosphate synthase thereby
(EPSPS) enzyme conferring
increased
tolerance to
glyphosate
herbicide
plrv_orf1 Potato Leaf Putative replicase Confers
Roll Virus domain of the potato leaf resistance to
(PLRV) roll virus (PLRV) potato leaf roll
virus (PLRV)
through gene
silencing
mechanism
plrv_orf2 Potato Leaf Putative helicase Confers
Roll Virus domain of the potato leaf resistance to
(PLRV) roll virus (PLRV) potato leaf roll
virus (PLRV)
through gene
silencing
mechanism
SP951 RB Solanum Late blight-resistant Broad spectrum
bulbocastanum protein resistance against
Phytophthora
infestans races
W8 asn1 Solanum Double-stranded RNA Designed to
tuberosum generate dsRNA
to downregulate
Asn1 transcripts
which lowers
asparagine
formation
ppo5 Solanum Double-stranded RNA Designed to
verrucosum generate dsRNA
to downregulate
Ppo5 transcripts
which lowers
black spot bruise
development
(continued)

Crop/Even/ Gene
Rpi-vnt1 Solanum Late blight-resistant Confers
venturii protein resistance to
potato late blight
PhL Solanum Double-stranded RNA Designed to
to downregulate
PhL transcripts
which lowers
reducing sugars
R1 Solanum Double-stranded RNA Designed to
to downregulate
R1 transcripts
which lowers
reducing sugars
Vlnv Solanum Double-stranded RNA Downregulates
tuberosum VInv transcripts
which lowers
reducing sugars
Innate® asn1 Solanum Double-stranded RNA Designed to
Acclimate tuberosum generate dsRNA
to down-regulate
Asn1 transcripts
which lowers
asparagine
formation
ppo5 Solanum Double-stranded RNA Designed to
verrucosum generate dsRNA
to downregulate
Ppo5 transcripts
which lowers
black spot bruise
development
Rpi-vnt1 Solanum Late blight-resistant Confers
venturii protein resistance to
potato late blight
R1 Solanum Double-stranded RNA Designed to
to downregulate
R1 transcripts
which lowers
reducing sugars
Vlnv Solanum Double-stranded RNA Downregulates
tuberosum VInv transcripts
which lowers
reducing sugars
(continued)

Crop/Even/ Gene
Squash (Cucurbita pepo)
CZW3 cmv_cp Cucumber Coat protein of Confers
Mosaic cucumber mosaic resistance to
Cucumovirus cucumovirus (CMV) cucumber mosaic
(CMV) cucumovirus
(CMV) through
“pathogen-
derived
resistance”
mechanism
zymv_cp Zucchini Coat protein of zucchini Confers
Yellow Mosaic yellow mosaic potyvirus resistance to
Potyvirus (ZYMV) zucchini yellow
(ZYMV) mosaic potyvirus
(ZYMV) through
“pathogen-
derived
resistance”
mechanism
wmv_cp Watermelon Coat protein of Confers
Mosaic watermelon mosaic resistance to
Potyvirus potyvirus 2 (WMV2) watermelon
2 (WMV2) mosaic potyvirus
2 (WMV2)
through
“pathogen-
derived
resistance”
mechanism
ZW20 zymv_cp Zucchini Coat protein of zucchini Confers
Yellow Mosaic yellow mosaic potyvirus resistance to
Potyvirus (ZYMV) zucchini yellow
(ZYMV) mosaic potyvirus
(ZYMV) through
“pathogen-
derived
resistance”
mechanism
wmv_cp Watermelon Coat protein of Confers
Mosaic watermelon mosaic resistance to
Potyvirus potyvirus 2 (WMV2) watermelon
2 (WMV2) mosaic potyvirus
2 (WMV2)
through
“pathogen-
derived
resistance”
mechanism
(continued)

Crop/Even/ Gene
Sweet pepper (Capsicum annuum)
PK-SP01 cmv_cp Cucumber Coat protein of Confers
Mosaic cucumber mosaic resistance to
Cucumovirus cucumovirus (CMV) cucumber mosaic
(CMV) cucumovirus
(CMV) through
“pathogen-
derived
resistance”
mechanism
Tomato (Lycopersicon esculentum)
PK- cmv_cp Cucumber Coat protein of Confers
TM8805R Mosaic cucumber mosaic resistance to
(8805R) Cucumovirus cucumovirus (CMV) cucumber mosaic
(CMV) cucumovirus
(CMV) through
“pathogen-
derived
resistance”
mechanism
Source: ISAAA’s GM Approval Database (2019)
Expression of toxic protein gene in many plant species for control of insect is another
successful example of transgenic approach in resistance breeding.
Transgenic breeding approaches have been successful in transferring different
genes conferring resistance to different pathogens in plant species. In addition, great
stride has been made in developing insect-pest resistant genotypes in different crop
species. Molecular genetic studies over past decades have originated new tools for
breeding programmes of crop plants. Innumerous genetic engineering techniques
were developed and applied to generate genetically modified crop varieties with
superior agricultural characteristics, including new traits that do not occur naturally
in the species. In the further refinement of GM technology, molecular techniques
promising to develop ‘transgene free’ crops have been introduced in plant breeding
programmes. These new techniques are collectively called Genome Editing (GE).
GE is changing the way of producing genetically modified organisms since it
produces specific genetic changes within a genome, with no transgene manipulation.
GE refers to platforms that use site-specific nucleases (SSNs) that can introduce
DNA lesions at a specific genomic position. Several novel GE systems based on
SSNs were developed: Zinc Finger Nucleases (ZFNs), Transcription Activator-Like
Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palin-
dromic Repeats (CRISPR/ Cas9). Because of its high efficiency and relatively low
cost, CRISPR/Cas9-based genome editing system have become the most popular
choice of plant molecular biologists for functional studies of plant genes. The
CRISPR-Cas9 system is a plant breeding innovation that uses site-directed nucleases

to target and modify DNA with great accuracy. Developed in 2012 by scientists from
the University of California, Berkeley, CRISPR-Cas9 has received a lot of attention
in recent years due to its range of applications in modifying plant genomes for
altering yield, architecture and tolerance/resistance to biotic and abiotic stress.
Advances in genome editing tools have opened new ways to achieve the improve-
ment of resistance in crops. In the recent past, the CRISPR/Cas system has been
employed to respond to several agricultural challenges, including improved biotic
stress resistance (Arora and Narula 2017). The application of CRISPR/Cas tools has
mainly been explored against virus infection, followed by efforts to improve fungal
and bacterial disease resistance. Recent experimental findings of many research
groups indicate genome editing technology as an effective tool in developing
tolerant genotypes without altering genetic make-up of plants (Borrelli et al. 2018;
Langner et al. 2018; Andolfo et al. 2016).
10.7 Wild Relatives in Biotic Stress Tolerance
10.7.1 Wheat
The species belonging to primary, secondary and tertiary gene pools of Triticeae
species are rich source of genes for improvement of traits pertaining to biotic stress
tolerance. Introgression of alleles from nearly 52 related species have already been
done for improvement of wheat for different traits (Wulff and Moscou 2014).
Fusarium head blight resistance has been reported by Oliver et al. (2007) in
Triticum dicoccoides. A diploid progenitor of bread wheat Triticum monococcum
has also been found to be the source of resistance genes to a number of fungal
diseases of wheat. Yao et al. (2007) discovered the genes for traits responsible for
imparting resistance against powdery mildew in Triticum monococcum, while
Sodkiewicz et al. (2008) identified resistant genes for leaf rust. An adult plant
resistance (APR) gene for stripe rust and leaf rust have been transferred from T.
monococcum to bread wheat by means of marker-assisted selection, and a leaf rust-
resistant gene have also been transferred to PBW343 (Singh et al. 2007). A race
specific stem rust resistance gene, Sr35, has also been identified in T. monococcum.
Moseman et al. (1984) identified T. monococcum as a source of powdery mildew
resistance. Aegilops tauschii, another diploid progenitor contributing D genome to
hexaploid wheat, serves as donor for Russian leaf rust resistance gene (Lr21)
(Yumurtaci 2015), race specific yellow rust resistance gene Yr28 and an adult
plant resistance gene Lr22a. Additionally, Bockus et al. (2012) identified
A. tauschii as a source of blast resistance in wheat. Introgression of stem rust
resistance genes found in T. turgidum and T. dicoccum into bread wheat have also
been reported as early as the 1930s by McFadden. T. dicoccum have also been
identified as donor source for Rmg7 gene conferring resistance to Triticum isolates of
Pyricularia oryzae by Tagle et al. (2015). Aegilops geniculata, an allotetraploid
relative of wheat, was found to be the source of barley yellow dwarf virus and
powdery mildew resistance of wheat (Yumurtaci 2015). The 6P chromosome of

A. cristatum is responsible for improving resistance to powdery mildew and barley
yellow dwarf virus besides enhancing some yield-contributing traits as number of
kernels and grain weight in wheat (Wang et al. 2011). Cruz et al. (2016) found that
2NS/2AS translocation from Aegilops ventricosa conferred resistance to wheat blast
disease. T.dicoccoides and Triticum carthlicum have also been reported as source of
genes conferring resistance to powdery mildew by Moseman et al. (1984). Vertical
and horizontal resistance genes have also been identified from diverse sources, viz.,
Yr5 from Triticum spelta, Lr9 from Aegilops umbellulata conferring vertical resis-
tance and Yr36 from Triticum diccocoides conferring horizontal resistance against
rust disease of wheat. Multiple disease resistance have been found in a number of
wild relative as Sr36/Pm6 from Triticum timopheevi, Pm8/Sr31/Lr26/Yr9 from rye
and Lr19/Sr25, Sr24/Lr24, Sr26 from A. elongatum and Pch1 and Sr38/Lr37/Lr17
from Aegilops ventricosa (Wulff and Moscou 2014). Wan et al. (1997) identified
Fusarium head blight resistance genes in different genera, viz., Agropyron, Elymus
and Hystrix as well as in related wheat species, namely T. monococcum,
T. timopheevi and T. militinae (Cai et al. 2005). Introgression of segments of alien
chromosome from wild relatives into wheat genetic background has been found to
improve tolerance to different disease. The 7DL.7Ag translocation from a wild
relative Lophopyrum elongatum carrying Lr19 gene was done by Monneveux
et al. (2008) which conferred leaf rust resistance to wheat. Similarly, genes for
stem rust resistance (Waines and Ehdaie 2007) and powdery mildew (Yediay et al.
2010) have also been successfully introgressed from rye into wheat germplasm lines.
10.7.2 Rice
Genus Oryza of the graminae family constitutes a total of 24 species. Out of these
24 species, O. sativa L. and O. glaberrima are the only cultivated species of genus,
Oryza while the remaining 22 are wild species distributed worldwide (Khush 1997;
Vaughan 1989). Depending on the ease of transfer of genes to their cultivated
counterparts, the wild species are divided into three complexes, i.e., O. sativa
complex, O. officinalis complex and O. meyeriana and O. ridleyi complex
(Morishima and Oka 1960). Khush (1997) later renamed these complexes as the
primary, secondary and tertiary gene pool of rice. Wild species of rice are a rich
source of economically valuable traits on account of being grown in diverse climate
and lack of artificial selection. Some of the donors of these traits are mentioned in
forthcoming section.
Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae is reported
to be one of the most destructive diseases of rice worldwide. However, the utilization
of two resistant genes namely, Xa3 and Xa4, have enhanced BLB resistance, but due
to continuous evolution of new pathogen strains, the need to discover new sources of
resistance became inevitable. Two new resistant genes (Xa21 and Xa 23) were
identified in wild rice (Song et al. 1995; Zhou et al. 2011). Xa21 have been identified
in Oryza longistaminata and was transferred to rice variety IR72 in 1990 to mark the
first ever example of utilization of rice wild relative for crop improvement. Positional
cloning of Xa21 gene was done, and it was found to encode receptor kinase-like
protein (Song et al. 1995). The gene was soon after tagged with molecular markers
and transferred into various famous rice cultivars by means of marker-assisted
breeding to produce BLB-resistant varieties. The resistance conferred by Xa21 was
soon broken down as many new virulent strains of BLB pathogen evolved in China.
Therefore, the need to identify novel resistant genes providing durable resistance
against BLB emerged. O. longistaminata along with O. rufipogon have also been
identified as a potential source of rice tungro virus and have been utilized to produce
a number of virus-tolerant rice lines. Rice blast caused by Magnaporthe oryzae is yet
another economically important fungal disease of rice. The disease was first reported
from the United States and since then has been reported in about 85 rice-growing
countries worldwide (Wang et al. 2014). The source of resistance for this disease has
been searched in wild relative of rice and to date, about 100 resistance genes and
more than 350 QTLs have been identified providing resistance against rice blast
(Wang et al. 2014; Ashkani et al. 2015; Vasudevan et al. 2015). 96% of the total
identified R genes have been found in japonica and indica cultivars while only 4% of
them are reported to be contributed by crop wild relatives (Wang et al. 2014)
including O. rhizomatis, O. minuta and O. autraliensis. Resistance genes have
been introgressed into susceptible lines to breed for blast tolerance (Sharma et al.
2012; Wang et al. 2014; Ashkani et al. 2015). Another blast resistance gene, Pi33,
identified in O. rufipogon has also been transferred into IR64, producing a blast-
resistant variety (Ballini et al. 2007). Three R gene clusters viz., Piz, Pik and Pita,
were discovered on chromosomes 6, 11 and 12, respectively. Besides providing blast
resistance, O. rufigon has also been identified as a source of a single, dominant
BLB-resistant gene Xa23 which provides efficient broad spectrum resistance at all
crop growth stages and was responsible for providing resistance against all
20 known strains of the pathogen (Zhang et al. 2001; Zhang and Xie 2014).
O. nivara also serves as source of a novel BLB-resistant gene, Xa38 (Ellur et al.
2016). Rice crop is affected by more than 20 viral disease with the most damaging
being grassy stunt disease; caused by rice grassy stunt virus (RGSV), and rice tungro
disease; caused by infection of rice tungro bacilliform virus (RTBV) and rice tungro
spherical virus (RTSV). BPH acts as vector for transmission of RGSV, while green
leafhopper serves as vector for rice tungro disease. A single dominant gene, Gs was
identified in O. nivara responsible for imparting resistance against RGSV. Resis-
tance against RGSV has been introgressed into a number of germplasm lines from
O. nivara (Leung et al. 2002).
10.7.3 Maize
Quantitative trait loci (QTLs) responsible for providing resistance against a number
of diseases were discovered in maize wild relatives. Lennon et al. (2016) conducted a
study where he utilized a population of near isogenic lines derived from cross
between maize and teosinte (Zea mays ssp. parviglumis) for QTL mapping of gray
leaf spot (GLS). A GLS resistance QTL was thus identified in bin 4.07. Chavan and
Smith (2014) reported that teosinte can also be used as donor of resistance to corn
smut disease. Z. diploperennis was reported to be a source of resistance to southern
corn leaf blight, northern corn leaf blight and corn leaf spot disease. Wei et al. (2001)
studied maize Z. diploperennis crosses and using genomic in situ hybridization
identified specific segments derived from Z. diploperennis imparting resistance to
above mentioned three fungal diseases. A higher level of resistance was reported
against a number of viral and mycoplasmal diseases of maize viz., maize chlorotic
mottle virus, maize bushy stunt mycoplasma, maize streak virus, maize stripe and
rayadofino virus and maize chlorotic dwarf virus in Z. diploperennis by Nault and
Findley (1982). Maazou et al. (2017) reported Z. mays ssp. mexicana to be the donor
for Fusarium and downy mildew resistance. Tripsacum dactyloides is also reported
to be a source of many resistance alleles which when transferred into maize genetic
background have historically helped to overcome various disease epidemics in
maize. One of the examples was the transfer of blight-resistant alleles into commer-
cial corn lines which resolved the problem of corn blight in the United States
(Maxted and Kell 2009). The introgression of Ht3, a northern leaf blight resistance
genes (Hooker 1981) and Rp1td, a novel rust resistance genes (Bergquist 1981) from
eastern gamagrass into maize genetic background remain the success stories of
utilization of wild relatives to breed for disease resiliency in crop plants. Hajjar
and Hodgkin (2007) have also reported the utilization of introgression from
Tripsacum to breed for Helminthosporium and Puccinia resistance in maize.
10.7.4 Soybean
Genus Glycine was divided into two subgenera, Glycine and Soja. Cultivated
soybean Glycine max and its wild progenitor G. soja make up the subgenus Glycine.
The subgenera Soja is composed of seven perennial wild, diploid, perennial species,
G. canescens, G. clandestine, G. falcate, G. latifolia, G. latrobeana, G. tabacina and
G. tomentella. Although only G. soja has been utilized in past for manipulation by
plant breeders, now wild relatives belonging to subgenus Soja are also being actively
utilized as donors of different agronomically useful traits in soybean breeding
programmes.
Soybean rust is one of the major diseases affecting soybean production world-
wide. It is caused by pathogen Phakopsora pachyrhizi. Hartman et al. (1992)
screened 294 accessions belonging to 17 Glycine species and identified numerous
rust-resistant sources, viz., G. clandestine, G. canescens, G. argyrea, G. tabacina,
G. microphylla, G. latifolia and G. tomentella. Partial resistance to Sclerotinia stem
rot or white moulds have also been reported in G. tabacina and G. tomentella
(Hartman et al. 2000). Several accessions of G. tomentella and G. canescens were
also reported showing resistance to powdery mildew disease and sudden death
syndrome (Mignucci and Chamberlain 1978; Hartman et al. 2000).
10.7.5 Chickpea
Genus Cicer is composed of a total of 43 species which includes 34 wild perennial

species, 8 wild annual species and 1 annual cultivated species. All the species are
self-pollinated and diploid (Ladizinsky and Adler 1976; Singh and Ocampo 1997).
Two wild species, C. microphyllum and Cicer songaricum, and one cultivated
species, C. arietinum, are reported to be grown under Indian conditions.
Phytophthora root rot- (Phytophthora medicaginis) resistant germplasm lines in
chickpea have been developed using C. echinospermum as a donor source of
resistant alleles (Knights et al. 2008). Resistance to botrytis grey mould has been
derived from C. echinospermum and C. reticulatum (Singh et al. 1984; Jaiswal et al.
1986; Singh and Ocampo 1997; Singh et al. 2005), C. judaicum (Chaturvedi and
Nadarajan 2010) and C. pinnatifidum (Kaur et al. 2013). Some species of wild
relative, C. bijugum, C. reticulatum, C. pinnatifidum and C. echinospermum also
show resistance to multiple stress conditions (Kumar et al. 2011) as
C. echinospermum harbours resistance for both pod borer and Phytophthora root
rot (Knights et al. 2008).
10.7.6 Pigeonpea
The genus Cajanus comprises of 32 species (van der Maesen 1986). Mallikarjuna
et al. (2010) divided pigeon pea wild relatives into primary, secondary, tertiary and
quaternary gene pools. Primary gene pool of pigeonpea constitutes Cajanus cajan
and its land races. The secondary gene pool comprises of 10 wild species,
C. cajanifolius, C. lineatus, C. lanceolatus, C. laticepalus, C. albicans,
C. reticulatus, C. sericeus, C. scarabaeoides, C. trinervius and C. acutifolius.
Tertiary gene pool is composed of C. goensis, C. heynei, C. kerstingii, C. mollis,
C. rugosus, C. volubilis, C. platycarpus, C. niveus, C. gandiflorus, C. crassicaulis,
C. rugosus, C. elongates, C. villosus, C. confertiflorus, C. visidus, C. aromaticus,
C. crassicaulis, C. lanuginosus, C. pubescens, C. cinereus, C. marmoratus,
C. mareebensis.C. lanuginosus and C. pubescens, while quaternary gene pool is
composed of Flemingia, Rhynchosia, Dunbaria, Erisema Paracalyx, Adenodolichos,
Bolusafra, Carissoa, Chrysoscias and Baukea.
Pigeonpea is prone to attack by a number of diseases. Resistance in cultivated
gene pool is limited (Pande et al. 2011), therefore wild relatives need to be
investigated. With the objective to investigate wild relatives for identification of
sources resistant to different diseases so as to utilize prebreeding to introgress genes
from wild Cajanus species belonging to secondary and tertiary gene pool in order to
expand the primary gene pool of pigeonpea, a number of experiments were
undertaken at International Crops Research Institute for the Semi-Arid Tropics
(ICRISAT) in Patancheru, India (Sharma and Upadhyay 2016). The experiments
involved advanced backcross population derived from interspecific crosses between
C. cajanifolius, C. acutifolius and C. scarabaeoides belonging to cross-compatible
secondary genepool and C. platycarpus belonging to cross-incompatible tertiary
gene pool as donors and cultivated pigeonpea as recipient. Embryo rescue technique
was utilized to recover population involving tertiary gene pool species. The
populations derived from interspecific crosses were screened for resistance to wilt
and sterility mosaic disease. Two inbred lines derived from C. platycarpus and
15 inbred lines derived from C. acutifolius displayed combined resistance to both
diseases. Phytopthora-resistant inbred lines were also recovered from the population
derived from cross between C. acutifolius and cultivated chickpea. Additionally, a
number of other wild species belonging to secondary and tertiary gene pool of
pigeonpea were also reported to be having resistant traits against a number of
other destructive diseases (Sharma and Upadhyay 2016). For example C. albicans,
C. cajanifolius, C. ineatus, C. scarabaeoides and C. sericeus belonging to secondary
gene pool; C. platycarpus, C. volubilis belonging to tertiary gene pool and
C. sericeus belonging to secondary gene pool were found to be resistant to alternaria
blight and phytophthora blight, respectively.
Apart from the crops described above, wild relatives have also been used as
excellent sources of resistance against various biotic stresses in many crop species
(Table 10.3).
10.8 Conclusion
Plants are the most important creatures on the earth, working like processing
machine that convert one form of ‘solar’ energy into another form on which all the
living organisms depend directly or indirectly for food, shelter and other require-
ment. Thus, higher productivity in terms of quantum and quality and easy and
adequate access to everyone is the only objective of growing plants. High yielding
genotypes have been created using classical as well as molecular breeding
approaches that are under cultivation. Genetic potential of the improved genotypes
are actually not harnessed at farmers’ field due to many reasons, among those biotic
stresses being one of the major causes. Due to excess and extreme nature of parasitic
load, total crop failures have been noted in many crops in the past which led to large
scale damage and consequently famines in many parts of the world. Uses of
synthetic chemicals have been effective in control of pathogens across the different
crops species. This approach is however adding additional burden and also it causes
many environmental and health hazards, in addition to polluting natural resources.
Gene-based resistance strategy seems to be the most economical and also environ-
ment friendly. Dynamic nature of the pathogens however reminds about breakdown
of resistance which requires availability of diverse pool of resistance sources that can
be used as potential source of donor in resistance-breeding programmes. Wild
relatives though have been used in the past, now more systematic investigation is
needed for identification of novel sources of resistant genes considering the change
in environmental parameters due to climate change. Furthermore, other species can
Table 10.3 Crop species, their wild relatives and resistant traits contributed
Crop Wild relative Trait References
Pearl P. glaucum subsp. monodii Rust resistance Hanna et al.
millet (1985)
P. glaucum subsp. monodii Leaf spot Hanna et al.
(1985)
P. orientale Pest Dujardin and
Hanna (1987)
P. pedicellatum Trin. and Downy mildew Dujardin and
P. polystachion Hanna (1989)
Pennisetum glaucum subsp. Striga spp. Hanna et al.
monodii (1985)
Barley Hordeum spontaneum Fusarium spp. Chen et al.
(2013)
Hordeum spontaneum Leaf stripe Biselli et al.
(2010)
Hordeum spontaneum Powdery mildew Schmalenbach
et al. (2008)
Hordeum spontaneum Leaf rust Schmalenbach
et al. (2008)
Hordeum spontaneum Leaf scald Friedt et al.
(2011)
H. bulbosum Powdery mildew, leaf rust Morrell and
and leaf scald Clegg (2011)
Oat Avena strigosa Leaf rust resistance Lehnhoff et al.
(2013)
Avena barbata Powdery mildew Swarbreck
et al. (2011)
Common Phaseolus coccineus Anthracnose, rootrots, white Sharmaand
bean mould, and bean yellow Rana (2012)
mosaic virus
P. coccineus and P. dumosus Angular leaf spot resistance Mahuku et al.
(2003)
P. coccineus and P. dumosus Anthracnose resistance Mahuku et al.
(2002)
P. acutifolius Common blight resistance Singh and
Muñoz (1999)
P. coccineus Root rots resistance Silbernagel
and Hannan
(1992)
P. coccineus Bean golden yellow mosaic Osorno et al.
virus (2003)
Vigna spp. V. vexillata Cowpea pod sucking bug, Kaur et al.
bruchids (2018)
Vigna tribolata, Yellow mosaic virus Kaur et al.
V. mungo var. sylvestris, (2018)
V. radiate var. sublobata
(continued)

Crop Wild relative Trait References
Lentil Lens orientalis, Rust, powdery mildew Singh et al.
L. odomensis and L. ervoides (2013)
Lens ervoides Anthracnose Tullu et al.
(2013)
Groundnut Arachis stenosperma Root-knot nematode Leal-Bertioli
et al. (2016)
F2 (A.duranensis x Late leaf spot resistance Leal-Bertioli
A. stenosperma) et al. (2009)
Brassica B. fructiculosa Cabbage aphid, cabbage root Pink et al.
fly (2003)
Linseed L. grandiflorum Linseed bud fly, Kaur et al.
Alternaria blight (2018)
Sesame Sesamum laciniatum Leaf phyllode Kaur et al.
(2018)
S. malabaricum Powdery mildew Kaur et al.
(2018)
S. mulyanum Powdery mildew Kaur et al.
(2018)
S. alatum Powdery mildew Kaur et al.
(2018)
also be evaluated for either allelic divergence or for entirely new gene. Effective use
of wild and wide genetic resources in resistance-breeding programme is possible
only by integrating genomic tools in resistance-breeding programmes. Different
genomic tools will be helpful in identification of genes and their validation followed
by their introgression in desired genotypes. Minor changes in nucleotide sequence of
a gene, such as single nucleotide change, addition/deletion of few nucleotide
sequences, have now been known to make large differences in expression profile
of one or many associated genes. Recently developed genome editing technologies
have entered with great stride in the boundary of plant breeding and have been
proved to be effective experimentally in precise modification of genome affecting
different functions including disease resistance. Furthermore, perfection and preci-
sion of genome editing (GE) followed by resolving other unresolved issues like
inheritance and recombination of edited sequence may help in the future in develop-
ment of better resisting genotypes. Genotypes loaded with resistance function
developed either by classical approaches or molecular approaches are great input
to the farmers in increasing production and productivity of crops. Resistance func-
tion of the genotypes can be elevated probably by using biological and physical
measures of disease control.
References
Andolfo G, Iovieno P, Frusciante L, Ercolano MR (2016) Genome editing technologies for
enhancing plant disease resistance. Front Plant Sci 7:1813
Arora L, Narula A (2017) Gene editing and crop improvement using CRISPR-Cas9 system. Front
Plant Sci 8:1932
Ashkani S, Rafii MY, Shabanimofrad M, Miah G, Sahebi M, Azizi P, Tanweer FA, Akhtar MS,
Nasehi A (2015) Molecular breeding strategy and challenges towards improvement of blast
disease resistance in rice crop. Front Pl Sci 6:886
Ballini E, Berruyer R, Morel JB, Lebrun MH, Notteghem JL, Tharreau D (2007) Modern elite rice
varieties of the ‘green revolution’ have retained a large introgression from wild rice around the
Pi33 rice blast resistance locus. New Phytologist 175:340–350
Bergquist R (1981) Transfer from Tripsacum dactyloides to corn of a major gene locus conditioning
resistance to Puccinia sorghi. Phytopathology 71:518–520
Biselli C, Urso S, Bernardo L, Tondelli A, Tacconi G, Martino V, Grando S, Val VG (2010)
Identification and mapping of the leaf stripe resistance gene Rdg1a in Hordeum spontaneum.
Theor Appl Genet 120:1207–1218
Blakeslee AF (1904) Sexual reproduction in the mucorineae. Proc Am Acad Arts Sci 40:205–319
Bockus W, Cruz C, Kalia B, Gill B, Stack J, Pedley K (2012) Reaction of selected accessions of
Aegilops tauschii to wheat blast, 2011. Pl Dis Manag Rep 6:CF005
Borlaug NE (1959) The use of multilineal or composite varieties to control airborne epidemic
diseases of selfpollinated crop plants. Proceedings international wheat genetics symposium. vol
1958. Winnipeg, Manitoba, pp 12–26
Borrelli VMG, Brambilla V, Rogowsky P, Marocco A, Lanubile A (2018) The enhancement of
plant disease resistance using CRISPR/Cas9 technology. Front Plant Sci 9:1245
Cai X, Chen PD, Xu SS, Oliver RE, Chen X (2005) Utilization of alien genes to enhance Fusarium
head blight resistance in wheat: a review. Euphytica 142:309–318
Chaturvedi SK, Nadarajan N (2010) Genetic enhancement for grain yield in chickpea:
accomplishments and resetting research agenda. Elect J Pl Breed 1:611–615
Chavan S, Smith SM (2014) A rapid and efficient method for assessing pathogenicity of Ustilago
maydis on maize and teosinte lines. J Visu Exper 83:e50712
Chen G, Liu Y, Ma J, Zheng Z, Wei Y, McIntyre CL, Zheng YL, Liu C (2013) A novel and major
quantitative trait locus for Fusarium crown rot resistance in a genotype of wild barley (Hordeum
spontaneum L.). PLoS ONE 8:e58040
Choudhary K, Choudhary OP, Shekhawat NS (2008) Marker assisted selection: a novel approach
for crop improvement. Am Eurasian J Agric 1:26–30
Collard BCY, Mackill DJ (2008) Marker-assisted selection: an approach for precision plant
breeding in the twenty-first century. Phil Trans R Soc B 363:557–572
Collinge DB, Loud OS, Thordal-Christensen H (2008) What are the prospects for genetically
engineered, disease resistant plants. European J Pl Path 121:217–231
Cruz CD, Peterson GL, Bockus WW, Kankanala P, Dubcovsky J, Jordan KW (2016) The 2NS
translocation from Aegilops ventricosa confers resistance to the Triticum pathotype of
Magnaporthe oryzae. Crop Sci 56:990–1000
Das G, Rao GJN (2015) Molecular marker assisted gene stacking for biotic and abiotic stress
resistance genes in an elite rice cultivar. Front Plant Sci 6:698. https://doi.org/10.3389/fpls.
2015.00698
Dujardin M, Hanna W (1987) Inducing male fertility in crosses between pearl millet and
Pennisetum orientale. Rich Crop Sci 27:65–68
Dujardin M, Hanna WW (1989) Crossability of pearl millet with wild Pennisetum species. Crop Sci
29:77–80
Duvick DN, Smith JSC, Cooper M (2004) Long term selection in a commercial hybrid
maizebreeding program. In: Janick I (ed) Plant breeding reviews. Part 2, vol 24. Wiley,
Ellur RK, Khanna A, Bhowmick PK, Vinod K, Nagarajan M, Mondal KK (2016) Marker-aided
incorporation of Xa38, a novel bacterial blight resistance gene, in PB1121 and comparison of its
resistance spectrum with xa13+ Xa21. Scien Rep 6:29188
Flor HH (1955) Host-parasite interaction in flax rust: its genetics and other implications. Phytopa-
thology 45:680–685
Flor HH (1956) The complementary genic systems in flax and flax rust. Adv Genet 8:29–54
Friedt W, Horsley RD, Harvey BL, Poulsen DME, Lance RCM, Ceccarelli S, Grando S, Capettini F
(2011) Barley breeding history, progress, objectives, and technology. In: Ullrich SE (ed) Barley:
production, improvement, and uses. Wiley-Blackwell, Oxford, pp 160–220
Hajjar R, Hodgkin T (2007) The use of wild relatives in crop improvement: a survey of
developments over the last 20 years. Euphytica 156:1–13
Hanna WW, Wells HD, Burton GW (1985) Dominant gene for rust resistance in pearl millet. J
Hered 76:134
Hartman GL, Wang TC, Hymowitz T (1992) Sources of resistance to soybean rust in perennial
glycine species. Plant Dis 76:396–399
Hartman G, Gardner M, Hymowitz T, Naidoo G (2000) Evaluation of perennial species for
resistance to soybean fungal pathogens that cause sclerotinia stem rot and sudden death
syndrome. Crop Sci 40:545–549
Hooker AL (1981) Resistance to Helminthosporium turcicum from Tripsacum floridanum
incorporated into corn. Maize Genet Coop Newsl 55:87–88
Horsch RB, Fraley RT, Rogers SG, Sanders PR, Lloyd A (1984) Inheritance of functional foreign
genes in plants. Science 223:496–498
ISAAA’s GM Approval Database (2019) http://www.isaaa.org/gmapprovaldatabase
Islam MR, Shepherd KW (1991) Present status of genetics of resistance in flax. Euphytica
55:255–267
Jaiswal HK, Singh BD, Singh AK, Singh RM (1986) Introgression of genes for yield and yield traits
from C. reticulatum into C. arietinum. Int Chickpea Newsl 14:5–8
Jensen NF (1952) Intra-varietal diversification in oat breeding. Agron J 44:30–34
Jordan IK, Rogozin IB, Glazko GV, Koonin EV (2003) Origin of a substantial fraction of human
regulatory sequences from transposable elements. Trends Genet 19:68–72. https://doi.org/10.
1016/S0168-9525(02)00006-9
Johnson T, Newtin M (1940) Mendelian inheritance of certain pathogenic characters of Puccinia
graminis tritici. Canad J Res Sec C Bot Sci 18:599–611
Jorgensen JH (1990) Disease and pest resistance genes. Coor Rep Barley Genet Newsl 20:89
Kaur L, Sirari A, Kumar D, Sandhu JS, Singh S, Singh I (2013) Harnessing ascochyta blight and
botrytis grey mould resistance in chickpea through interspecific hybridization. Phytopath Medit
52:157–165
Khan MA, Naeem M, Iqbal M (2014) Breeding approaches for bacterial leaf blight resistance in rice
(Oryza sativa L.), current status and future directions. Eur J Plant Pathol 139:27–37. https://doi.
org/10.1007/s10658-014-0377-x
Khan MH, Dar ZA, Dar SA (2015) Breeding strategies for improving rice yield-a review. Agric Sci
6:467–478. https://doi.org/10.4236/as.2015.65046
Khush GS (1997) Origin, dispersal, cultivation and variation of rice. Plant Mol Biol 35:25–34
Knights EJ, Southwell RJ, Schwinghamer MW, Harden S (2008) Resistance to Phytophthora
medicaginis Hansen and Maxwell in wild Cicer species and its use in breeding root rot resistant
chickpea (Cicer arietinum L.). J Agricul Res 59:383–387
Koch MF, Parlevliet JE (1991) Genetic analysis of, and selection for, factors affecting quantitative
resistance to Xanthomonas campestris pv oryzae in rice. Euphytica 53:235–245
Kumar J, Choudhary AK, Solanki RK, Pratap A (2011) Towards marker-assisted selection in
pulses: a review. Plant Breed 130:297–313
Kaur V, Shubha KP, Manju MS (2018) Role of crop wild relatives in crop improvement under
hanging climatic conditions. In: Yadav PK, Kumar S, Kumar S, Yadav RC (eds) Crop
improvement for sustainability. Daya Publishing House, New Delhi, pp 13–35
Ladizinsky G, Adler A (1976) The origin of chickpea Cicer arietinum L. Euphytica 25:211–217.
https://doi.org/10.1007/BF00041547
Langner T, Kamoun S, Belhaj K (2018) CRISPR crops: plant genome editing toward disease
resistance. Annu Rev Phytopathol 56:2221–2234
Leal-Bertioli SC, José AC, Alves-Freitas DM, Moretzsohn MC, Guimarães PM, Nielen S, Vidigal
BS, Pereira RW, Pike J, Fávero AP (2009) Identification of candidate genome regions
controlling disease resistance in Arachis. BMC Pl Biol 9:112
Leal-Bertioli SC, Moretzsohn MC, Roberts PA, Ballén-Taborda C, Borba TC, Valdisser PA,
Vianello RP, Araújo ACG, Guimarães PM, Bertioli DJ (2016) Genetic mapping of resistance
to Meloidogyne arenaria in Arachis stenosperma: a new source of nematode resistance for
peanut. Gen Geno Gen 6:377–390
Lehnhoff EA, Keith BK, Dyer WE, Menalled FD (2013) Impact of biotic and abiotic stresses on the
competitive ability of multiple herbicide resistant wild oat (Avena fatua). PLoS ONE 8:64478
Lennon JR, Krakowsky M, Goodman M, Flint-Garcia S, Balint-Kurti PJ (2016) Identification of
alleles conferring resistance to gray leaf spot in maize derived from its wild progenitor species
teosinte. Crop Sci 56:209–218
Leung H, Hettel GP, Cantrell RP (2002) International rice research institute: roles and challenges as
we enter the genomics era. Trends Plant Sci 7:139–142
Maazou ARS, Qiu J, Mu J, Liu Z (2017) Utilization of wild relatives for maize (Zea mays L.)
improvement. African J Plant Sci 11:105–113
Magar MM, Rani CVD, Anuradha G (2014) Marker assisted selection for bacterial leaf blight
resistance in segregating populations of Cottondora sannalu. Int J Appl Sci Biotechnol
2:229–237. https://doi.org/10.3126/ijasbt.v2i3.10570
Mallikarjuna N, Saxena KB, Jadhav DR. (2010) Cajanus.Wild Crop Relativ: Genomic Breed Res.
21–33
Maxted N, Kell SP (2009) Establishment of a global network for the in situ conservation of crop
wild relatives: status and needs. Food and Agriculture Organization of the United
NationsCommission on Genetic Resources for Food and Agriculture, Rome
McFadden ES (1930) A successful transfer of emmer characters to vulgare wheat. J Amer Soc Agro
22:1020–1034
Mcintosh RA (1996) Breeding wheat for resistance to biotic stress. In: Braun HJ, Altay F, Kronstad
WE, Beniwal SPS, McNab A (eds) Wheat: prospects for global improvement, Proceedings of
the 5th international wheat conference, vol 1997. Kluwer Academic Publishers, Dordrecht, pp
71–86
Mignucci JS, Chamberain D (1978) Interactions of Microsphaera diffusa with soybeans and other
legumes. Phytopathology 68:169–173
Monneveux P, Reynolds MP, Aguilar JG, Singh RP, Weber WE (2008) Effects of the 7DL.7Ag
translocation from Lophopyrum elongatum on wheat yield and related morphophysiological
traits under different environments. Plant Breeding 122:379–384
Morishima H, Oka HI (1960) The pattern of interspecific variation in the genus Oryza: its
quantitative representation by statistical methods. Evolution 14:153–165
Morrell PL, Clegg MT (2011) Hordeum. In: Kole C (ed) Wild crop relatives: genomic and breeding
resources. Springer, Berlin, pp 309–319
Moseman JG, Nevo E, Morshidy MAE, Zohary D (1984) Resistance of Triticum dicoccoidesto
infection with Erysiphe graminis tritici. Euphytica 33:41–47
Nault L, Findley W (1982) Zea diploperennis: a primitive relative offers new traits to improve corn.
Desert Plants 3:203–205
Oliver RE, Stack RW, Miller JD, Cai X (2007) Reaction of wild emmer wheat accessions to
Fusarium head blight. Crop Sci 47:893–897
Orton WA (1900) The wilt disease of cotton and its control. US department of agriculture, division
of vegetable physiology and pathology. Bulletin 27
Pande S, Sharma M, Mangala UN, Ghosh R, Sundaresan G (2011) Phytophthora blight of

pigeonpea [Cajanus cajan(L.) Millsp.]: an updating review of biology, pathogenicity and
disease management. Crop Prot 30:951–957
Parlevliet JE (1979) Components of resistance that reduce the rate of epidemic development. Annu
Parlevliet JE (2002) Durability of resistance against fungal, bacterial and viral pathogens; present
situation. Euphytica 124:147–156. https://doi.org/10.1023/A:1015601731446
Pink DAC, Kift NB, Ellis PR, McClemant SJ, Lynn J, Tatchell GM (2003) Genetic control of
resistance to aphid Brevicoryne brassicae in the wild species Brassica fruticulosa. Plant Breed
122:24–29
Pradhan SK, Nayak DK, Mohanty S, Behera L, Barik SR, Pandit E et al (2015) Pyramiding of three
bacterial blight resistance genes for broad-spectrum resistance in deep water rice variety,
Jalmagna. Rice 8:19. https://doi.org/10.1186/s12284-015-0051-8
Rafique MZ, Zia M, Rashid H, Chaudhary MF, Chaudhry Z (2010) Comparison of transgenic plant
production for bacterial blight resistance in Pakistani local rice (Oryza sativa L.) cultivars. Afr J
Biotechnol 9:1892–1904. https://doi.org/10.5897/AJB09.868
Reif JC, Melchinger AE, Xia XC, Warburton ML, Hoisington DA, Vasal SK, Beck D, Bohn M,
Frisch M (2003) Use of SSRs for establishing heterotic groups in subtropical maize. Theor Appl
Genet 107:947–957. https://doi.org/10.1007/s00122-003-1333-x
Ribaut JM, de Vicente MC, Delannay X (2010) Molecular breeding in developing countries:
challenges and perspectives. Curr Opinion Plant Bio 13:1–6
Saxena KMS, Hooker AL (1968) On the structure of a gene for disease resistance in maize. Proc Nat
Acad Sci USA 61:1300–1303
Schmalenbach I, Körber N, Pillen K (2008) Selecting a set of wild barley introgression lines and
verification of QTL effects for resistance to powdery mildew and leaf rust. Theoretical Appl Gen
117:1093–1106
Sharma SK, Rana JC (2012) Strategies for the conservation of crops wild relatives: Indian context.
In: Sharma AK, Ray D, Ghosh SN (eds) Biological diversity – origin, evolution and conserva-
tion. Viva Books Private Limited, New Delhi, pp 433–468
Sharma S, Upadhyay HD (2016) Pre-breeding to expand primary genepool through introgression of
genes from wild cajanus species for pigeonpea improvement. Legume Perspect 11:17–20
Sharma T, Rai A, Gupta S, Vijayan J, Devanna B, Ray S (2012) Rice blast management through
host-plant resistance: retrospect and prospects. Agricul Res 1:37–52
Shamsudin NAA, Swamy BPM, Ratnam W, Cruz MTS, Raman A, Kumar A (2016) Marker
assisted pyramiding of drought yield QTLs into a popular Malaysian rice cultivar, MR219.
BMC Genet 17:30. https://doi.org/10.1186/s12863-016-0334-0
Singh KB, Ocampo B (1997) Exploitation of wild cicer species for yield improvement in chickpea.
Theor Appl Gen 95:418–423
Singh BD, Jaiswal HK, Singh RM, Singh AK (1984) Isolation of early flowering recombinants
from the interspecific cross between Cicer arietinum and C. reticulatum. Int Chickpea Newsl
11:14
Singh S, Gumber RK, Joshi N, Singh K (2005) Introgression from wild Cicer reticulatum to
cultivated chickpea for productivity and disease resistance. Plant Breed 124:477–480
Singh K, Chhuneja P, Ghai M, Kaur S, Goel RK, Bains NS, Keller B, Dhaliwal HS (2007)
Molecular mapping of leaf and stripe rust resistance genes. In: Buck et al. (eds)
T. Monococcum and their transfer to hexaploid wheat. Wheat production in stressed
environments, Springer, Cham, pp 779–786
Singh M, Rana MK, Kumar K, Bisht IS, Dutta M, Gautam NK, Sarker A, Bansal KC (2013)
Broadening the genetic base of lentil cultivars through inter-subspecific and interspecific crosses
of Lens taxa. Plant Breed 132:667–675
Sleper DA, Poehlman JM (2006) Breed field crops. Crop Sci 47:900–901
Smith EF (1896) A bacterial disease of the tomato, eggplant and Irish potato (Bacillus solanacearum
Nov. sp.). USDA. Bulletin 12:1
Smith CM, Havlickova H, Starkey S, Gill BS, Holubec V (2004) Identification of Aegilops
germplasm with multiple aphid resistance. Euphytica 135:265–273
Sodkiewicz W, Strzembicka A, Apolinarska B (2008) Chromosomal location in triticale of leaf rust
resistance genes introduced from Triticum monococcum. Plant Breed 127:364–367
Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Ronald P (1995) A receptor kinase-like
protein encoded by the rice disease resistance gene, Xa21. Science 270:1804–1806
Suh JP, Cho YC, Won YJ, Ahn EK, Baek MK, Kim MK, Kim BK, Jena KK (2015) Development of
resistant gene-pyramided Japonica rice for multiple biotic stresses using molecular marker-
assisted selection. Plant Breed Biotechnol 3:333–345. https://doi.org/10.9787/PBB.2015.3.4.
333
Swarbreck SM, Lindquist AE, Ackerly DD, Andersen GL (2011) Analysis of leaf and root
transcriptomes of soil grown Avena barbata plants. Plant Cell Physio 52:317–332
Tagle AG, Chuma I, Tosa Y (2015) Rmg7, a new gene for resistance to Triticum isolates of
Pyricularia oryzae identified in tetraploid wheat. Phytopathology 105:495–499
Tullu A, Bett K, Banniza S, Vail S, Vandenberg A (2013) Widening the genetic base of cultivated
lentil through hybridization of Lens culinaris “Eston” and L. ervoides accession IG72815. Can J
Plant Sci 93:1037–1047
Van der Maesen LJG (1986) Cajanus DC. and Atylosia W. & A. (Leguminosae). Agricultural
University, Wageningen Papers 85–4 (1985). Agricultural University, Wageningen, pp 1–225
Vasudevan K, Gruissem W, Bhullar NK (2015) Identification of novel alleles of the rice blast
resistance gene Pi54. Sci Rep 5:15678
Vaughan D (1989) The genus Oryza L.: current status of taxonomy, IRRI Paper Series. Number
138. International Rice Research Institute, Manila, p 21
Vencovsky R, Ramalho MAP (2006) Contribuicoes do melhoramento genético no Brasil. In:
Paterniani E (ed) Ciência, agricultura e sociedade, Contributions of Plant Breeding in Brazil.
Embrapa, Brasília, DF, pp 41–74
Waines JG, Ehdaie B (2007) Domestication and crop physiology: roots of green-revolution wheat.
Annales Botanici 100:991–998
Wan YF, Yen C, Yang JL (1997) Sources of resistance to head scab in Triticum. Euphytica
94:31–36
Wang J, Liu W, Wang H, Li L, Wu J, Yang X (2011) QTL mapping of yield-related traits in the
wheat germplasm 3228. Euphytica 177:277–292
Wang X, Lee S, Wang J, Ma J, Bianco T, Jia Y (2014) Current advances on genetic resistance to rice
blast disease. In: Yan W, Bao J (eds) Rice—germplasm, genetics and improvement. InTech,
Rijeka, pp 195–217
Wei WH, Qin R, Song YC, Guo LQ, Gu MG (2001) Comparative analyses of disease resistant and
nonresistant lines from maize Zea diploperennis by GISH. Bot bulletin Acad Sinica
42:109–114
Wijerathna YMAM (2015) Marker assisted selection: biotechnology tool for rice molecular breed-
ing. Adv Crop Sci Technol 3:187. https://doi.org/10.4172/2329-8863.1000187
Wulff BBH, Moscou MJ (2014) Strategies for transferring resistance to wheat: from wide crosses to
GM cassettes. Front Plant Sci 5:692
Xu J (2013) Pyramiding of two BPH resistance genes and Stv-bi gene using marker-assisted
selection in japonica rice. Crop Breed Appl Biotechnol 13:99–106. https://doi.org/10.1590/
S1984-70332013000200001
Yabuuchi A, Kosako Y, Yano L, Hotta H, Nishiuchi Y (1996) Validation of the publication of new
names and new combinations previously effectively published outside the IJSB Int. J Syst
Bacteriol 46:625–626
Yadav S, Anuradha G, Kumar RR, Vemireddy LR, Sudhakar R, Donempudi K, Venkata D,
Jabeen F, Narasimhan YK, Marathi B, Siddiq EA (2015) Identification of QTLs and possible
candidate genes conferring sheath blight resistance in rice (Oryza sativa L.). Springer Plus
4:175. https://doi.org/10.1186/s40064-015-0954-2
Yao G, Zhang J, Yang L, Xu H, Jiang Y, Xiong L, Zhang C, Zhang Z, Ma Z, Sorrells ME (2007)

Genetic mapping of two powdery mildew resistance genes in einkorn (Triticum monococcum
L.) accessions. Theoretical Appl Gen 114:351–358
Yellareddygari SKR, Reddy MS, Kloepper JW, Lawrence KS, Fadamiro H (2014) Rice sheath
blight: a review of disease and pathogen management approaches. J Plant Pathol Microb 5:241.
https://doi.org/10.4172/2157-7471.1000241
Yediay F, Baloch F, Kilian B, Ozkan H (2010) Testing of rye-specific markers located on 1RS
chromosome and distribution of 1AL.RS and 1BL.RS translocations in Turkish wheat (Triticum
aestivum L. durum Desf.) varieties and landraces. Gen Resour Crop Evol 57:119–129
Young ND (1996) QTL mapping and quantitative disease resistance in plants. Ann Rev Phytopath
34:479–501
Yumurtaci A (2015) Utilization of wild relatives of wheat, barley, maize and oat in developing
abiotic and biotic stress tolerant new varieties. Emirates J Food Agricul 27:1–23
Zhang F, Xie J (2014) Genes and QTLs resistant to biotic and abiotic stresses from wild rice and
their applications in cultivar improvements. In: Bao J (ed) Rice-Germplasm, genetics and
improvement. IntechOpen, London, pp 59–78
Zhang Q, Wang C, Zhao K, Zhao Y, Caslana V, Zhu X (2001) The effectiveness of advanced rice
lines with new resistance gene Xa23 to rice bacterial blight. Rice Genetics Newsl 18:71–72
Zhou L, Wang JK, Yi Q, Wang YZ, Zhu YG, Zhang ZH (2007) Quantitative trait loci for seedling
vigor in rice under field conditions. Field Crop Res 100:294–301. https://doi.org/10.1016/j.fcr.
2006.08.003
Zhou YL, Uzokwe VNE, Zhang CH, Cheng LR, Wang L, Chen K, Gao XQ, Sun Y, Chen JJ, Zhu
LH, Zhang Q, Ali J, Xu JL, Li ZK (2011) Improvement of bacterial blight resistance of hybrid
rice in China using the Xa23 gene derived from wild rice (Oryza rufipogon). Crop Prot
30:637–644
New-Generation Fungicides for Sustainable
Production and Disease Suppression 11
Dele Omoyele Adeniyi, Deepshikha Kunwar,
Lelia Nkechinyere Dongo, David Adedayo Animasaun,
and T. Aravind
Abstract
In modern-day agriculture, with the population increasing at an alarming rate, it’s

difficult to increase the production with the ever-decreasing land and water
resources. The plant diseases alone account for more that 15–20% of the yield
loss caused by the various biotic and abiotic factors. Efficient management of
these plant pathogens will help in increasing the productivity of the crop and lead
to enhanced production. Among the various disease management strategies, the
chemical control using the fungicides has been the most widely adopted method
for fungal disease management. There have been many adverse effects on the
environment and human health associated with the use of the traditional
fungicides that were less efficient and broad spectrum. In order to overcome the
limitation of these fungicides, many new fungicides with novel mode of action
have been developed in the past two decades. These so-called new-generation
fungicides are highly efficient even at low doses, are more target specific, and
leave no or very less residue on the produce. This chapter reiterates the different
new-generation fungicides along with their mode of action and target pathogens.
D. O. Adeniyi (*)
Department of Plant Biology, University of Ilorin, Ilorin, Kwara State, Nigeria
Plant Pathology Section, Cocoa Research Institute of Nigeria, Ibadan, Nigeria
D. Kunwar · T. Aravind
Department of Plant Pathology, G B Pant University of Agriculture &Technology, Pantnagar,
Uttarakhand, India
L. N. Dongo
Plant Pathology Section, Cocoa Research Institute of Nigeria, Ibadan, Nigeria
D. A. Animasaun
Department of Plant Biology, University of Ilorin, Ilorin, Kwara State, Nigeria

https://doi.org/10.1007/978-981-15-6275-4_11
250 D. O. Adeniyi et al.
Keywords
New-generation fungicides · Novel · Mode of action · Disease management
11.1 Introduction
Agriculture has provided the pertinent origin of subsistence for human societies over
the years, and this has provided means of survival to about 50% of the population of
the world till now. Greater percentage of the world populations are directly or
indirectly dependent on agricultural produce and allied activities for their sustenance
and survival. With the ever-increasing population, it has become increasingly
difficult to enhance our production with limited area. There are many factors
which influence the food production like biotic and abiotic factors. The major factor
which influences food production is plant diseases. Crop losses due to plant diseases
constitute the most significant constraint worldwide to increasing productivity and
total food production. Crop loss due to diseases, weeds, and pest is about 10–30% of
crop production (Kumar and Gupta 2012; Kumar 2009). In the history of plant
diseases, there were many diseases which took form of epidemic such as the Irish
Famine and Bengal Famine which occurs due to rice brown spot. Like these there are
many diseases which are of historical significance and cause impact on human life.
To minimize these crop losses, chemical methods for management of plant diseases
using fungicides are more preferred by the farming community.
11.2 Key Methods for Management of Plant Diseases
1. Regulatory measures
2. Cultural methods
3. Biological methods
4. Chemical control
There are different management strategies which can be adopted from sowing to
harvesting stages of crop. Although different management strategies are present, still
we rely more on chemicals for crop disease management due to their high efficiency
and comparatively lower CB ratio. Diseases in plants are caused by a number of
microorganisms which include fungi, bacteria, viruses, viroids, and phytoplasma;
among all these, a large number of diseases are caused by fungi, and to manage these
fungal diseases, different fungicides are used (Dehne et al. 2007). Use of chemical
fungicides in agriculture was started in the nineteenth century when effectiveness of
copper fungicides was discovered against seed-borne bunt by B. Prevost. Since the
nineteenth century, many fungicides were discovered for plant disease management
(Tables 11.1, 11.2, 11.3 and 11.4). In the past two decades, several novel fungicides
were also discovered which are better than earlier fungicides in their mode of action
and are more effective under low doses (Sajad et al. 2017).
11 New-Generation Fungicides for Sustainable Production and Disease Suppression 251
Table 11.1 History of fungicides

Year Fungicides
1637 Brine
1755 Arsenic
1760 Copper sulfate
1824 Sulfur (dust)
1833 Lime sulfur
1885 Bordeaux mixture
1934 Dithiocarbamates
1951–1960 Captan
1971–1980 Dicarboximides, sterol biosynthesis inhibitors, phenylamides, alkyl phosphonates,
carbamates, isoxazoles, tricyclazoles, cyanoacetamide-oximes, melanin
biosynthesis inhibitors
1991–2005 Strobilurins, phenylpyrroles, anilinopyrimidines, spiroxamines, probenazole,
benzothiadiazole, phenylpyridylanines, quinolines
Table 11.2 First-generation fungicides

Fungicide class Active ingredient
Dithiocarbamate Thiram, zineb, nabam, maneb, mancozeb
Phthalimide Captan, captafol
Triazine Anilazine
Guanidine Dodine
Tin compounds TPTA, TPTH
Chloronitro benzenes PCNB
Table 11.3 Second-generation fungicides (1966–1976)

Oxathiins Carboxin and oxycarboxin
Benzimidazoles Thiabendazole, benomyl
Hydroxypyrimidines Ethirimol, dimethirimol
Dicarboximides Iprodione, vinclozolin, procymidone
Table 11.4 Third-generation fungicides (1977–1990)

Phenylamides Metalaxyl, ofurace, oxadixyl
Sterol biosynthesis Triazoles, imidazoles, piperazines, piperidines, pyrimidines,
inhibitors morpholines
Carbamates Prothiocarb, propamocarb
11.3 Classification of Fungicides on the Basis of Generations
11.3.1 Fourth-Generation/New-Generation Fungicides (Novel

Modes of Action)
1. Strobilurins
2. Melanin biosynthesis inhibitors
3. Phenylpyrroles
4. Anilinopyrimidines
5. Phenoxyquinolines
6. Spiroketalamines
7. Benzamides
8. Oxazolidinediones
11.3.1.1 Strobilurins
Strobilurins are new class of fungicides which are isolated from wood-decaying
basidiomycete fungus Strobilurus tenacellus. Strobilurins’ group of fungicides
inhibits mitochondrial respiration in fungi. At Qo site of the cytochrome bc1 com-
plex, oxidation of ubiquinol is blocked which is situated at the inner mitochondrial
membrane of fungi (Knight et al. 1997). These fungicides are now referred to as QoI
fungicides. These are world’s biggest selling fungicides (Bartlett et al. 2002). These
classes of fungicides are of broad-spectrum activity and are active against many
pathogens. The application of these classes of fungicides is recommended for seed
treatment, foliar treatment, and furrow application. The commercial strobilurins are
azoxystrobin, kresoxim-methyl, metominostrobin, trifloxystrobin, pyraclostrobin,
picoxystrobin, etc. Azoxystrobin was patented by Zeneca in 1988 which was
followed by other two compounds which are kresoxim-methyl and trifloxystrobin.
Kresoxim-methyl exhibits excellent eradicant properties against powdery mildew
(Gold et al. 1995). One advantage of this class of fungicide which makes it more
important is that it is effective against the fungal strains which developed resistance
against phenylamide, DMIs, dicarboximide, and benzimidazoles.
11.3.1.2 Melanin Biosynthesis Inhibitors

This group of fungicides prevents melanin biosynthesis in appressoria of Pyricularia
oryzae and prevents penetration of pathogen. MBI are effective against rice blast
disease (Koichiro et al. 2003). They act on enzymes dihydroxynaphthalene (DHN)
melanin in biosynthesis pathway through fusion of five isoprenyl units, two sets of
alternating reduction, and dehydration steps and polymerization of
1,8-dihydroxynaphthalene. In this process, fungicides like phthalide, tricyclazole,
and pyroquilon inhibit the reduction step, and carpropamid inhibits the dehydration
step. MBI prevents pathogen to enter into the host epidermis; these are commonly
anti-penetrants in their mode of action. Melanization of appressorial walls is very
important for successful penetration of pathogen into the host epidermis. MBI
inhibition in melanin synthesis provides excellent control of pathogens P. grisea
and Colletotrichum spp. also.
11.3.1.3 Phenylpyrroles
Phenylpyrroles are based on pyrrolnitrin, which are secondary metabolites produced
by Pseudomonas pyrrocinia having antifungal properties (Floss et al. 1971).
Pyrrolnitrin is instable under light; therefore, it is unsuitable to use practically in
disease control (Corran et al. 2008). Phenylpyrroles inhibit all stages of fungal
development like spore germination, germ tube elongation, and mycelial growth
(Leroux et al. 1992). Optimization of photostability of this compound results in the
development of two commercial fungicides fenpiclonil and fludioxonil which are
used as seed dressing and foliar spray, respectively. Primary targets of
phenylpyrroles are uncertain; they appear to affect glucose phosphorylation.
11.3.1.4 Anilinopyrimidines
Anilinopyrimidines are also known as pyrimidinamines, which are broad-spectrum
fungicides. This class of fungicide can be used in a variety of crops. Mepanipyrim
and pyrimethanil are active against Botrytis cinerea on grapevine and other fruits and
Venturia inaequalis on apples (Neumann et al. 1992). Another such compound,
cyprodinil, of this class has additional activity against Pseudocercosporella
herpotrichoides, Erysiphe graminis, Helminthosporium gramineum, Pyrenophora
teres, and Septoria nodorum on cereals (Heye et al. 1994). Anilinopyrimidines are
considered to be involved in methionine biosynthesis inhibition; biochemical mode
of action is still uncertain (Leroux 1996). They are single-site inhibitors in the amino
acid biosynthesis pathway and also affect the secretion of hydrolytic enzymes during
penetration of the target pathogens into plant tissue. In fungi Neurospora crassa and
Aspergillus nidulans, the cystathionine pathway has been established as the major
route of homocysteine and methionine biosynthesis; within this pathway,
cystathionine β-lyase which catalyzes the synthesis of homocysteine from
cystathionine was identified as a target site (Masner et al. 1994).
11.3.1.5 Phenoxyquinolines
Phenoxyquinolines consist of protectant fungicide (Longhurst et al. 1996).
Quinoxyfen was introduced in 1996 for control of powdery mildew of cereals and
grapevine. Quinoxyfen disrupts signaling processes which are crucial for growth and
development; this fungicide changes in the early development stages of powdery
mildew. It also interferes with conidia germination and appressorium formation in
the life cycle of target fungi. Phenoxyquinolines act on dihydroorotate dehydroge-
nase in pyrimidine biosynthesis pathway (Knight et al. 1997).
11.3.1.6 Spiroketalamines
In spiroketalamines, the representative compound is spiroxamine which was
introduced in 1996. Spiroxamine is a novel ergosterol biosynthesis inhibitor (that
is essential for the organization and functions of structure) (Dutzmann et al. 1996). It
controls powdery mildew of cereals and grapes. Spiroketalamines shows cross-
resistance with morpholines and piperidines.
11.3.1.7 Benzamides
The benzamide class is also called anti-tubulin fungicide which includes zoxamide.
Zoxamide fungicide is a β-tubulin inhibitor. Benzamides have demonstrated their
potential for the control of oomycete pathogens and interfere with microtubule
skeleton and arrest nuclear division similar to benzimidazoles (Young and Richard
2001). It exhibits high activity against a broad spectrum of oomycetes such as
Phytophthora infestans, Plasmopara viticola, and various Pythium spp. (Chao
et al. 2011). It also acts against certain non-oomycete fungi such as Venturia spp.,
Sclerotinia spp., Mycosphaerella spp., Botrytis spp., and Monilinia spp. (Young and
Richard 2001).
11.3.1.8 Oxazolidinediones
This class of fungicide is represented by famoxadone. This class has a broad-
spectrum activity and belongs to bc1 complex Q0I family (Abrue et al. 2006).
Famoxadone acts as protectant compound. It is used against late blight of potato
and downy mildew of grapes (Thind 2012). This compound inhibits pathogen by
inhibiting activity of ubiquinol cytochrome C oxidoreductase (Sajad et al. 2017).
Famoxate is a newly developed fungicide useful for preventative and curative
control of fungal diseases in crops. This new class of fungicide acts on the catalytic
function of mitochondrial cytochrome of bc1 (Douglas et al. 1999).
11.3.1.9 Recently Developed New-Generation Fungicides in Nigeria

In Nigeria, some of the recently developed new-generation fungicides/active
ingredients on crops and farmlands have been registered for use, while some others
are currently under evaluation for different diseases. Few of the new-generation
fungicides registered in Nigeria are for use against major diseases of cash and annual
crops, fruits, and vegetables. And some of these new-generation fungicides are as
follows:
1. Cabrio Duo (pyraclostrobin and dimethomorph), Pergado (metalaxyl-M +

mandipropamid), Ridomil Gold Plus (metalaxyl-M, cuprous oxide), and Red
Force (copper(I) oxide + metalaxyl-M) against black pod disease of cocoa caused
by Phytophthora species
2. Camazeb (carbendazim + mancozeb) against twig and inflorescence dieback of
cashew and pod rot of cacao
3. Ridomil Gold MZ (metalaxyl-M + mancozeb) against oomycete diseases in
beans, cucurbit, lettuce, onions, spinach, pepper, pineapple, rubber, oil palm,
citrus, potatoes, soybeans, sugar beet, tobacco, and tomatoes
4. Ortiva Top (azoxystrobin, difenoconazole) against early blight, black scurf, black
dot in potatoes, powdery mildew, leaf spots, early blight, anthracnose in
tomatoes, leaf and neck blast in rice, and gray leaf spot, smuts, and rusts in grains
and sorghum
5. Fungicare (azoxystrobin, difenoconazole) against rust disease; powdery mildew
in rice, maize, and wheat; leaf spot; early and late blight in potato, tomato,
cucumber, pepper, onion, and carrot; black spot; leaf rust; stem rot and powdery
mildew in soybeans, cowpea, and groundnut; and disease complexes in mango,

citrus, cashew, guava, banana, and ornamental plants
There is significant improvement in the formulations of the new fungicides,

which are also less phytotoxic and produce safe crops. Due to their novel mode of
action and specificity, their demand and use are increasing day by day.
11.4 Conclusion
For plant disease management, chemicals are very important because they prevent
plant diseases. In spite of the advantages of chemicals in food production, there are
also disadvantages of their use. Due to inadequate use of chemicals, many pathogens
had developed resistance against many chemicals. Moreover, another major disad-
vantage of using these chemicals is their negative effects on the environment,
humans, and other living organisms. So to overcome these negative effects, some
new classes of fungicides are developed, which are better than those old chemicals in
many ways in that these new compounds which have originated from different
approaches such as traditional random screening and from natural products are
expected to provide better disease control options. These are ecologically safe and
show good efficacy at much lower doses. These require lesser treatment per season
compared to earlier compounds. Since they possess novel mode of action, there are
fewer chances of cross-resistance to previous fungicides. These are some of the
advantages of new-generation fungicides which will help in food production without
causing harm to the environment.
11.5 Future Prospects
For management of plant diseases, there are many methods which are adopted in the
field, and fungicides will always play a very important role in this management.
Nowadays, it is very important to use chemicals wisely because there are many
chemicals which have developed resistance against many pathogens, so it will be
always challenge for us to use chemicals in a selective and judicious manner.
New-generation fungicides which are derived from natural origin are less harmful
to the environment and are novel in their action, so in the future more new novel
fungicides should be developed so that we can overcome the problems of resistance
and environment pollution from earlier chemicals. Development of ecologically
safer molecules with broader range of target pathogens can be done. To minimize
the resistance risk development and use of combination products with multisite
action can be a good option for disease management. These novel action fungicides
will be obtained by screening of natural-based products.
Acknowledgment The authors wish to appreciate the support provided by Mr Ismail Akanmu of
Jubaili Agrotec Limited, Nigeria, Mr Adewole Fatokun of BASF Nigeria Limited, and Mr Akeem
Abimbola of Syngenta, Nigeria.
References
Abreu SM, Caboni P, Pirisi FM, Garau VL (2006) Residues of the fungicide famoxadone in grapes
and its fate during wine production. Food Addit Contam 23:1–3
Bartlett WB, Clough JM, Godwin JR, Hall AA, Mick H, Bob PD (2002) The strobilurin fungicides.
Pest Manag Sci 58:649–662
Chao Y, Chantal H, Vladimir V, Yantai G (2011) Fungicide: modes of action and possible impact
on non-target microorganisms. Ecology 21:1–8
Corran A, Knau G, Zeun R (2008) Fungicides acting on signal transduction. In: Krämer W,
Schirmer U (eds) Modern crop protection compounds. Wiley-VCH Verlag GmbH, Weinheim,
pp 561–580
Dehne HW, Deising HB, Gisi U, Kuck KH, Russell PE, Lyr H (2007) Modern fungicides and
antifungal compounds. 15th International Rein hard in brunn symposium, vol 52. Intercept,
Andover, pp 107–157
Douglas BJ, Robert SL, John JB, Keith ED, Stephen O (1999) Mode of action of famoxadone.
Pestic Sci 55:105–118
Dutzmann S, Berg D, Clausen NE, Kramer W, Kuck KH (1996) A novel foliar fungicide with a
particular activity against powdery mildew. Proc Brighton Crop Prot Conf Pests Dis 1:47–52
Floss HG, Manni PE, Hamill RL, Mabe JA (1971) Further studies on the biosynthesis of
pyrrolnitrin from tryptophan by Pseudomonas. Biochem Biophys Res Commun 45:781–787
Gold RE, Ammerman E, Kohle H, Leinhos GM, Lorenz G (1995) The synthetic strobilurin BAS
490 F: profile of a modern fungicide. 84a: pp 79–92
Heye UJ, Speich J, Siegle H, Wohlhauser R, Hubele A (1994) CGA 219417- A novel broad-
spectrum fungicide. Proc Brighton Crop Prot Conf Pests Dis 1:501–508
Knight SC, Anthon VM, Brady AM, Greenland AJ, Heaney SP (1997) Rationale and perspectives
on the development of fungicides. Annu Rev Phytopathol 35:349–372
Koichiro K, Makiichi T, Tsutomu S, Kozo N (2003) Diagnosis of dehydratase inhibitors in melanin
biosynthesis inhibitor (MBI-D) resistance by primer-introduced restriction enzyme analysis in
scytalone dehydratase gene of Magnaporthegrisea. Pest Manag Sci 59:843–846
Kumar S (2009) Plant disease management in India: advances and challenges. Afr J Agric Res 9
(15):1207–1217
Kumar S, Gupta O (2012) Expanding dimensions of plant pathology. JNKVV Res J 46:286–293
Leroux P (1996) Recent developments in the mode of action of fungicides. Pestic Sci 47:191–197
Leroux P, Lanen C, Fritz R (1992) Similarities in the antifungal activities of fenpiclonil, iprodione
and tolclofos-methyl against Botrytis cinerea and Fusarium nivale. Pestic Sci 36:255–261
Longhurst, C., Dixon, K. and Mayer, A. (1996) DE‐795—a novel fung‐icide for the control of
powdery mildew in cereals. In Proceedings of the Brighton Crop Protection Conference—Pests
and Diseases. pp. 21 – 26. Farnham, UK: British Crop Protection Council.
Masner P, Muster P, Schmid J (1994) Possible methionine biosynthesis inhibition by
pyrimidinamine fungicides. Pestic Sci 42:163–166
Neumann GL, Winter EH, Pittis JE (1992) Pyrimethanil: a new fungicide. Proc Brighton Crop Prot
Conf Pests Dis 1:395–402
Sajad N, Wasim H, Mohammad SD (2017) New generation fungicides in disease management of
horticultural crops. Indian Hortic J 7:01–07
Thind TS (2012) New generation fungicides. National symposium on plant health management
annual meeting of IPS, NZ September 28–29. pp 148–149
Young DH, Richard AS (2001) Mode of action of Zoxamide, a new Oomycete fungicide. Pestic
Biochem Physiol 69:100–111
Toxicity of Organophosphate Pesticide
on Soil Microorganism: Risk Assessments 12
Strategies
Durgesh Kumar Jaiswal, Ram Krishna, Saurabh Singh, Tarun Belwal,

Jay Prakash Verma, and Janardan Yadav
Abstract
Synthetic organophosphate pesticide (Op) is used extensively in modern agricul-

ture by farmers to enhance crop yield under a limited agriculture land resource as
well as food for the increasing population worldwide. Further, OP is increasing
due to more adaptation and resistance of varieties of pests in agricultural crops.
Recently, the toxicity of OPs has increased in the soil ecosystem so that some
microbes may be survived and some may be lost. Therefore, soil fertility and
health may be disturbed due to more toxicity of OPs. Currently, pesticide-
resistant microbes can be one alternative for boosting the agricultural productivity
as well as enhancing soil fertility and health. Therefore, in this chapter, we
attempt to explore the risk assessment methodology and techniques as well as
challenges regarding toxicity of OPs on the soil ecosystem and plant-microbe
interaction function. Also, we attempt to explore the advance analytical
techniques and molecular tools, like culture-dependent and culture-independent
methods for measuring tolerance of multiple pollutants at the microbial commu-
nity level and ecological disturbance.
D. K. Jaiswal · R. Krishna · S. Singh · J. P. Verma (*)

Department of Environment & Sustainable Development, Institute of Environment and Sustainable
Development, Banaras Hindu University, Varanasi, Uttar Pradesh, India
e-mail: durgesh.jaiswal@9gmail.com; jpv.iesd@bhu.ac.in
T. Belwal
College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agri-Food
Processing, Zhejiang University, Hangzhou, People’s Republic of China
J. Yadav
Department of Soil Science and Agricultural Chemistry, Institute of Agriculture Science, Banaras
Hindu University, Varanasi, UP, India

https://doi.org/10.1007/978-981-15-6275-4_12
258 D. K. Jaiswal et al.
Keywords
Organophosphate pesticide · Plant growth-promoting microbes (PGPM) ·

Toxicity assessment methodologies · Culture-independent and -dependent
analyses
12.1 Introduction
Soil microbes are the most important factor in determining good soil and plant
health. The microbial diversity of the soil helps in carrying out several vital functions
necessary for the well-being of plants. Microbes act as mediators and feed the plants
in the very literal sense. Many nutrients present in the soil are in unavailable form,
where these microbes come to the rescue of plants. Based on the type of environment
they inhabit, they can be classified into various types. There are many free-living
microbes, symbiotic microbes, soil inhabiting microbes, endophytic microbes, and
so on. Many microbes that live in association with the plants or around them may not
function at all until its requirement comes. There are many microbes, which help
mitigate various plant stresses and promote plant growth; also, they are called as
plant growth-promoting microbes (PGPM) (Table 12.1). There are many microbes
that live in association with the plants to provide beneficial effects to them. The most
commonly known are the Rhizobium and Bradyrhizobium (mostly belonging to
α-proteobacteria), which live in symbiotic association with the root nodules of the
leguminous plants and carry out nitrogen fixation (Appelbaum 2018). These help in
fixing the atmospheric nitrogen unavailable to plants to organic forms of nitrogen,
which is readily available to them. These microbes facilitate plant growth, by
providing nitrogen to plants, and in turn are benefitted from the plants. This is
vital as it provides substantial economic and environmental benefits. Mineral solu-
bilization is also an important function performed by the soil microbes, which
facilitate plants with the uptake of essential nutrients, such as phosphate solubiliza-
tion, potassium solubilization, and many others. Phosphate-solubilizing microbes
(bacteria and fungi) play an important role in P bioavailability and are used to
increase the phosphorous uptake in plants. These microbes help in solubilizing
inorganic P into soluble forms. This process is achieved through many mechanisms,
the usual of which is the production of organic acids by the phosphate-solubilizing
bacteria (mostly Gram-negative), which leads to the dissolution of mineral glucose
to gluconic acid. These organic acids act as good chelating agents of Ca2+ ions and
thus release phosphates from calcium phosphate (Shenoy and Kalagudi 2005).
These bacterial species include Enterobacter sp., Pantoea sp., Klebsiella sp., and
many others. Apart from the solubilization of inorganic calcium phosphates, iron and
aluminum phosphates are also solubilized by phosphate-solubilizing microbes.
Potassium, another essential nutrient for the plants, is also facilitated to plants by
some capable soil microbes. The main sources of potassium in soil include
K-feldspar, muscovite, biotite phlogopite, and mica. The microbes, such as Pseudo-
monas spp., Bacillus spp., and Burkholderia spp., can be potassium-solubilizing
12 Toxicity of Organophosphate Pesticide on Soil Microorganism: Risk Assessments. . . 259
Table 12.1 List of beneficial soil microbes (BSM) with their plant growth–promoting activities
and activity against different stresses
Name of microbe Objective Application References
Biofertilizer
B. amyloliquefaciens Screening, plant growth Promoted banana Gamez et al.
Bs006 and promotion, and root growth similarly or even (2019)
P. fluorescens Ps006 colonization pattern of slightly superior to
two rhizobacteria on 100% chemical
banana cv. Williams fertilization
(Musa acuminata Colla)
Bacillus licheniformis Molecular Strains BHUJP-P4 and Jaiswal
(BHUJP-P3) and characterization of BHUJP-P3 showed et al. (2019)
Bacillus cereus monocrotophos- and higher EPS, IAA, PSB,
(BHUJP-P4) chlorpyrifos-tolerant HCN, and ammonia
bacterial strain for production
enhancing seed
germination of
vegetable crops
Achromobacter, Purple corn-associated N2 fixation, phosphate Castellano-
Bacillus, Lysinibacillus, rhizobacteria with solubilization, indole Hinojosa
Paenibacillus, potential for plant acetic and siderophore et al. (2018)
Pseudomonas, and growth promotion production,
Stenotrophomonas, 1-aminocyclopropane-
1-carboxylic acid
deaminase activity and
biocontrol abilities,
significant increases in
root and shoot dry
weight, total C and N
contents of the plants
Bacillus spec. strain Isolation and All strains significantly Rahmoune
Bt04, Pseudomonas, characterization of the improved plant growth et al. (2017)
Lysinibacillus three new PGPR and of the plant species
fusiformis strain Lf89 their effects on the tested, and some strains
growth of Arabidopsis produce a shift in the
and Datura plants C/N ratio in A. thaliana
Pseudomonas Evaluation of Phytohormones, Kumar et al.
fluorescens spp. fluorescent siderophores, HCN, (2012)
Pseudomonas spp. with proteases, chitinases,
single and multiple cellulases, ammonia,
PGPR traits for plant and exopolysaccharide
growth promotion of production and
sorghum in combination phosphate solubilization
with AM fungi or antagonistic activity
Pantoea, Serratia, Plant growth promotion Produce siderophores Viruel et al.
Enterobacter, and traits of phosphobacteria and indoles (2014)
Pseudomonas isolated from Puna,
Argentina
Pseudomonas Organic acid production Production of organic Vyas and
fluorescens in vitro and plant growth acids during inorganic Gulati
promotion in maize phosphate solubilization (2009)
(continued)

under controlled and influence on plant
environment by growth
phosphate-solubilizing
fluorescent
Pseudomonas
Bacillus megaterium Plant growth promotion Promoted growth of Ortíz-Castro
by Bacillus megaterium A. thaliana and et al. (2008)
involves cytokinin P. vulgaris seedlings
signaling
Bacillus subtilis and Comparison of plant B. subtilis and Adesemoye
Pseudomonas growth promotion with P. aeruginosa increased et al. (2008)
aeruginosa Pseudomonas 31% for tomato, 36%
aeruginosa and Bacillus and 29% for okra, and
subtilis in three 83% and 40% for
vegetables African spinach,
respectively
Biocontrol agent
Stenotrophomonas Endophytic bacteria Suppress tomato Rania et al.
sp. str. S33 and from Datura metel for Fusarium wilt disease (2016)
Pseudomonas sp. str. plant growth promotion caused by Fusarium
S85 and bioprotection oxysporum f. sp.
against Fusarium wilt in lycopersici (FOL) and to
tomato enhance plant growth
Bacillus Multiple effects of Dose-response studies Asari et al.
amyloliquefaciens Bacillus with UCMB5113 on MS (2016)
subsp. plantarum strains amyloliquefaciens agar with or without root
volatile compounds: exudates showed
plant growth promotion significant plant growth
and growth inhibition of promotion even at low
phytopathogens levels of bacteria.
Bacillus VOC
antagonized growth of
several fungal pathogens
in vitro
Pseudomonas Evaluation of plant Suppressed growth of Verma et al.
aeruginosa and growth-promoting phytopathogen, e.g., (2014)
Trichoderma harzianum activities of microbial Fusarium oxysporum
strains and their effect and Rhizoctonia solani
on growth and yield of
chickpea (Cicer
arietinum L.) in India
Bacillus subtilis CAS15, The siderophore- B. subtilis CAS15 Yu et al.
producing bacterium, induced systemic (2011)
Bacillus subtilis CAS15, resistance to Fusarium
has a biocontrol effect wilt in pepper. Iron
on Fusarium wilt and supplementation
promotes the growth of reduced this biocontrol
pepper effect
(continued)

B. amyloliquefaciens Comparative analysis of Stimulates plant growth Chen et al.
FZB42 the complete genome and produces secondary (2007)
sequence of the plant metabolites
growth-promoting
bacterium Bacillus
amyloliquefaciens
FZB42
Bacillus thuringiensis Bacillus thuringiensis Induces plant growth Raddadi
beyond insect promotion and can also et al. (2007)
biocontrol: plant growth be used as a biofertilizer
promotion and biosafety
of polyvalent strains
Abiotic stress
Bacillus subtilis strain Bacillus subtilis strain The application of strain Woo et al.
GOT9 GOT9 confers enhanced GOT9 led to the (2020)
tolerance to drought and enhancement of drought
salt stresses in and salt stress tolerance
Arabidopsis thaliana in Arabidopsis thaliana
and Brassica campestris and Brassica campestris
Trichoderma Phosphate solubilization T. koningiopsis employs Tandon
koningiopsis (NBRI- by Trichoderma different mechanisms of et al. (2020)
PR5) koningiopsis (NBRI- P solubilization in
PR5) under abiotic stress different stress
conditions conditions, and
therefore, it can be used
in the management of
stressed soils
P. mendocina, Saline soil microbiome: All are indole acetic acid Hingole and
A. beijerinckii, a rich source of and ammonia producers, Pathak
P. stutzeri, halotolerant PGPR and they also solubilize (2016)
B. subtilis the phosphate under 4%
NaCl concentration
P. simiae AU Putative bacterial Lower Na accumulation, Vaishnav
volatile-mediated higher proline contents and
growth in soybean Choudhary
(Glycine max L. Merrill) (2019)
and expression of
induced proteins under
salt stress
Burkholderia, Resistance to drought Enhance root length of Thijs et al.
Variovorax, Bacillus, stress, cold, nutrient Arabidopsis under (2014)
Pseudomonas and starvation, 2,4-DNT 2,4-DNT stress,
Ralstonia species (dinitrotoluene) stress doubling of the main
root length
bacteria, potassium-dissolving bacteria or potassium-solubilizing rhizobacteria

(Pandey et al. 2019). The bioavailability of potassium is induced by the production
of organic acids, such as citric acid, oxalic acid, succinic acid, tartaric acid, and malic
acid (Saritha and Tollamagudu et al. 2019). These microbes can be used as
bioinoculants under the prospect of sustainable agriculture practice.
Further, there are many soil microbes, which help plant mitigate different types of
abiotic stresses majorly drought and salinity stress. There many microbes not only
help in mitigating the stress but also in promoting the growth of plants through plant
growth-promoting (PGP) activity (Vurukonda et al. 2016). Drought stress coping is
achieved by the production of phytohormones, which leads to adaptations, such as
partial closure of stomata, to prevent excessive transpiration. Likewise, for salinity
stress, there are many bacterial strains, which produce IAA, I3B, ABA, ACC, and
many other chemicals, which directly or indirectly lead to help in mitigating the
adverse effects of the stress (Table 12.1).
12.1.1 Plant Growth-Promoting Microorganism: As Biofertilizer
As has been discussed briefly that the soil microbes help convert insoluble and
unavailable form of minerals to soluble, organic, and available forms. The major soil
enrichment products or fertilizers use the addition of NPK (nitrogen, phosphorus,
and potassium) to the soil to promote any kind of plant growth and the desired
product yield. Though the NPK is abundantly present in the surrounding environ-
ment of the plant, they are unable to acquire it. Hence, with soil microbes converting
the unavailable forms to available forms, this problem can be solved and are called as
biofertilizers. These microbes show plant growth-promoting properties and hence
are called as PGPR. The nitrogen requirement is fulfilled by the nitrogen-fixing
microbes, which may either be free-living (nonsymbiotic) or in symbiotic relation-
ship with the plants. The microbes living in symbiotic relationship with the plants are
generally rhizobia (Mus et al. 2016). The free-living nitrogen fixers include Azoto-
bacter, Clostridium, and cyanobacteria. The process includes conversion of atmo-
spheric nitrogen to ammonia, which is then converted into amino acids and other
nitrogenous compounds. Phosphorous is another major nutrient required for the
growth of plants. It is involved in various key mechanisms of the plants, such as
cell division and development, photosynthesis, signal transduction, and biosynthesis
of macromolecules (Sengupta and Gunri 2015).
The presence of insoluble phosphates in soil allows soil microbes to solubilize it
and make it available to plants, a process called as phosphate solubilization, and the
microbes involved phosphate-solubilizing microbes. The mechanism of solubiliza-
tion of the insoluble phosphates, such as tricalcium phosphate (Ca3PO4)2, aluminum
phosphate (Al3PO4), and iron phosphate (Fe3PO4), to soluble forms includes the
production of organic acids, such as malic acid and gluconic acid (Enterobacter
sp. FS-11) (Shahid et al. 2012.), citric acid, fumaric acid, formic acid, oxalic acid,
acetic acid, isobutyric acid, valeric acid, isovaleric acid, and tartaric acid, produced
by bacterial species, such as Serratia marcescens, Delftia, Chryseobacterium, and
Phyllobacterium myrsinacearum (Khan et al. 2014). The fungal species producing
such acids include Aspergillus niger FS1, Penicillium canescens FS23,
Eupenicillium ludwigii FS27, Penicillium islandicum FS30, Aspergillus awamori
S19, T. flavus, T. helices, P. purpurogenum, and P. janthinellum, which release

organic acids for solubilizing inorganic phosphates (Mendes et al. 2013; Jain et al.
2012; Scervino et al. 2010). Other mechanisms through which insoluble P can be
converted to soluble P, i.e., without the production of organic acids, are chelation
and reduction processes, which are useful in the management of plant pathogens.
Furthermore, H+ pump in Penicillium rugulosum and inorganic acids, such as HCl,
HNO3, and H2SO4, by some bacterial species is also known to cause solubilization
of inorganic phosphate. Next is the uptake of available phosphorous by plants
mediated by the enzymes of soil microbes.
The complex organic P compounds are further mineralized by soil enzymes, such
as acid phosphatase (commonly found in fungi) and alkaline phosphatase, which are
considered as the principal mechanism for mineralization of soil organic P. An
enzyme called phytase leads to the production of P from phytic acid. Phytate is a
complex organic compound in soil, and hence its degradation is common in
phosphate-solubilizing microbes. After nitrogen and phosphorous, potassium is the
third macronutrient essential for the growth of the plants. Absorbed in the form of
K+, it is required in the early developmental stages of the plant. Out of the total
whose concentration generally exceeds 20,000 ppm, only 1–2% is readily available
to plants. This is where potassium-solubilizing microbes come to the rescue. Soil
microbes, such as Acidithiobacillus, Burkholderia, and Pseudomonas, have the
ability of solubilize phosphate from the fixed forms in the soil. Though the exact
mechanism of solubilization remains lagging, activities like acid hydrolysis, through
the production of organic acids, and chelation and reduction reactions are attributed
to be the reason behind the process. The mechanism through which potassium is
taken up by plants is either through high-affinity transport system (HATS) or
low-affinity transport system (LATS). In high-affinity mechanism, the influx of K+
is driven by the outflux of H+ over the plasma membrane mediated by proton
ATPase. In the LATS mechanism, electrogenic uniport of K+ takes place by the
ATP-driven efflux of H+ (Sharma et al. 2016). In the plant cell, the cytosol concen-
tration of K+ ranges between 40 and 200 mM, which presents a challenge for K+
uptake by plants when low concentrations in the soil are present. The low potassium
concentration in the soil demands ATP-driven process as the flux needs to be driven
against the gradient. The solubilization of K from different insoluble minerals in soil
is dependent also on the proton concentration of the soil or soil solution. Other
factors include lowering of pH, enhancing chelation of cations bound to K, and
acidolysis around the microorganism’s present.
12.1.2 Biocontrol Agents
Biopesticides refer to the use of biocontrol agents for the management of the crops
and controlling pests rather than using chemical pesticides. The major difference
between the biopesticides and biocontrol agents is that the former is passive in nature
while the latter is active in nature and seeks out pest to kill them, such as parasitoids,
predators, and many species of entomopathogenic nematodes. Biopesticides cover a
wide range of agents, such as microbial pesticides, entomopathogens, plant-

incorporated protectants, and biochemical pesticides. Here, we will be focusing on
the microbial pesticides that are used to manage and control pests. The microbial
pesticides include bacteria, virus, and fungi, which attack specific pests. Many
Bacillus and Pseudomonas strains are known to act biopesticides. Bacillus
thuringiensis (Bt) is considered to have a market share of about 95% of the total
microbial pesticides. The mechanism through which microbial pesticides act are
through the formation of spores and crystals, which are used against several agricul-
tural and horticultural crops. The effectiveness of the of microbial pesticides depends
upon the broad range of conditions that they survive in, such as temperature, shelf
life during storage, and moisture content. Considering these parameters, the Bacillus
thuringiensis dominates the market of microbial pesticides as it is more effective at
high temperatures and has higher shelf life during storage (Vimala Devi et al. 2019).
12.1.3 Alleviating Abiotic Stress
There are many microbial species which help in alleviating the abiotic stress, such as
drought and salinity stress. Plant growth-promoting bacteria are known to produce
plant hormones, like gibberellins, auxin, and precursor of several hormones, such as
ethylene and other volatile organic compounds. Under abiotic stress conditions, two
major plants hormones are secreted as a primary response which are ethylene
and abscisic acid. Ethylene is secreted primarily as a response toward salinity stress
and abscisic acid as a response toward drought stress. Ethylene retards plant growth
and development, while abscisic acid induces the formation of lateral roots in the
plants. While the formation of lateral roots in plants is beneficial, the effect of
ethylene is not beneficial for the plant in any sense. Several microbes are also
known to produce ACC (aminocyclopropane-1-carboxylic acid), which is a precur-
sor of ethylene and thus reduces the retardation of growth in plants under stress. The
ACC deaminase activity is also known to induce modifications in the root tip and its
surface area, thereby making it resistant to stress conditions by promoting nutrient
acquisition. Other plant growth-promoting activities of bacteria under salinity stress
include the production of auxins, IAA (indole acetic acid) and I3B (indole-3-butyric
acid), and other beneficial substances, such as extra-polymeric substances and
antioxidants. The microbial species include Bacillus spp., Pseudomonas spp.,
Frankia spp., Nocardia spp., Streptomyces spp., and many others.
12.2 Organophosphates
The organophosphate (OP) pesticides, likewise called organophosphorus pesticides,

are a group of chemical substance, which are all esters of phosphoric acid with
aliphatic, phenyl, and heterocyclic utilitarian gatherings (Kumar et al. 2016). Organ-
ophosphate compounds are characterized in the main groups of herbicides,
insecticides, and fungicide. In the 1970s, first-time organophosphate pesticides
were introducing after banned of organochlorine due to high toxicity and their
persistent rate high in soil. Grube et al. (2011) reported OPs widely used pesticide
in the world. American agriculture witnessed consumption of 334 million pounds of
organophosphate insecticides from 2001 to 2007.
The commonly used organophosphates in India are chlorpyrifos, phorate,
profenofos, dichlorvos, quinalphos, acephate, triazofos, monocrotophos, and mala-
thion, (Table 12.2). The responsible authority for registering pesticides for use on
crops to control pests and weeds is the Central Insecticides Board and Registration
Committee (CIBRC), which falls under the Ministry of Agriculture and Farmer
Welfare.
Organophosphate is liable for a large number of deaths worldwide due to their
unregulated utilization and their easy accessibility (Buckley et al. 2004; Gunnell
et al. 2007). So, also, toxicity of pesticides could be a lot higher for developing
countries like India, where countless farmers don’t have any information about the
toxicity of applies of pesticides, and they don’t use any defensive gear during the
spraying of pesticide (Banerjee et al. 2014). Therefore, farmers face occupational
risk while applying pesticides onto the farming field (Mamane et al. 2015; Muñoz-
Quezada et al. 2016; Rastogi et al. 2010). The occupational hazard of pesticides isn’t
limited to just the farmers and furthermore influences the family members of farmers
and individuals living close to farming field. In a study by Rastogi et al. (2010), the
Table 12.2 Most consumed organophosphate insecticide in the country (during 2017–2018)
S. No. Organophosphate insecticide Quantity (in metric tons)
1 Chlorpyrifos 477.90
2 Phorate 480.24
3 Profenofos 300.25
4 Dichlorvos 287.11
5 Quinalphos 242.37
6 Acephate 168.77
7 Triazofos 151.21
8 Monocrotophos 140.30
9 Malathion 103.00
10 Dimethoate 91.94
11 Phosphamidon 45.15
12 Ethion 20.73
13 Methyl parathion 19.27
14 Temefos 18.00
15 Fenitrothion 16.50
16 Oxydemeton-methyl 13.80
17 Phenthoate 11.20
18 Phosalone 3.56
19 Fenthion 3.00
20 Diazinon 1.00
Source: http://ppqs.gov.in/statistical-database
high recurrence of neurologic manifestations was found in kids, having a place with
the groups of farmers dealing with and utilizing OPs bug sprays, which presented
occupationally to different organophosphate bug sprays (chlorpyrifos, diazinon,
dichlorvos, ethyl parathion, fenthion, malathion, methyl parathion).
12.2.1 Organophosphate Insecticide Pollution and Contamination

Status
Amidst the growing Indian population and proportional food demand till 2024,
under the pressures of limited agricultural land and attack of pest infestation, the
need of food grains, vegetables, and fruits is foreseen to be multiplied from 2.5 to
5 times. Therefore, to achieve the projected goal, it is forecasted that pesticide
application will also increase by at least 2–3 times in the forthcoming years (Dar
et al. 2020). The widespread contamination of OP, due to their excessive use, has
been revealed in soil, sediments, water, fruits, vegetables, tea, and human fluids by
residual analysis of these pesticides (Table 12.2). Pesticide contamination of water,
air, and food chain to ultimately reach the human body occurs mainly due its
transport through the soil via means of leaching, runoff, transfer, interflow, and
subsurface drainage (Abrahams 2002).
Vegetables, being an essential dietary component with higher consumption rates
across the world, have been under extensive OPs application due to their higher
vulnerability to pest infestations. Thus, a higher application rate of pesticides in
agriculture has resulted in residual contamination of vegetables and fruits with OPs.
Several studies to assess pesticide contamination in vegetables, like beetroot, bitter
gourd, brinjal, cabbage, capsicum, carrot, cauliflower, chili, cucumber, French bean,
jackfruit, knolkhol, ladyfinger, mustard, okra, onion, pea, potato, radish, spinach,
tomato, etc., have reported residual concentration of different OPs, such acephate,
anilofos, chlorpyrifos, diazinon, dichlorvos, dimethoate, fenitrothion,
phosphamidon, profenofos, phorate, malathion, quinalphos, and monocrotophos,
to be above their respective MRL levels by application of different techniques,
viz., GC-ECD, GC-NPD, FPD, and LCMS/MS techniques (Mukherjee 2003;
Kumari et al. 2003, 2004; Srivastava et al. 2011; Gowda and Somashekar 2012;
Sinha et al. 2012). On the contrary, some studies have reported residual
concentrations of different OPs, such as methyl parathion, chlorpyrifos, malathion,
monocrotophos, and others, to be below their respective MRL in vegetables (Bhanti
and Taneja 2007; Mandal and Singh 2010; Chandra 2014); however, continuous
consumption of these vegetables may result in the bioaccumulation of these
pesticides with fatal consequences. Singh and Gupta (2002) found different
vegetables collected from agricultural farms and vegetable markets, having OP
contamination (e.g., chlorpyrifos, dimethoate, quinalphos, and monocrotophos)
from below to above their residual limits. Apart from pesticide contamination in
vegetables, fruits were also found to be contaminated with different OPs.
Harinathareddy et al. (2014) found different fruit samples obtained from an agricul-
tural farm in Andhra Pradesh, India, to be contaminated with various OPs. Another
study by REF showed contamination of different OPs, such as chlorpyrifos-methyl,

diazinon, malathion, monocrotophos, profenofos, and pirimiphos-methyl, in fruits
and vegetables (apple, grapes, strawberry, watermelon, bell pepper, cabbage, carrot,
cucumber, eggplant, potato, tomato, and zucchini consumed commonly in Kuwait).
Contamination with different OPs is not just limited to fruits and vegetables; they
have also been detected in other food items, such as butter, ghee, tea, honey, soft
drinks, juices, etc., and in specific animal tissues. A pesticide, chlorpyrifos, was
found to be above its maximum residual limit in butter and ghee samples, collected
on a random basis from different rural and urban locations in Haryana, India (Kumari
2005). Contamination with highly hazardous, class Ib pesticide monocrotophos,
classified by the WHO, which is also likely to be mutagenic and neurotoxic, was
observed in tea samples manufactured by different companies, like Tata, Hindustan
Unilever, Golden Tips, Goodricke, Royal Girnar, Kho Cha, and Wagh Bakri, from
all across India with a concentration ranging from 0.026 to 0.270 mg/kg, even when
its application on tea is not allowed (Greenpeace India report 2014). Another
pesticide, ethion, was also detected in 22 tea samples, resulting from its direct
application on tea (Greenpeace India report 2014). Seenivasan and Muraleedharan
(2011) reported contamination of only 0.5% tea samples out of the collected
912 samples, from various districts of Tamil Nadu, India, with residues of pesticides
ethion, quinalphos, and others, however, below their respective MRL values.
The presence of organophosphate residues in fish, bovine milk, human blood,
urine, and breast milk has been reported across the world, even though OPs are less
persistent with a lesser potential of bioaccumulation as compared to other classes of
pesticides. Several studies have shown contamination of OPs in humans and animals
due to their exposure to contaminated water, air, soil, food, etc. Detection of
chlorpyrifos, phosphamide, monocrotophos, and malathion has been reported in
human blood samples obtained from different villages of Punjab, India, indicating
toward their longer persistence in human body (CSE report 2005). Residues of
another OP, chlorpyrifos, have also been detected in blood samples of chick (Gallus
gallus), fish (Rita rita Ham.), goat (Capra hircus Linn.), and humans in Uttar
Pradesh, India, due to their transport to other ecosystems. The maximum level of
the residue was found in fish followed by chick, goat, and humans (Singh et al.
2008). Sanghi (2003) reported contamination of breast milk samples with chlorpyri-
fos, malathion, and methyl parathion residues in poor women belonging to the age
group of 19 to 45 years from Madhya Pradesh, India. The presence of OP residues in
bovine milk samples is linked to the consumption of fodder or herbaceous vegetation
contaminated with pesticides, by the cows (Fagnani 2011). Contamination of bovine
milk with residues of methyl parathion, a highly hazardous (class Ia) pesticide, was
reported in samples collected from rural and urban dairies of Allahabad, India
(Srivastava et al. 2008). The presence of methyl parathion residues was higher in
samples obtained from the diary in rural area, possibly due to extensive agricultural
activities and unsafe handling methods in rural areas.
12.2.2 Fate of Organophosphosphate Insecticide
After pesticides are applied to the crops in the agricultural field, it is not utilized
completely; instead, they get distributed into the biosphere (i.e., atmosphere, litho-
sphere, and hydrosphere), depending on various physical and biological factors.
Some portion of pesticide may volatize into the atmosphere, depending on the
temperature, wind speed, humidity, and atmospheric stability (Van Den Berg et al.
1999). Other pesticide residues may reach to the water bodies due to spray drift,
volatilization and precipitation, surface runoff, and groundwater leaching (Carter
2000). Their distribution on land is determined by factors, such as surface prepara-
tion, soil structure, clay content of soil, moisture content of soil, type of irrigation,
pesticide group, time of application of pesticide, and rainfall (Flury, 1996). The
factors controlling the fate of pesticides in the environment are either biotic or abiotic
in nature. The physical processes, like transportation, adsorption, and desorption,
control the transformation of pesticide once it enters into the soil. Finally, pesticides
also affect flora and fauna, decrease biological diversity, and contaminate the food
chain (Ribeiro et al. 2005).
12.3 Impact of Pesticide and Its Toxicity
12.3.1 Soil Microbe’s Biomass
The stability and their toxicity of pesticide in soil are determined by its chemical
composition and soil structure (Laabs et al. 2007). The presence of pesticide residue
pollution in soil are available to contact with soil rhizospheric and non-rhizospheric
microbial community and disturbed their biochemical and physiochemical behavior
(Singh and Walker, 2006). The foremost essential marker of microbial activities in
soil is microbial biomass, which shows the dimension microbial growth, nutrient
availability, and its biotransformation and different ecological phenomena (Schultz
and Urban 2008). Many studies on the toxicity of pesticide on microbial biomass are
positive, negative, or neutrals. It is depending on the different nature of pesticide
structure (Pampulha and Oliveira 2006). According to Wang et al. (2006), some soil
microbes can utilize the pesticide as a carbon source for their growth but for other
soil beneficial microbes (like, PGPMs) may decrease their population. Also, it can
disturb the microbial and their functional diversity. But, in some studies, pesticides
induce the fictional diversity but reduce the microbial diversity (Wang et al. 2006;
Pampulha and Oliveira 2006). Also, pesticides periodically show reversible positive
or reversible repressing effects on soil microbes (Pampulha and Oliveira 2006). This
implies either evoked the biodegradation of pesticide by soil microorganism or
change in microbial diversity.
12.3.2 Soil Enzymatic Activities
Severable-biological reactions in soil are catalyzed by different soil enzymes, and

also, they act as a critical ecological indicator and marker of various ecological
processes of soil ecosystem (Antonious 2003; Jaiswal and Verma 2018). Soil
enzymes have defined the status of agricultural field soil, like fertility (Antonious
2003), quality (Bucket and Dick 1998), and pollution status (Kucharski et al 2010;
Schaffer 1993). Soil enzymes, presented within the live microbial cells, remotely
appended to the cell layer and discharged extracellularly in soil system or bounded
with the dead microbial cells, soil matrix, soil colloids, or humic materials (May-
anglambam et al. 2005). The activity of the enzyme is depending upon the soil
physico-biological properties such as organic matter content (Kandeler and Gerber
1988), moisture (Bergstrom et al. 1998), and temperature (Tscherko et al. 2001), and
pollutants present in the soil. But, changes in the trends of pesticide application,
under conventional agricultural practices, are responsible for increasing the ratio of
pesticide residues in the soil as compared to the recommended dose. Therefore, the
presence of pesticide residues in the soil can interact with soil enzymes, and it’s
characterized by their response as positive, negative, and neutral effects. Many
studies on the interaction of pesticide and soil enzymes observed negative responses
on soil enzymes, such as alkaline phosphatase, hydrolase, phosphomonoesterase,
acid phosphatase, urease, and dehydrogenase (Caceres et al. 2009; Monkiedje and
Spiteller 2002). Another study showed the positive response of pesticides on the soil
enzyme properties (Fragoeiro and Magan 2008; Defo et al. 2011). Bolton et al.
(1985) have been reported the dehydrogenase soil enzyme, characterized in intracel-
lular microbial enzyme, and it is a significant marker of soil microbial activity.
According to Jastrzebska (2011), pesticides show all types of impacts that are
neutral, positive, and negative on dehydrogenase. There is an immense range of
research considered that reports the effect of pesticides on dehydrogenase activity.
Mayanglambam et al. (2005) observed 30% inhibition in the activity of the dehy-
drogenase enzyme after 15 days, under the toxicity of organophosphate insecticide
quinalphos. Additionally, the loss of enzyme activities shows recovery after 90 days
of treatment; the conceivable reason for this could be the acclimatization of soil
microorganisms toward quinalphos. Similarly, Bayer et al. (1982) observed that
dehydrogenase activities reduced in alluvial soil treated with a recommended dose of
different organophosphate insecticide methidathion, methoxatin parathion, after six
weeks of incubation. Pozo et al. (1995) found that the application of chlorpyrifos at
2–10 kgha-1 reduced the dehydrogenase activity; however, the activity recovered
after 14 days to control levels. Another most important soil enzyme phosphatase is a
group of enzymes – mono-, di- and, tri-esterase, pyrophosphatases, and
phosphamides, which hydrolyze both esters and anhydrides of phosphoric acid
and convert the organic phosphate to inorganic phosphate (Hussain et al. 2009).
Phosphomonoesters are the most common group of phosphatases in the soil
(De Cesare et al. 2000), which include two subgroups, enzyme-like acid phosphatase
and alkaline phosphatase, that work at different pH range (Hussain et al. 2009). In
the phosphorus cycle, the phosphatase enzyme is playing a significant role in
increasing the availability phosphorus to plant (Schneider et al. 2001). The

rhizospheric zone is rich in a high concentration of phosphate, along with low
content of humus (Tarafdar et al. 2001). The toxicity of insecticides on acid and
alkaline phosphatase activities affects in a different manner due to the structural
difference between soil microbial communities (Klose et al. 2006; Srinivasulu and
Rangaswamy 2014). Sikora et al. (1990) observed the negative, positive, or neutral
effect on the acid phosphatase activity in the loamy soil under the toxicity of
organophosphate groups of insecticides chlorpyrifos, terbufo, and fonofos. But in
the case of fungicide, toxicity showed positive effect on acid phosphatase and
negative effect on alkaline phosphatase. In the nitrogen cycle, urease plays a vital
role and hydrolyzes urea into CO2 and ammonia. Urease enzyme is abundantly
present intra- and extracellularly in both plants and soil microbes but it rapidly
degrades in soil, when extracted from plant and microbes. This surely shows urease
activities in soil fundamentally contributed by extracellular urease immobilized on
organic soil (Beri et al. 1978; Zantua and Bremner 1977). Urease, especially, has
seen a broad concentration because of the essentialness of its substrate urea in
agriculture farming. Urease activity is a significant marker to evaluate the soil
contamination condition, since urease function in soil diminishes as pesticides
meddle with the urease enzyme and decline urea hydrolysis (Antonious, 2003;
Srinivasulu and Rangaswamy 2014). Several studies have indicated the influence
of different pesticides on urease in a positive, negative, or unbiased way (Antonious
2003;Ingram et al. 2005). Lethbridge and Burns 1976 observed that the urease
activity decline by 40–50% after 60 days in a sandy clam soil and silt loam soil,
due to the presence residues of organophophate groups pesticide phosphorothioates,
fenitrothion, malathion, and phorate at the concentration range 50 to 1000 mg kg-1.
In contrast, another study showed that urease activity increased in profenofos-treated
soil (6.4 and 38.4 μg g1) for 6 weeks (Abdel-Mallek et al. 1994). In outline, the
physicochemical properties of both soil and pesticide residues and natural conditions
govern the toxicity of pesticide on the soil beneficial microbes (Dick et al. 2000).
12.3.3 Plant Growth-Promoting Microorganism and Biofertilizer
Repeated application of pesticide on vegetable and crops against the control of pest
infestation, as results unwanted pesticides residues reached to the soil, where that
impose their toxicity on rhizospheric microbes like PGPMs (plant growth promotion
microorganism) and also suppress their beneficial properties (IAA, production,
phosphorus and Zn solubilization, etc.) (Ahemad and Khan 2012a). PGPMs are
defined and categorized on the based totally over the feature and their mechanism
(direct and indirect) that consists of out; like inoculum or consortium of PGPMs are
used as biofertilizer, which improve the bioavailability soil nutrients (N, P, K, Ca, Zn
& S) to plants by solubilizing and mineralizing natural organic compounds;
phytostimulators, that stimulate the plant and root growth or development by
released plant hormones; biocontrol agent, which act as a biopesticide against
different pest and weed infestation by producing antibiotics and antifungal
metabolites. Several soil microbe’s species, such as Bacillus (Jung and Kim, 2003),
Pseudomonads (Lemanceau and Alabouvette 1993; Raaijmakers et al. 2002),
Rhizobacteria and Bradyrhizobium (Chaintreuil et al. 2000), Acetobacter (Sevilla
et al. 2001), and Klebsiella (El-Khawas and Adachi, 1999), showed the plant
growth-promoting properties, like fixing biological nitrogen, producing of plant
hormones (auxins, cytokinins, gibberellins, abscisic acid), facilitating the availabil-
ity of soil nutrients, and tolerance toward toxicity of pesticides (Upadhyay et al.
2009; Yasouri 2003). However, several studies (in vitro and in vivo) have reported
the interference and toxicity of organophosphate groups of pesticide on plant
growth-promoting microorganisms and also on their interaction with the plant
(Verma et al. 2014). Several studies have found the toxicity of pesticide on plant
growth-promoting microorganisms and their properties; Shaheen and Sundari 2013
showed that PGPM strains are more sensitive at 100 ppm of monocrotophos
insecticide. Ahemad and Khan (2012b), found that under pesticide stress (metribuzin
and glyphosate, imidacloprid and thiamethoxam, hexaconazole, metalaxyl, and
kitazin), all plant growth-promoting traits of Mesorhizobium sp. strain MRC4 have
progressively declined with an increase in their concentration. Khan et al. (2009)
observed that nodulation in chickpea and rhizobacterial population suppressed under
the toxicity of chlorpyrifos (Pyrifos 40% EC). Sepperumal et al. (2016) showed that
the growth of Klebsiella pneumoniae declined by increasing the dose of phorate.
Kumar et al. 2019 found that siderophore properties of strains P. fluorescens (NCIM-
5096), R. leguminosarum (NCIM-2749), B. brevis (NCIM-2532), A. vinelandii
(NCIM-2821), and S. typhimurium (NCIM-2501) decreased under the dose toxicity
of phorate, acephate, monocrotophos, and glyphosate.
12.3.4 Human Health
According to Bajgar (2004), organophosphate showed as highly toxic nerve agents,

which inhibit the cholinergic nerve signaling, and simultaneously, it also catalyzes
the neurotransmitter cholinesterase, resulting in an increased level of acetylcholine.
The chemical acetylcholine is responsible for the lesions of the different parts of the
brain. The clinical sign of organophosphate (OP) poisoning is frequently
characterized into receptor-specific manifestation, time-onset-precise manifestation,
and organ-specific manifestation. In serious insecticide poisoning, patients have
muscarinic signs, like nausea, reflex, drooling, blurred vision, etc. (Agrawal and
Sharma 2010). There are a variety of cases of insecticide chemical toxicity in India
(Gupta et al. 2006; Shivakumar et al. 2013; Parmar et al. 2014). The first case of
insecticide toxicity of organophosphate insecticide in India was reported from the
state of Kerala in 1958, wherever quite more than a hundred individuals affected to
the poisoning of parathion by overwhelming of contaminated wheat flour (Gupta
2004). The occupational toxicity of OPs was reported as symptoms and sign of
neurotoxic poisoning in the state of Andhra Pradesh, by accidentally exposed of the
farmer in a cotton-filled during mixing and refiling spraying tank (Mancini et al.
2005). Likewise, another poising of OP case was reported in Saran District of Bihar,
in 2013, in which 23 children were dead after taking the foods of midday meal that
were contaminated with monocrotophos (BBC Report 2013).
12.4 Analytical Techniques for the Determination

of Organophosphate Pesticides in Environmental Samples
The happening to different coherent explanatory methods has enabled us to accumu-

late the information and choose the structure of the recently orchestrated atoms.
Eventually, incessant assessment is being coordinated to uncover the ligand and
detailing media for organophosphate pesticide identification by means of various
spectroscopic systems like UV-Vis (Elgailani and Alghamdi 2018), Fourier-
transform infrared (FTIR) spectroscopic (Buvaneswari et al. 2017.), NMR spectros-
copy (Dahiya et al. 2020), X-ray diffraction (Wang and Liu 2016) mass spectrometry
(Alnedhary et al. 2020; Liu et al. 2005), and electrochemical (Gong et al. 2009). The
detection and estimation of organophosphate pesticides have been reported in water
and vegetables by using analytical methods (Tables 12.3 and 12.4).
12.5 Assessments of Organophosphate Pesticide Toxicity

to Microorganisms
Soil is a living entity which has diverse micro- and macrofauna as well as flora.
These soil organisms are an integral part of agricultural ecosystems, essential for the
maintenance of healthy productive soils. Among the soil microflora, plant growth-
prompting rhizobacteria (PGPR) promotes plant growth and development in differ-
ent ways (Krishna et al. 2019; Verma et al. 2018). Nevertheless, extensive use of
plant protection chemicals had led to an accumulation of a huge amount of residues,
adversely affecting the environmental health as well as ecosystem services. Among
the different classes of pesticides used, organophosphate (OP), a phosphoric acid
derivative, is widely used in highly toxic heterogeneous compound. Globally, about
140 OP compounds are being used as pesticides and plant growth promoters (Kang
et al. 2006). These agents cause a specific and irreversible inhibition of acetylcho-
linesterase, a vital enzyme responsible for the degradation of acetylcholine in the
nerve terminal (Bakry et al. 2006). Though pesticides have been developed with
target organism specificity, often nontarget species are affected by their application.
Being persistent, its accumulation in the environment had led to a substantial soil
health hazard and toxicity to soil microflora, resulting in altered soil microbial
diversity and biomass. Hence, assessment for soil toxicity of OP compounds is a
vital component of agricultural environment pollution monitoring. Assessment of
biological effects by utilizing a quick, sensitive, and economic method can indicate
precise information on OP ecotoxicity. In recent decades, toxicity of OP compounds,
by assessing the microbial diversity and biomass, has gained momentum due to
advantages, like short life cycles, inexpensiveness, less time-consuming, and sensi-
tivity to various toxic chemicals (Hassan et al. 2016; Su et al. 2011; Tothill and
Table 12.3 List of organophosphate pesticide residues in vegetable samples in India
12
Rang of
Environmental Filed organophosphorus Technique
sample Crops location Detected organophosphate residues (μg g1) used References
Vegetable Brinjal, chili, Jaipur, Monocrotophos, quinalphos, 0.03–0.10; GC-NPD Singh and
cauliflower, bitter gourd, Rajasthan dimethoate, chlorpyrifos 0.02–0.08; Gupta
tomato, cucumber, okra 0.08–0.09; (2002)
0.01–0.06
Brinjal, Hisar, Dimethoate, malathion, fenitrothion, 0.002–0.208; GC-ECD, Kumari et al.
cabbage Haryana monocrotophos, phosphamidon, 0.003–0.016; GC-NPD (2003)
cauliflower, quinalphos, chlorpyrifos 0.013–0.024;
pea grain, tomato, 0.033–0.051;
potato 0.045–1.284;
0.009–0.048;
0.001–0.047
Brinjal, Hisar, Monocrotophos, 0.005–0.435; GC-ECD Kumari et al.
okra, Haryana cypermethrin, 0.005–0.275; and (2004)
cauliflower, quinalphos, 0.003–0.278; GC-NPD
cabbage, malathion, 0.103–0.309;
pea chlorpyrifos, 0.001–0.094;
methyl parathion, 0.018–0.026
Cauliflower Punjab Quinalphos, 0.00–0.20; GC-ECD, Mandal and
methyl parathion, 0.00–0.24; GC-FTD, Singh (2010)
chlorpyrifos, 0.00–0.20; GC-MS
monocrotophos, 0.00–0.20;
cypermethrin, 0.00–0.40;
deltamethrin 0.00–0.20;
Eggplant, cabbage, Delhi Cypermethrin, 0.008–0.025; GC-ECD Mukherjee
tomato, cauliflower, fenvalerate, 0.005–0.075; (2003)
chili, malathion 0.011–0.015;
okra, mustard,
brinjal, okra, Samastipur, Cypermethrin, 0.014–0.450; GLC- Singh and
cauliflower, cabbage, and deltamethrin, 0.032–0.162; ECD and Singh (2006)
Toxicity of Organophosphate Pesticide on Soil Microorganism: Risk Assessments. . . 273
and green chili Nalanda quinalphos, fenvalerate, 0.010–0.33; GLC-TID

chlorpyrifos, 0.00–0.294;
(continued)
274
Rang of
Environmental Filed organophosphorus Technique
sample Crops location Detected organophosphate residues (μg g1) used References
districts, ethion 0.00–0.058;
Bihar malathion 0.015–5.064;
0.00–0.040
Okra, Muzaffarpur Dimethoate, 0.560–0.058; GC- ECD; Sah et al.
brinjal, cauliflower, District, cypermethrin, 0.112–0.407; GC-TID (2018)
cabbage Bihar quinalphos, 0.024–0.462;
fenvalerate, 0.081–0.588;
chlorpyrifos 0.057–0.121;
methyl parathion 0.00–0.462;
malathion 0.279–0.713;
Onion, cucumber, Lucknow Anilofos, dichlorvos, 0.01–0.014; GC-ECD/ Srivastava
beetroot, spinach, radish, City, Uttar dimethoate, and 0.011–0.020; NPD et al. (2011)
cauliflower, Pradesh malathion 0.008–0.042;
cabbage 0.090–0.151
Eggplant, ladyfinger, Hyderabad Chlorpyrifos, Triazofos, 2.49–178.87; LC-MS/ Sinha et al.
cauliflower, cabbage, fenitrothion, 0.491–3.014; MS (2012)
tomato, acephate 12.10–53.90;
chili 2.48–2.48
Brinjal, cucumber, okra, Kothapalli, Monocrotophos, 0.001–0.044; GC-MS Ranga Rao
ridge gourd, and tomato Andhra chlorpyrifos, 0.001–5.154; et al. (2009)
Pradesh cypermethrin, 001 - 0.352
Beans, Kolar Acephate, 80.7–89.2; GLC- Gowda and
brinjal, cabbage, District, chlorpyrifos, 83.4–94.6; ECD; Somashekar
carrot Karnataka dichlorvos, 87.2–96.6; GLC- (2012)
monocrotophos 86.9–92.7;
phorate 83.4–92.1;
profenofos 80.0–88.5;
D. K. Jaiswal et al.
12
cypermethrin 83.4–93.6;
fenvalerate 77.4–90.3
Brinjal, okra, green chili, Andaman Chlorpyrifos, 0.019–1.379; GC/MS Swarnam
crucifers, cucurbits Islands profenofos, 0.042–1.136; and
monocrotophos, 0.023–1.696; Velmurugan
triazofos, 0.023–1.140; (2013)
ethion, 0.083–0.509;
dimethoate, 0.00–0.298;
acephate 0.00–0.345
FAO/WHO MRL values (μg g1): Monocrotophos, 0.2; phosphamidon; 0.2–0.5; chlorpyrifos, 0.01; cypermethrin, 0.2; quinalphos, 0.1–0.2; malathion, 3.0;
methyl parathion, 1.0; dimethoate, 2.0
Table 12.4 List of analytical techniques for the determination of organophosphate pesticides in environmental samples
276
OPs Analytical methods Samples Objective References

Acephate UV-VIS Coriandrum sativum Analytical methods for the determination of Elgailani and
spectrophotometry (leaves), acephate pesticide residues in some vegetables Alghamdi
Petroselinum crispumand, (2018)
Eruca sativa
Chlorpyrifos Waste discharges Validation of UV spectrophotometric and HPLC Zalat et al.
methods for quantitative determination of (2014)
chlorpyrifos
Malathion Water, Spectrophotometric determination of malathion Venugopal
cauliflower, (an organophosphorus insecticide) with potassium et al. (2012)
potato, spinach bromate
Malathion, Cauliflower, A rapid spectrophotometric assay of some Mathew et al.
dimethoate, cabbage, organophosphorus pesticide residues in vegetable (2007)
phorate spinach samples
Monocrotophos Fourier-transform infrared Paddy field soil Screening of efficient monocrotophos degrading Buvaneswari
(FTIR) bacterial isolates from paddy field soil of Sivaganga et al. (2017)
spectroscopic District, Tamil Nadu, India
Dimethoate Aqueous solution of gold Adsorption of organophosphate pesticide Momić et al.
nanospheres and nanorods dimethoate on gold nanospheres and nanorods (2016)
Chlorpyrifos, Liquid broth Simultaneous degradation of organophosphorus Abraham et al.
monocrotophos and organochlorine pesticides by bacterial (2014)
consortium
Chlorpyrifos Liquid broth Analysis of chlorpyrifos degradation by Kocuria Neti and
sp. using GC and FTIR Zakkula (2013)
Acephate Commercial acephate Extraction, UV-visible, FTIR, NMR spectroscopic Upadhay et al.
study of acephate and effect of pH (2013)
Organophosphate Biological sample Mandal nanoparticle-based electrochemical Liu et al.
pesticides immunosensor for the detection of phosphorylated (2008)
acetylcholinesterase: an exposure biomarker of
organophosphate pesticides and nerve agents
12
Chlorpyrifos and Nuclear magnetic Biological sample In vitro interaction of organophosphate metabolites Dahiya et al.
parathion resonance (NMR) with bovine serum albumin: a comparative 1H (2020)
NMR, fluorescence, and molecular docking
analysis
Organophosphate Biological sample 1H nuclear magnetic resonance (NMR) Alam et al.
pesticides metabolomic study of chronic organophosphate (2012)
exposure in rats
Methyl parathion Sorbed on clays NMR investigation of the behavior of an Seger and
organothiophosphate pesticide, methyl parathion, Maciel (2006)
sorbed on clays
Dichlorvos Lab study Dichlorvos degradation studied by 31 P-NM Benoit-
Marquié et al.
(2004)
Chlorpyrifos Lab study Removal of chlorpyrifos, an insecticide using Vigneshwaran
metal-free heterogeneous graphitic carbon nitride et al. (2019)
(g-C3N4) incorporated chitosan as catalyst:
photocatalytic and adsorption studies
Methyl parathion X-ray diffraction (XRD) Lab study Methyl parathion detection in vegetables and fruits Govindasamy
using silver@ graphene nanoribbons et al. (2017)
nanocomposite modified screen printed electrode
Diazinon Lab study Diazinon degradation by a novel strain Ralstonia Wang et al.
sp. DI-3 and X-ray (2006)
crystal structure determination of the metabolite of
diazinon
Chlorpyrifos Lab study Photocatalytic degradation of organophosphate Khan et al.
pesticides (chlorpyrifos) using synthesized zinc (2015)
(continued)
278
OPs Analytical methods Samples Objective References

oxide nanoparticle by membrane filtration reactor
under UV irradiation
Organophosphorus Lab study Crystallization and preliminary X-ray diffraction Gotthard et al.
compounds analysis of the organophosphorus hydrolase (2013)
OPHC2 from Pseudomonas pseudoalcaligenes
Methyl parathion and Lab study Computational interaction analysis of Sharma, et al.
parathion organophosphorus pesticides with different (2011)
metabolic proteins in humans
Dimethoate Gas chromatography-mass Lab study Optimization and efficiency comparison of Alnedhary
spectrometry (GC-MS) dispersive and cartridge solid phase extraction et al. (2020)
cleanup techniques in the analysis of pesticide
residues in some vegetables using gas
chromatography-mass spectrometry
Organophosphate Citrus aurantium Selective pressurized extraction as single-step Santos et al.
esters extraction and cleanup for the determination of (2020)
organophosphate ester flame retardant in Citrus
aurantium leaves by gas chromatography-tandem
mass spectrometry
Parathion, Soil samples Chemometric-assisted ultrasound leaching-solid Ahmadi et al.
methyl parathion, phase extraction followed by dispersive- (2015)
disulfoton solidification liquid-liquid microextraction for
determination of organophosphorus pesticides in
soil samples
Organophosphate Dust and soil Analysis of organophosphate esters in dust, soil, Jian-Xia et al.
esters (OPEs) and sediment samples using gas chromatography (2014)
coupled with mass spectrometry
Organophosphate Soil Application of Twisselmann extraction, SPME, and Mihajlovic
esters GC-MS to assess input sources for et al. (2011)
organophosphate esters into soil
12
Malathion, isoprocarb Liquid chromatography- Tomato, apple, carrot, and Simultaneous determination of carbamate and Liu et al.
mass spectrometry cabbage organophosphorus pesticides in fruits and (2005)
(LC-MS) vegetables by liquid chromatography-mass
spectrometry
Methyl parathion Electrochemical Bioassay Lab study Electrochemical biosensing of methyl parathion Gong et al.
pesticide based on acetylcholinesterase (2009)
immobilized onto Au-polypyrrole interlaced
network-like nanocomposite
Paraoxon, methyl Lab study Electrochemical stripping analysis of Liu et al.
parathion, and organophosphate pesticides and nerve agents (2005)
fenitrothion
Parathion methyl Food samples Screening of food samples for carbamate and Del Carlo et al.
organophosphate pesticides using an (2004)
electrochemical bioassay
Turner 1996b). Moreover, most microorganisms have similar biochemical pathways

as that of higher organisms and hence quickly respond to the changes in soil
ecosystem. Consequently, for screening pesticide toxicity, numerous microbial
techniques have been evolved and utilized. According to Tothill and Turner
(1996a), microbial-mediated bioassays utilized various mechanisms based on:
• The ability of these microorganisms to convert carbon, sulfur, and nitrogen

• The enzymatic activity like ATP utilization, dehydrogenases, acid phosphatase
and alkaline phosphatase, etc.
• Growth, mortality, and photosynthetic activity of microbes
• Glucose uptake
• Oxygen consumption
• Luminescence output
For example: Dehydrogenase-based bioassays utilize specific dyes like methy-

lene blue, resazurin, and triphenyltetrazolium chloride, as acceptors of electron, and
change color in their reduced form Tothill and Turner (1996a). Adenosine
triphosphates (ATPs) are the basis of all cellular activities, which are found in all
living cells and quickly break down upon microbial death. ATP toxicity assay is
based on the change in ATP content under pesticide exposure. ATP luminescence as
a toxicity detection method is established on luciferin-luciferase enzyme-mediated
reaction, wherein luminescence produced is proportional to the amount of ATP
present (Dalzell and Christofi 2002). In order to evaluate the toxicity of chemicals,
activated sludge respiration inhibition was found to be an effective method (ISO
2007; OECD 2014). Bacterial respiration is commonly measured as the oxygen
uptake rate. The respiration rates of samples of activated sludge fed with synthetic
sewage within an enclosed cell containing oxygen electrode are measured after
3 hours of contact period (OECD, 2014). Liao et al. (2001) identified biosensor
based on respiration rate inhibition for wastewater toxicity using oxygen-sensitive
bacterial isolates obtained from activated sludge. When bacterial respiration was
inhibited due to toxicity, more oxygen was able to cross the biosensor membrane,
which resulted in change in the oxidation-reduction rates. The response time of the
biosensor was shown to be approximately 8 min. dos Santos et al. (2002) developed
an activated sludge respirometry-based assay using 96-well microplates. In this
micro-assay, respiration was indirectly quantified by using a dye (tetrazolium violet)
mixed with sludge bacteria in the wells of the microplate. As the result of redox
reaction, tetrazolium violet dye was chemically reduced by bacterial respiration and
produced deep purple color. Hence, the dye color was used as an indicator of sludge
respiration. An incubation period of 24 h was needed, which limits its use for effluent
toxicity monitoring, wherein a quick response is required. Likewise, to evaluate
nitrification-inhibiting substances in industrial wastewaters, similar bioassay was
used (Svenson et al. 2000). Conversion rates of ammonium-nitrogen and nitrite-
nitrogen were used to calculate the nitrification inhibition levels in Nitrosomonas
and Nitrobacter assays, respectively. Luminescent microorganisms have been used
in the production of several toxicity test biosensors. The bioluminescence inhibition
assay is based on marine Gram-negative bacteria, Vibrio fischeri or Photobacterium

phosphoreum. The specific strain, V. fischeri NRRL B- 11177, has been widely used
to estimate acute toxicity. Several commercial test kits, i.e., Microtox, LUMIStox,
and ToxAlert, based on this strain are available (Shijin, 2004). The test relies on the
change in bacterial luminescence upon the exposure to toxic chemicals. The biolu-
minescence inhibition of V. fischeri has been standardized (OECD, 2014), and the
test kits are commercially available in different versions. The advantages of these
toxicity tests include short analyzing period and simplicity of operation. The follow-
ing are the methodology in brief, which can be used to analyze the pesticide toxicity
on microorganisms.
12.5.1 Culture-Independent Analyses
The microbial community diversity in pesticide-contaminated soil can be analyzed

by a culture-independent approach. This is achieved by cloning of small subunit of
ribosomal RNA (SSU rRNA) gene (Zhang et al. 2006). In this technique, microbial
DNA isolated from both the pesticide-contaminated and non-contaminated soil
sample and PCR are performed using 16S rRNA gene-specific primers. The
amplicons are sequenced, and sequence phylogenetic analysis is carried out, com-
paring both samples. The indication of dominant phylotypes demonstrates consider-
able change in bacterial communities.
12.5.1.1 Biomass Estimation: Counting

To evaluate the impact of pesticide on the bacterial population, biomass estimation
technique is applied. According to the method of Frostegård and Bååth (1996), the
bacteria from soil are isolated following the method of Bååth et al. (1992), which is
the modification form of Faegri et al. (1977). The collected 2.5–10 grams of powered
soil sample add in 100ml of distilled water and mixed properly by Omni mixer
(1 rain, 80% of full speed) and after centrifugation of soil solution at 5 C for 10 min
at 750 g. The supernatant was collected and filtered with the help of glass wool; the
sediments redissolved in 100 ml of distilled water and further centrifuged at 5 C for
10 min at 750 g. From this, a 2 ml of sample was taken out to count the bacteria from
the resulting 200 ml solution containing bacteria. After this, the remaining solution
was centrifuged at 13000 g for 20 min. The pellet was collected and redissolved in
3 ml of citrate buffer. The sonication of dissolved pellets for 15 min from different
soil samples was conducted to break up the bacterial aggregates. After this, a 100 μl
bacterial solution was taken to microscopical count. The bacterial count values were
calculated per gram organic matter (measured as loss on ignition at 600 ~ for 4 h).
For the microscopical count, bacterial suspension was diluted using filter sterilized
HAc buffer (0.15 M; pH 4.0), and acridine orange dye is mixed (0.001% working
concentration). After 5 min of incubation, the suspension is filtered via a 0.2 μg black
nucleopore filter, and with the help of fluorescence microscope, stained bacteria are
counted. The bacterial suspension and pellet can be stored in formalin to count the
bacteria after adding an appropriate amount of HAc buffer and acridine orange dye.
12.5.1.2 Phospholipid Fatty Acid (PLFA) Analysis

The extraction of phospholipid fatty acid (PLFA) can be carried out by using the
method developed by Frostegård et al. (1993); in this method, 1.0 g of soil (wet mss)
is extracted in a one-phase mixture having chloroform, methanol, and citrate buffer
(1: 2:0.8, v/v/v). After this, the extract gets separated into two phases; the phase
containing lipid is dried under N2 stream and kept at 20 C. The lipid component is
fractionated on columns having silicic acid into neutral, glycolipid, and phospho-
lipid, including polar lipids. The fraction of phospholipid is dried under N2 stream
and stored for fatty acid methyl ester preparations. Methyl nonadecanoate is mixed
to the fractions of phosphor lipid as standard (internal). After this, the samples are
carried out for alkaline metanalysis as by Dowling et al. (1986), and the separation of
fatty acid methyl esters are carried out by using gas chromatograph (GC), having a
flame ionization detector. Various types of capillary columns having different
polarities can be used. The programming of temperature can be set as follows:
initially, 80 C for 1 min increases with 20 C per min to 160 C, and after this,
increase at 5 C per min to 270 C final temperature for 10 min. The fatty acid methyl
ester’s relative retention times are compared with standards.
12.5.2 Culture-Dependent Analyses
There are several established conventional methods which can be used for the
quantitative analysis of soil microbial characteristics, including mineralization and
respiration tests (Iyyemperumal et al. 2007; Kähkönen et al. 2007), microbial
biomass quantification (Brookes et al. 1985), growth analysis and patterns of
substrate use by utilizing standardized Biolog® plates (e.g., Stefanowicz et al.
2009; Wainwright 1978), and community-level physiological profiling (CLPP)
(e.g., Yang et al. 2006). Soil microbial metabolic activities can be quantified by
recording the production of CO2 (Iyyemperumal et al. 2007; Kähkönen et al. 2007)
and also with the help of different enzyme assay (Wainwright 1978), Wallenstein
and Weintraub 2008). Pesticide toxic impact on microbes is generally analyzed by
measuring the functional responses in monitored soils, like the overall microbial
activity rates (Wainwright 1978) or respiration (Burton and Lanza 1985), or individ-
ual bacterium test in laboratory (Bitton and Koopman 1992). However, key culture-
dependent techniques for analyzing pesticide impact like compatibility in petri dish,
technical grade pesticide in soil and plating, ester-linked fatty acid methyl esters, and
fluorescein diacetate hydrolyzing activity are described here. In these techniques,
various specific enzymes, or combinations of different enzymes have to apply,
access to the impact of pesticides on bacterial communities. Previously, amidase,
alkaline phosphatase, asparaginase protease, dehydrogenase, and urease activities
have been characterized to analyze mancozeb fungicide (Rasool and Reshi 2010);
activity of catalase for pyrethroids and organophosphorus pesticides (Shiyin et al.
2004); and dehydrogenase, phosphatase, and urease for the insecticides
propiconazole, profenofos, and pretilachlor (Kalam et al. 2004).
12.5.2.1 Technical Grade Pesticide in Soil and Plating

In agriculture, the pesticide is used in different dose or concentrations and also in
combination with other pesticides; these pesticides affect the microbial population,
their interactions, and microbial biomass. The impact of different concentration of
pesticide on soil microbes can be analyzed by the method earlier developed by
Radivojević et al. (2011). In this method, the soil is collected from top to 10 cm and
dried; after drying, the soil sample is sieved with a sieve having a mesh pore size
5 mm and kept at 4 C. At the time of use, the soil is dried for 24 hours at room
temperature. The different pesticide solutions of technical grade substances are
prepared in distilled water. After this, the solution concentration was pipetted to
1 kg of soil surface followed by rotation for 30 min at rotator stirrer prior to
homogenization. The homogenized soil is portioned into pots. Soil without treatment
is kept as control. After this, the pots are incubated in a controlled chamber at
20 2 C, 50% air and soil humidity, and 12/12 h photoperiod. The sample is being
collected after the application of pesticide at the desired intervals (e.g., 7, 14, and
30 days). The culture-dependent microbes are analyzed by soil dilution plate
method, with the help of tryptic soy agar and Czapek agar, respectively, for bacteria
and fungi and incubated at 28 C for 3 and 5 days, respectively, for bacteria and
fungi; after this, the colonies are being counted. The determination of microbial
biomass C is carried out by the fumigation-extraction as described by Vance et al.
(1987). The fumigation of samples is done with non-alcohol chloroform for 24 hours
under wet conditions. The carbon is extracted with a 100 ml 0.5 M of potassium
sulfate solution (K2SO4) and its titration performed with 0.0333 M Mohr salt
((NH4)2Fe(SO4)2) solution containing phenylantranil acid to determine the
contents. Control soil sample has also followed the same procedure. The
microbiological biomass carbon (MBC) is calculated by the formula given by
Jenkinson et al. (1979): MBC ¼ C extracted 0.33, where C was extracted from
fumigated and *non-fumigated samples. The results are expressed in μg C g1 soil.
12.5.2.2 Ester-Linked Fatty Acid Methyl Esters (EL-FAME)

Ester-linked fatty acid methyl esters (EL-FAME) also applied to analyze the impact
of pesticides on microbial diversity; this analysis can be performed by ester-linked
fatty acid methyl ester (EL-FAME) extraction method. In this process, as described
by Schutter and Dick (2000), alkaline methanolysis of ester-linked fatty acid carried
out leaving the free fatty acid, which involves four reagents and steps. In the
El-FAME analysis, firstly taken 3 gram of soil sample and kept in a 35 mL
Teflon-coated, caped glass centrifuge tube and mixed with the 0.2 M KOH prepared
in methanol and stored for 1 hour at 37 C. And then intermittent vortexing done after
every 10 min to release the ester-linked fatty acids and its methylation. In the second
step, 3 ml of acetic acid (1.0 M) is mixed for pH neutralization in the tube. The
FAME is separated into an organic phase by mixing 10 ml hexane and centrifuging
at 480Xg for 10 min. After this, in the third step, the hexane layer is collected in a
fresh glass test tube, and evaporation of hexane is carried out under N2 stream. In the
final, fourth step, FAME is being dissolved in 0.5 mL of 1:1 hexane-methyl tert-
butyl ether and transferred to a GC vial for analysis. The gas chromatography can be
carried out in a Hewlett-Packard 5890 Series II (Palo Alto, CA) equipped with an HP
Ultra 2 capillary column (5% diphenyl-95% dimethylpolysiloxane, 25 m by 0.2) and
an ionization detector flame. The ramping temperature setup ranges from 170 to
270 C at 5 C per min, with 2 min at 270 C between samples to clean the column.
The identification of fatty acid being carried out and their relative peak can be
determined by using MIS Aerobe chromatographic program and peak naming
table as supplied by the supplier. To describe FAMEs, the standard nomenclature
should be used.
12.5.2.3 PCR-Denaturing/Temperature Gradient Gel Electrophoresis

DNA fragments of similar length and different base-pairing sequence can be
analyzed using either denaturation gradient gel electrophoresis (DGGE) or tempera-
ture gradient gel electrophoresis (TGGE). From the soil sample, DNA is isolated and
PCR amplified with sequence-specific 16S or 18S rRNA universal primers (Krishna
et al. 2019; Ferreira et al. 2009). Further based on the mobility of DNA fragments on
partially melted acrylamide gels, amplicons are separated along with gradients of
linear DNA denaturants, such as urea and formamide. Change in the melting
property of DNA is attributed to any variation in the amplicon sequence, due to
which separation takes place in denaturing gradient gel. Within the nucleotide
strand, melting occurs at different points having the same melting temperatures.
Based on the concentration of denaturants, sequence variation of fragments will
prevent migration at different points in the gel. Theoretically, DGGE can separate
DNA fragments, which shows single-base-pair difference (Miller et al. 1999). Also,
TGGE works on the same principle; the only difference is the use of temperature
gradient for denaturation instead of chemical. Both DGGE and TGGE techniques are
quick, reliable, reproducible, and cost-effective compared to other techniques and
can analyze numerous samples at the same time and also track the changes in
microbial population under any harsh conditions or stimuli.
12.5.2.4 Terminal Restriction Fragment Length Polymorphisms

(T-RFLP)
Terminal restriction fragment length polymorphisms (T-RFLP) is a modified version
of RFLP, an alternate technique for rapid microbial diversity analysis in different
ecosystems (Thies et al. 2007). This technique works on a similar principle of RFLP
except for fluorescent dye labeling of one PCR primer, such as TET (4,7,20,70-
tetrachloro-6-carboxyfluorescein) or 6-FAM (phosphoramidite fluorochrome
5-carboxyfluorescein). The PCR is performed using l6S rDNA universal primers,
one of which is labeled with fluorescent dye. The fluorescent-labeled terminal RFLP
(FLT_RFLP) patterns can be prepared by digesting the labeled PCR product with
restriction enzymes. The fragments are then separated using gel electrophoresis in an
automated sequence analyzer. The operational taxonomic unit (OTU) is prepared by
counting the unique DNA fragments, and OTU frequency was computed. To
measure species diversity and similarities as well as evenness between samples,
banding patterns were used. However, this technique might mislead the actual
microbial diversity, because in most case the most dominant species are detected
because of the availability in large amount of DNA template (Liu et al. 1997),
whereas the incomplete or partial digestion of DNA by restriction enzymes may
result in diversity overestimation. Further, universal primers are unable to amplify
the whole sequences from bacterial and archaeal domains, and the primers are
designed from the existing 16S rRNA and internal transcribed spacer (ITS) sequence
databases, which have sequences from culturable microbes. Hence, it doesn’t repre-
sent the true diversity of the microbial sample. Moreover, different restriction
enzymes will generate dissimilar fingerprints of microbial community. T-RFLP is
a unique technique used for comparing relationships among different microbial
samples and has also been used for the measurement of spatial as well as temporal
changes in the communities of bacteria to analyze complicated microbial
communities, detect and monitor populations, and assess arbuscular mycorrhizal
fungi diversity in rhizosphere.
12.5.2.5 Single-Strand Conformation Polymorphisms (SSCP)

The single-strand conformation polymorphism (SSCP) technique works on
electrophoresis-based discrimination of DNA sequences and allows differentiation
of DNA fragments of equal length but with varied nucleotide sequences. This
technique was initially developed to distinguish point mutations or novel
polymorphisms in DNA fragments. In this technique, single-stranded DNA
fragments were separated in a polyacrylamide gel, wherein variation in the mobility
of DNA fragments was due to its secondary structure (i.e., heteroduplex). This
technique is helpful in analyzing the genetic diversity of microbes. Sometimes,
multiple bands are produced for the same DNA sequence on the gel caused by the
presence of multiple stable conformations of some single-stranded DNA. However,
this technique does not require a GC clamp or gradient gel construction. It has been
also used for the study of rhizosphere community diversity of bacteria and fungi
(Stach et al. 2001).
12.6 Future Perspectives and Challenges
Food security for a rapidly growing global population and consequent higher rates of
food consumption has resulted in increased production and application of pesticides
in agricultural sector. Different modes of pesticide classification are generally based
on their chemical composition, characteristics, target pest and mode of action, and
entry. Organophosphates find a wide usage as compared to other classes of
pesticides due to their efficiency and degradation. In spite of several number of
studies to decipher the effect of pesticide application on soil ecosystem, there is
lesser clarity in understanding the role of pesticides to cause disturbance in the soil
environment due to a wide variation in the results obtained from these studies. It may
be that certain pesticides’ residues serve as a source of carbon or energy for the
microorganisms following their microbial degradation and assimilation, while many
reports state adverse effects of these residues on soil microflora also. Therefore, the
effect of pesticides on microbes and associated soil nutrient transformations, enzyme
activities, and other biochemical processes is not definitely conclusive because of the
differences in the levels of toxicity exhibited by the different classes of pesticides.
Additionally, the effect of pesticides on soil biological activities is also defined by
other contributing factors, such as soil physicochemical properties, nature, concen-
tration and activity of pesticide used, and metabolites produced in soil from the
resulting metabolic activities. Disturbance in biochemical equilibria affecting the
fertility and productivity of soil occurs mostly due to the long-term application of
pesticides. Thus, the understanding of fundamental mechanisms behind the different
microbial responses at the molecular level due to the application of pesticides could
provide great help in explaining the risk of pesticide contaminations. It will also help
to understand the subsequent adverse effects on microbial diversity, enzymatic
activities, and biochemical processes in soil. Therefore, to gain a comprehensive
understanding and quantify the net effect of pesticide application on soil biology and
biochemistry, induction of molecular techniques is very important in contrast to the
traditional methods.
References
Abdel-Mallek AY, Moharram AM, Abdel-Kader MI, Omar SA (1994) Effect of soil treatment with
the organophosphorus insecticide profenofos on the fungal flora and some microbial activities.
Microbiol Res 149:167–171
Abraham J, Silambarasan S, Logeswari P (2014) Simultaneous degradation of organophosphorus
and organochlorine pesticides by bacterial consortium. J Taiwan Inst Chem E 45:2590–2596
Abrahams PW (2002) Soils: their implications to human health. Sci Total Environ 291:1–32
Adesemoye AO, Obini M, Ugoji EO (2008) Comparison of plant growth-promotion with Pseudo-
monas aeruginosa and Bacillus subtilis in three vegetables. Braz J Microbiol 39:423–426
Agrawal A, Sharma B (2010) Pesticides induced oxidative stress in mammalian systems. Int J Biol
Med Res 1(3):90–104
Ahemad M, Khan MS (2012a) Ecological assessment of biotoxicity of pesticides towards plant
growth promoting activities of pea (Pisum Sativum)-specific Rhizobium Sp. Strain MRP1.
Emirates J Food Agric 24:334–343
Ahemad M, Khan MS (2012b) Effects of pesticides on plant growth promoting traits of
Mesorhizobium strain MRC4. J Saudi Soc Agric Sci 11:63–71
Ahmadi K, Abdollahzadeh Y, Asadollahzadeh M, Hemmati A, Tavakoli H, Torkaman R (2015)
Chemometric assisted ultrasound leaching-solid phase extraction followed by dispersive-
solidification liquid–liquid microextraction for determination of organophosphorus pesticides
in soil samples. Talanta 137:167–173
Alam TM, Neerathilingam M, Alam MK, Volk DE, Ansari GA, Sarkar S, Luxon BA (2012) 1H
nuclear magnetic resonance (NMR) metabolomic study of chronic organophosphate exposure in
rats. Meta 2:479–495
Alnedhary AA, AL-Hammadi MM, Numan AA, Murshed FA (2020) Optimization and efficiency
comparison of dispersive and cartridge solid phase extraction cleanup techniques in the analysis
of pesticide residues in some vegetables using gas chromatography-mass spectrometry spec-
trometry. PSM Biol Res 5:40–54
Antonious GF (2003) Impact of soil management and two botanical insecticides on urease and
invertase activity. J Environ Sci Health B 38:479–488
Appelbaum E (2018) The rhizobium/Bradyrhizobium-legume symbiosis. In: Molecular biology of
symbiotic nitrogen fixation. CRC Press, Boca Raton, pp 131–158
Asari S, Matzén S, Petersen MA, Bejai S, Meijer J (2016) Multiple effects of Bacillus
amyloliquefaciens volatile compounds: plant growth promotion and growth inhibition of
phytopathogens. FEMS Microbiol Ecol 92:fiw070
Bååth E, Frostegård Å, Fritze H (1992) Soil bacterial biomass, activity, phospholipid fatty acid
pattern, and pH tolerance in an area polluted with alkaline dust deposition. Appl Environ
Microbiol 58:4026–4031
Bajgar J (2004) Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, pro-
phylaxis, and treatment. Adv Clin Chem 38:151–216
Bakry NM, El-Rashidy AH, Eldefrawi AT, Eldefrawi ME (2006) Direct action of
organophosphates anticholinesterase on nicotinic and muscarinic acetylcholine receptors. J
Biochem Toxicol 3:235–259
Banerjee I, Tripathi SK, Roy AS, Sengupta P (2014) Pesticide use pattern among farmers in a rural
district of West Bengal, India. J Nat Sci Biol Med 5:313
Bayer H, Mitterer M, Schinner F (1982) Einfluss von Insektiziden auf mikrobiogene prozessein
Ah-Materialien eineslandwirtsch aftlichgenutzten Bodens, Pedobiologia
BBC report (2013). https://www.bbc.com/news/world-asia-india-23353017
Bergstrom DW, Montreal CM, Millette JA, King DJ (1998) Spatial dependence of soil enzyme
activities along a slope. Soil Sci Soc Am J 62:1302–1308
Benoit-Marquié F, De Montety C, Gilard V, Martino R, Maurette MT, Malet-Martino M (2004)
Dichlorvos degradation studied by 31 P-NMR. Environ Chem Lett 2(2):93–97
Beri V, Goswami KP, Brar SS (1978) Urease activity and its Michaelis constant for soil systems.
Plant Soil 49:105–115
Bhanti M, Taneja A (2007) Contamination of vegetables of different seasons with
organophosphorous pesticides and related health risk assessment in northern India.
Chemosphere 69:63–68
Bitton G, Koopman B (1992) Bacterial and enzymatic bioassays for toxicity testing in the environ-
ment. In: Reviews of environmental contamination and toxicology. Springer, New York, pp
1–22
Bolton H, Elliot LF, Papendick RI, Bezdicek DF (1985) Soil microbial biomass and selected soil
enzymes activities: effect of fertilization and cropping practices. Soil Biol Biochem 17:297–302
Brookes PC, Landman A, Pruden G, Jenkinson DS (1985) Chloroform fumigation and the release
of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil.
Soil Biol Biochem 17:837–842
Buckley NA, Roberts D, Eddleston M (2004) Overcoming apathy in research on organophosphate
poisoning. BMJ 329:1231–1233
Bucket JZ, Dick RP (1998) Microbial and soil parameters in relation to N mineralization in soils of
diverse genesis under differing management systems. Biol Fertil Soils 27:430–438
Burton GA, Lanza GR (1985) Sediment microbial activity tests for the detection of toxicant
impacts. In: Aquatic toxicology and hazard assessment: Seventh symposium. ASTM Interna-
tional, Philadelphia
Buvaneswari G, Thenmozhi R, Nagasathya A, Thajuddin N (2017) Screening of efficient
monocrotophos degrading bacterial isolates from paddy field soil of Sivagangai District,
Tamil Nadu, India. J Environ Sci Technol 10:13–24
Carter A (2000) How pesticides get into water-and proposed reduction measures. Pesticide Outlook
11:149–156
Caceres TP, He WX, Megharaj M, Naidu R (2009) Effect of insecticide fenamiphos on soil
microbial activities in Australian and Ecuadorean soils. J Environ Sci Health C 44:13–17
Castellano-Hinojosa A, Pérez-Tapia V, Bedmar EJ, Santillana N (2018) Purple corn-associated
rhizobacteria with potential for plant growth promotion. J Appl Microbiol 124:1254–1264
Chandra S (2014) Effect of washing on residues of Chlorpyrifos and Monocrotophos in vegetables.
Int J Adv Res 2:744–750
Chaintreuil C, Giraud E, Prin Y, Lorquin J, Bâ A, Gillis M, de Lajudie P, Dreyfus B (2000)

Photosynthetic Bradyrhizobia as natural endophytes of the African wild rice Oryza
breviligulata. Appl Environ Microbiol 66:5437–5447
Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Junge H (2007)
Comparative analysis of the complete genome sequence of the plant growth–promoting bacte-
rium Bacillus amyloliquefaciens FZB42. Nature Biotech 25(9):1007–1014
CSE Report (2005) Analysis of pesticide residues in blood samples from villages of Punjab
CSE/PML/PR- 21/2005. http://indiaenvironmentportal.org.in/files/Punjab-blood-report.pdf
Dar MA, Kaushik G, Chiu JFV (2020) Pollution status and biodegradation of organophosphate
pesticides in the environment. In: Abatement of environmental pollutants, Elsevier, pp 25–66
Dahiya V, Anand BG, Kar K, Pal S (2020) In vitro interaction of organophosphate metabolites with
bovine serum albumin: a comparative 1H NMR, fluorescence and molecular docking analysis.
Pestic Biochem Physiol 163:39–50
Dalzell DJ, Christofi N (2002) An ATP luminescence method for direct toxicity assessment of
pollutants impacting on the activated sewage sludge process. Water Res 36:1493–1502
De Cesare F, Garzillo AMV, Buonocore V, Badalucco L (2000) Use of sonication for measuring
acid phosphatase activity in soil. Soil Biol Biochem 32:25–832
Defo MA, Njine T, Nola M, Beboua FS (2011) Microcosm study of the long-term effect of
endosulfan on enzyme and microbial activities on two agricultural soils of Yaounde-Cameroon.
Afr J Agric Res 6:2039–2050
Del Carlo M, Mascini M, Pepe A, Diletti G, Compagnone D (2004) Screening of food samples for
carbamate and organophosphate pesticides using an electrochemical bioassay. Food Chem
84:651–656
Vimala Devi PS, Duraimurugan P, Chandrika KSVP (2019) Bacillus thuringiensis-based
nanopesticides for crop protection. In: Nano-biopesticides today and future perspectives. Aca-
demic Press, London, pp 249–260
Dick WA, Cheng L, Wang P (2000) Soil acid and alkaline phosphatase activity as pH adjustment
indicators. Soil Biol Biochem 32:1915–1919
Dowling NJ, Widdel F, White DC (1986) Phospholipid ester-linked fatty acid biomarkers of
acetate-oxidizing sulphate-reducers and other sulphide-forming bacteria. Microbiology
132:1815–1825
Elgailani IEH, Alghamdi AAA (2018) Analytical methods for the determination of Acephate
pesticide residues in some vegetables. Rasayan J Chem 11:979–983
El-Khawas H, Adachi K (1999) Identification and quantification of auxins in culture media of
Azospirillum and Klebsiella and their effect on rice roots. Biol Fertil Soils 28:377–381
Fægri A, Torsvik VL, Goksøyr J (1977) Bacterial and fungal activities in soil: separation of bacteria
and fungi by a rapid fractionated centrifugation technique. Soil Biol Biochem 9:105–112
Fagnani R (2011) Organophosphorus and carbamates residues in milk and feedstuff supplied to
dairy cattle. Pesqui Vet Bras 31:598–502
Ferreira HL, Spilki FR, Santos MMABD, Almeida RSD, Arns CW (2009) Comparative evaluation
of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian
metapneumovirus subtype A. Ciência Rural 39(5):1445–1451
Fragoeiro S, Magan N (2008) Impact of Trametes versicolor and Phanerochaete chrysosporium on
differential breakdown of pesticide mixtures in soil microcosms at two water potentials and
associated respiration and enzyme activity. Int Biodeterior Biodegradation 62:376–383
Frostegård A, Bååth E (1996) The use of phospholipid fatty acid analysis to estimate bacterial and
fungal biomass in soil. Biol Fertil Soils 22:59–65
Frostegård Å, Tunlid A, Bååth E (1993) Phospholipid fatty acid composition, biomass, and activity
of microbial communities from two soil types experimentally exposed to different heavy metals.
Flury M (1996) Experimental evidence of transport of pesticides through field soils — a review. J
Environ Qual 25:25–45
Gamez R, Cardinale M, Montes M, Ramirez S, Schnell S, Rodriguez F (2019) Screening, plant

growth promotion and root colonization pattern of two rhizobacteria (Pseudomonas fluorescens
Ps006 and Bacillus amyloliquefaciens Bs006) on banana cv. Williams (Musa acuminata Colla).
Gong J, Wang L, Zhang L (2009) Electrochemical biosensing of methyl parathion pesticide based
on acetylcholinesterase immobilized onto au–polypyrrole interlaced network-like
nanocomposite. Biosens Bioelectron 24:2285–2288
Gotthard G, Hiblot J, Gonzalez D, Chabriere E, Elias M (2013) Crystallization and preliminary
X-ray diffraction analysis of the organophosphorus hydrolase OPHC2 from Pseudomonas
pseudoalcaligenes. Acta Crystallogr F 69:73–76
Govindasamy M, Mani V, Chen SM, Chen TW, Sundramoorthy AK (2017) Methyl parathion
detection in vegetables and fruits using silver@ graphene nanoribbons nanocomposite modified
screen printed electrode. Sci Rep 7:46471
Gowda SA, Somashekar RK (2012) Evaluation of pesticide residues in farmgate samples of
vegetables in Karnataka, India. Bull Environ Contam Toxcol 89:626–632
Greenpeace India report, Pesticide Residues in Tea Samples from India, August (2014). https://
www.greenpeace.org/...india/Global/india/image/2014/cocktail/download/Trouble
Grube A, Donaldson D, Kiely T, Wu L (2011) Pesticides industry sales and usage. US EPA,
Washington, DC
Gunnell D, Eddleston M, Phillips MR, Konradsen F (2007) The global distribution of fatal pesticide
self-poisoning: systematic review. BMC Public Health 7:357
Gupta PK (2004) Pesticide exposure—Indian scene. Toxicology 198:83–90
Gupta RC (2006) Classification and uses of organophosphates and carbamates. Toxicology of
organophosphate & Carbamate compounds. Academic Press Cambridge, pp. 5–24
Harinathareddy A, Prasad NBL, Devi LK (2014) Pesticide residues in vegetable and fruit samples
from Andhra Pradesh. India J Biol Chem Res 31:1005–1015
Hassan SH, Van Ginkel SW, Hussein MA, Abskharon R, Oh SE (2016) Toxicity assessment using
different bioassays and microbial biosensors. Environ Int 92:106–118
Hussain S, Siddique T, Saleem M, Arshad M, Khalid A (2009) Impact of pesticides on soil
microbial diversity, enzymes, and biochemical reactions. Advan Agron 102:159–200
Hingole SS, Pathak AP (2016) Saline soil microbiome: a rich source of halotolerant PGPR. J Crop
Sci Biotechnol 19:231–239
Ingram CW, Coyne MS, Williams DW (2005) Effects of commercial diazinon and imidacloprid on
microbial urease activity in soil and sod. J Environ Qual 34:1573–1580
ISO 8192 (2007) Water quality–test for inhibition of oxygen consumption by activated sludge for
carbonaceous and ammonium oxidation
Iyyemperumal K, Israel DW, Shi W (2007) Soil microbial biomass, activity and potential nitrogen
mineralization in a pasture: impact of stock camping activity. Soil Biol Biochem 39:149–157
Jain R, Saxena J, Sharma V (2012) Solubilization of inorganic phosphates by Aspergillus awamori
S19 isolated from rhizosphere soil of a semi-arid region. Ann Microbiol 62:725–735
Jaiswal DK, Verma JP (2018) Role of geomicrobiology and biogeochemistry for bioremediation to
clean the environment. Environ Anal Eco Stud 1:EAES.000505
Jaiswal DK, Verma JP, Krishna R, Gaurav AK, Yadav J (2019) Molecular characterization of
monocrotophos and chlorpyrifos tolerant bacterial strain for enhancing seed germination of
vegetable crops. Chemosphere 223:636–650
Jastrzebska E (2011) The effect of chlorpyrifos and teflubenzuron on the enzymatic activity of soil.
Pol J Environ Stud 20:903–910
Jenkinson DS, Davidson SA, Powlson DS (1979) Adenosine triphosphate and microbial biomass in
soil. Soil Biol Biochem 11:521–527
Jian-Xia LU, Wen JI, Sheng-Tao MA, Zhi-Qiang YU, Zhao WANG, Han LI, Jia-Mo FU (2014)
Analysis of organophosphate esters in dust, soil and sediment samples using gas chromatogra-
phy coupled with mass spectrometry. Chin J Anal Chem 42:859–865
Jung HK, Kim SD (2003) Purification and characterization of an antifungal antibiotic from Bacillus
megaterium KL 39, a biocontrol agent of red-pepper phytophthora blight disease. Kor J
Microbiol Biotechnol 31:235–241
Kähkönen MA, Tuomela M, Hatakka A (2007) Microbial activities in soils of a former sawmill
area. Chemosphere 67:521–526
Kalam A, Tah J, Mukherjee AK (2004) Pesticide effects on microbial population and soil enzyme
activities during vermicomposting of agricultural waste. J Environ Biol 25:201–208
Kandeler E, Gerber H (1988) Short-term assay of soil urease activity using colorimetric determina-
tion of ammonium. Biol Fertil Soil 6:68–72
Kang DG, Choi SS, Cha HJ (2006) Enhanced biodegradation of toxic organophosphate compounds
using recombinant Escherichia coli with sec pathway‐driven periplasmic secretion of organo-
phosphorus hydrolase. Biotechnol Prog 22(2):406–410
Khan H, Zeb A, Ali Z, Shah SM (2009) Impact of five insecticides on chickpea (Cicer arietinum L.)
nodulation, yield and nitrogen fixing rhizosperic bacteria. Soil Environ (Pakistan)
Khan MS, Zaidi A, Ahmad E (2014) Mechanism of phosphate solubilization and physiological
functions of phosphate-solubilizing microorganisms. In: Phosphate solubilizing
microorganisms. Springer, Cham, pp 31–62
Khan SH, Suriyaprabha R, Pathak B, Fulekar MH (2015) Photocatalytic degradation of organo-
phosphate pesticides (Chlorpyrifos) using synthesized zinc oxide nanoparticle by membrane
filtration reactor under UV irradiation. Front Nanosci Nanotechnol 1:23–2710
Klose S, Acosta-Martı’nez V, Ajwa HA (2006) Microbial community composition and enzyme
activities in a sandy loam soil after fumigation with methyl bromide or alternative biocides. Soil
Biol Biochem 38:1243–1254
Krishna R, Ansari WA, Verma JP, Singh M (2019) Modern molecular and omics tools for
understanding the plant growth-promoting rhizobacteria. In: Role of plant growth promoting
microorganisms in sustainable agriculture and nanotechnology. Woodhead Publishing,
Duxford, pp 39–53
Kucharski J, Tomkiel M, Boros E, Wyszkowska J (2010) The effect of soil contamination with
diesel oil and petrol on the nitrification process. J Elem 15(1):111–118
Kumar V, Singh S, Upadhyay N (2019) Effects of organophosphate pesticides on siderophore
producing soils microorganisms. Biocatal Agric Biotechnol 21:101359
Kumari B, Kumar R, Madan VK, Singh R, Singh J, Kathpal TS (2003) Magnitude of pesticidal
contamination in winter vegetables from Hisar, Haryana. Environ Monit Assess 87:311–318
Kumari B, Madan VK, Singh J, Singh S, Kathpal TS (2004) Monitoring of pesticidal contamination
of farmgate vegetables from Hisar. Environ Monit Assess 90
Kumari B (2005) Monitoring of butter and ghee (clarified butter fat) for pesticidal contamination
from cotton belt of Haryana, India. Environ Monit Assess 105:111–120
Kumar S, Kaushik G, Villarreal-Chiu JF (2016) Scenario of organophosphate pollution and toxicity
in India: a review. Environ Sci Pollut Res 23:9480–9491
Kumar GP, Kishore N, Amalraj ELD, Ahmed SKMH, Rasul A, Desai S (2012) Evaluation of
fluorescent Pseudomonas spp. with single and multiple PGPR traits for plant growth promotion
of sorghum in combination with AM fungi. Plant Growth Regul 67:133–140
Laabs V, Wehrhan A, Pinto A, Dores E, Amelung W (2007) Pesticide fate in tropical wetlands of
Brazil: an aquatic microcosm study under semi-field conditions. Chemosphere 67:975–989
Lemanceau P, Alabouvette C (1993) Suppression of Fusarium wilts by fluorescent pseudomonas:
mechanisms and applications. Biocontrol Sci Tech 3:219–234
Lethbridge G, Burns RG (1976) Inhibition of soil urease by organophosphorus insecticides. Soil
Biol Biochem 8(2):99–102
Liao JD, Wang SH, Hsu DJ (2001) Studies on the early detection of wastewater’s toxicity using a
microbial sensing system. Sensor Actuators B Chem 72:167–173
Liu G, Wang J, Barry R, Petersen C, Timchalk C, Gassman PL, Lin Y (2008) Nanoparticle-based
electrochemical immunosensor for the detection of phosphorylated acetylcholinesterase: an
exposure biomarker of organophosphate pesticides and nerve agents. Chem Eur J 14:9951–9959
Liu M, Hashi Y, Song Y, Lin JM (2005) Simultaneous determination of carbamate and organo-
phosphorus pesticides in fruits and vegetables by liquid chromatography–mass spectrometry. J
Chromatogr 1097:183–187
Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial diversity by
determining terminal restriction fragment length polymorphisms of genes encoding 16S
rRNA. Appl Environ Microbiol 63:4516–4522
Mamane A, Baldi I, Tessier JF, Raherison C, Bouvier G (2015) Occupational exposure to pesticides
and respiratory health. Eur Respir Rev 24(306–319):2015
Mancini F, Van Bruggen AH, Jiqqins JL, Ambatipudi AC, Murphy H (2005) Acute pesticide
poisoning among female and male cotton growers in India. Int J Occup Environ Health
11:221–232
Mandal K, Singh B (2010) Magnitude and frequency of pesticide residues in farmgate samples of
cauliflower in Punjab, India. Bull Environ Contam Toxicol 85:423–426
Mathew SB, Pillai AK, Gupta VK (2007) A rapid spectrophotometric assay of some organophos-
phorus pesticide residues in vegetable samples. Spectrochim Acta A Mol Biomol Spectrosc 67
(5):1430–1432
Mayanglambam T, Vig K, Singh DK (2005) Quinalphos persistence and leaching under field
conditions and effects of residues on dehydrogenase and alkaline phosphomonoesterases
activities in soil. Bull Environ Contam Toxicol 75:1067–1076
Mendes GO, Dias CS, Silva IR, Júnior JIR, Pereira OL, Costa MD (2013) Fungal rock phosphate
solubilization using sugarcane bagasse. World J Microbiol Biotechnol 29:3–50
Mihajlovic I, Miloradov MV, Fries E (2011) Application of Twisselmann extraction, SPME, and
GC-MS to assess input sources for organophosphate esters into soil. Environ Sci Technol
45:2264–2269
Miller KM, Ming TJ, Schulze AD, Withler RE (1999) Denaturing gradient gel electrophoresis
(DGGE): rapid and sensitive technique to screen nucleotide sequence variation in populations.
BioTechniques 27:1016–1030
Momić T, Pašti TL, Bogdanović U, Vodnik V, Mraković A, Rakočević Z, Vasić V (2016)
Adsorption of organophosphate pesticide dimethoate on gold nanospheres and nanorods. J
Nanomat
Monkiedje A, Spiteller M (2002) Effects of the phenylamide fungicides, mefenoxam and metalaxyl,
on the biological properties of sandy loam and sandy clay soil. Biol Fertil Soil 35:393–398
Mukherjee I (2003) Pesticides residues in vegetables in and around Delhi. Environ Monit Assess
86:265–271
Muñoz-Quezada MT, Lucero BA, Iglesias VP, Muñoz MP, Cornejo CA, Achu E, Baumert B,
Hanchey A, Concha C, Brito AM, Villalobos M (2016) Chronic exposure to organophosphate
(OP) pesticides and neuropsychological functioning in farm workers: a review. Int J Occup
Environ Health 22:68–79
Mus F, Crook MB, Garcia K, Costas AG, Geddes BA, Kouri ED, Paramasivan P, Ryu MH,
Oldroyd GE, Poole PS, Udvardi MK (2016) Symbiotic nitrogen fixation and the challenges to
its extension to nonlegumes. Appl Environ Microbiol 82:3698–3710
Neti N, Zakkula V (2013) Analysis of chlorpyrifos degradation by Kocuria sp. using GC and FTIR.
Curr Biotica 6:466–472
OECD (2014) Guidance on grouping of chemicals. OECD series on testing and assessment, 194
Ortíz-Castro R, Valencia-Cantero E, López-Bucio J (2008) Plant growth promotion by Bacillus
megaterium involves cytokinin signaling. Plant Signal Behav 3:263–265
Pampulha ME, Oliveira A (2006) Impact of an herbicide combination of bromoxynil and
prosulfuron on soil microorganisms. Curr Microbiol 53:238–243
Pandey A, Tripathi A, Srivastava P, Choudhary KK, Dikshit A (2019) Plant growth-promoting
microorganisms in sustainable agriculture. In: role of plant growth promoting microorganisms
in sustainable agriculture and nanotechnology. Woodhead Publishing, Duxford, pp 1–19
Parmar P, Rathod GB, Rathod S, Parikh A (2014) Organophosphorus compound poisoning
demographic profile in Gandhinagar, Gujarat. J Forensic Toxicol Pharmacol 3:1–4
Pozo C, Martinez-Toledo MV, Salmeron V, Rodelas B, Gonzalez-Lopez J (1995) Effect of

chlorpyrifos on soil microbial activity. Environ Toxicol Chem Int J 14(2):187–192
Raaijmakers JM, Vlami M, de Souza JT (2002) Antibiotic production by bacterial biocontrol
agents. Antonie Van Leeuwenhoek 81:537–547
Radivojević L, Šantrić L, Gajić-Umiljendić J (2011) Rimsulfuron in soil: effects on microbiological
properties under varying soil conditions. Pesticidi i fitomedicina 26:135–140
Raddadi N, Cherif A, Ouzari H, Marzorati M, Brusetti L, Boudabous A, Daffonchio D (2007)
Bacillus thuringiensis beyond insect biocontrol: plant growth promotion and biosafety of
polyvalent strains. Ann Microbiol 57:481–494
Rahmoune B, Morsli A, Khelifi-Slaoui M, Khelifi L, Strueh A, Erban A, van Dongen JT (2017)
Isolation and characterization of three new PGPR and their effects on the growth of Arabidopsis
and Datura plants. J Plant Interact 12:1–6
Ranga Rao GV, Sahrawat KL, Rao CS, Das B, Reddy KK, Bharath BS, Wani SP (2009) Insecticide
residues in vegetable crops grown in Kothapalli watershed, Andhra Pradesh, India: a case study.
Indian J Dryland Agric Res Dev 24:21–27
Rania ABA, Jabnoun-Khiareddine H, Nefzi A, Mokni-Tlili S, Daami-Remadi M (2016) Endo-
phytic bacteria from Datura metel for plant growth promotion and bioprotection against
Fusarium wilt in tomato. Biocontrol Sci Tech 26:1139–1165
Rastogi SK, Tripathi S, Ravishanker D (2010) A study of neurologic symptoms on exposure to
organophosphate pesticides in the children of agricultural workers. Indian J Occup Environ Med
14:54
Rasool N, Reshi ZA (2010) Effect of the fungicide Mancozeb at different application rates on
enzyme activities in a silt loam soil of the Kashmir Himalaya, India. Trop Ecol 51:199
Ribeiro CO, Vollaire Y, Sanchez-Chardi A, Roche H (2005) Bioaccumulation and the effects of
organochlorine pesticides, PAH and heavy metals in the eel (Anguilla anguilla) at the Camargue
nature reserve, France. Aquat Toxicol 74:53–69
Sah SB, Gupta RN, Kumar M, Mandal SK, Saha T, Singh SP (2018) Pesticide residues from farm
gate vegetable samples of vegetables in Bihar. Int J Curr Microbiol App Sci 7:4090–4096
Santos FSD, Moura NBA, Mazur N, Oliveira CD (2002) Heavy metal contamination in soil and
plants by intensive use of pesticides and fertilizers. In 29th symposium, 17th WCSS, Thailand,
pp 1441–1446
Sanghi R (2003) Organochlorine and organophosphorus pesticide residues in breast milk from
Bhopal, Madhya Pradesh, India. Hum Exp Toxicol 22:73–76
Santos JL, Malvar JL, Abril C, Martín J, Aparicio I, Alonso E. (2020) Selective pressurized
extraction as single-step extraction and clean-up for the determination of organophosphate
ester flame retardant in Citrus aurantium leaves by gas chromatography-tandem mass spec-
trometry. Anal Bioanal Chem 1-10
Saritha M, Tollamadugu NP (2019) The status of research and application of biofertilizers and
biopesticides: global scenario. In: Recent developments in applied microbiology and biochem-
istry. Academic Press, London, pp 195–207
Scervino JM, Mesa MP, Della Mónica I, Recchi M, Moreno NS, Godeas A (2010) Soil fungal
isolates produce different organic acid patterns involved in phosphate salts solubilization. Biol
Fertil Soils 46(7):755–763
Schäffer AN, D R E A S (1993) Pesticide effects on enzyme activities in the soil ecosystem. Soil
Biochem 8:273–340
Schneider K, Turrion MB, Grierson PF, Gallardo JF (2001) Phosphatase activity, microbial
phosphorus, and fine root growth in forest soils in the sierra de Gata, western Central Spain.
Biol Fertil Soil 34:151–155
Schutter ME, Dick RP (2000) Comparison of fatty acid methyl ester (FAME) methods for
characterizing microbial communities. Soil Sci Soc Am J 64:1659–1668
Schultz P, Urban NR (2008) Effects of bacterial dynamics on organic matter decomposition and
nutrient release from sediments: a modelling study. Ecol Modell 210:1–14
Seenivasan S, Muraleedharan N (2011) Survey on the pesticide residues in tea in South India.
Environ Monit Assess 176:365–371
Sengupta A, Gunri SK (2015) Microbial intervention in agriculture: an overview. African J
Microbiol Res 9(18):1215–1226
Sepperumal U, Dhivya N, Shanmugavalli R (2016) Phorate induced changes in the growth and
siderophore production by Klebsiella pneumoniae isolated from tomato crop field soil. Ann Biol
Res 7:49–58
Sevilla M, Burris RH, Gunapala N, Kennedy C (2001) Comparison of benefit to sugarcane plant
growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter
diazotrophicus wild-type and Nif– mutant strains. Mol Plant-Microbe Interact 14:358–366
Seger MR, Maciel GE (2006) NMR investigation of the behavior of an organ thiophosphate
pesticide, methyl parathion, sorbed on clays. Environ Sci Technol 40:552–558
Shaheen S, Sundari KS (2013) Exploring the applicability of PGPR to remediate residual organo-
phosphate and carbamate pesticides used in agriculture fields. Int J Agri Food Sci Technol 4
(10):947–954
Shahid M, Hameed S, Imran A, Ali S, van Elsas JD (2012) Root colonization and growth promotion
of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. fs-11. World J
Sharma A, Shankhdhar D, Shankhdhar SC (2016) Potassium-solubilizing microorganisms: mecha-
nism and their role in potassium solubilization and uptake. In: Potassium solubilizing
microorganisms for sustainable agriculture. Springer, New Delhi, pp 203–219
Sharma AK, Gaur K, Tiwari RK, Gaur MS (2011) Computational interaction analysis of organo-
phosphorus pesticides with different metabolic proteins in humans. J Biomed Res 25:335–347
Shenoy VV, Kalagudi GM (2005) Enhancing plant phosphorus use efficiency for sustainable
cropping. Biotechnol Adv 23:501–513
Shijin R (2004) Assessing wastewater toxicity to activated sludge: recent research and
developments. Environ Int 30:1151–1164
Shivakumar S, Mohammed Ishaq R, Raghavan K, Geetha S (2013) Organophosphorus poisoning–a
study of 165 cases from Chennai, India
Shiyin L, Lixiao N, Panying P, Cheng S, Liansheng W (2004) Effects of pesticides and their
hydrolysates on catalase activity in soil. Bull Environ Contam Toxicol 72:00–606
Singh B, Gupta A (2002) Monitoring of pesticide residues in farmgate and market samples of
vegetables in a semiarid, irrigated area. Bull Environ Contam Toxicol 68:747–751
Singh PB, Singh V, Nayak PK (2008) Pesticide residues and reproductive dysfunction in different
vertebrates from north India. Food Chem Toxicol 46:2533–2539
Singh BK, Walker A (2006) Microbial degradation of organophosphorus compounds. FEMS
Microbiol Rev 30(3):428–471
Singh SP, Singh NK (2006) Pesticide residues in farm gate vegetable samples in Bihar. Pest Manag
Hortic Ecosyst 12:152–155
Sinha SN, Rao MVV, Vasudev K (2012) Distribution of pesticides in different commonly used
vegetables from Hyderabad. India Food Res Int 45:161–169
Srivastava AK, Trivedi P, Srivastava MK, Lohani M, Srivastava LP (2011) Monitoring of pesticide
residues in market basket samples of vegetable from Lucknow City, India: QuEChERS method.
Environ Monit Assess 176:465–472
Srinivasulu M, Rangaswamy V (2014) Enzymes and pesticides. In: Gianfreda L, Rao AM (eds)
Enzymes in agricultural sciences. OMICS group e books, Foster city
Srivastava S, Narvi SS, Prasad SC (2008) Organochlorines and organophosphates in bovine milk
samples in Allahabad region. Int J Environ Res 2:165–168
Stach JE, Bathe S, Clapp JP, Burns RG (2001) PCR-SSCP comparison of 16S rDNA sequence
diversity in soil DNA obtained using different isolation and purification methods. FEMS
Microbiol Ecol 36:139–151
Stefanowicz AM, Niklińska M, Laskowski R (2009) Pollution-induced tolerance of soil bacterial

communities in meadow and forest ecosystems polluted with heavy metals. Eur J Soil Biol
45:363–369
Su L, Jia W, Hou C, Lei Y (2011) Microbial biosensors: a review. Biosens Bioelectron
26:1788–1799
Svenson A, Sandén B, Dalhammar G, Remberger M, Kaj L (2000) Toxicity identification and
evaluation of nitrification inhibitors in wastewaters. Environ Toxicol Int J 15:527–532
Swarnam TP, Velmurugan A (2013) Pesticide residues in vegetable samples from the Andaman
Islands, India. Environ Monit Assess 185:6119–6127
Tarafdar JC, Yadav RS, Meena SC (2001) Comparative efficiency of acid phosphatase originated
from plant and fungal source. J Soil Sci Plant Nutr 164:279–282
Tandon A, Fatima T, Shukla D, Tripathi P, Srivastava S, Singh PC (2020) Phosphate solubilization
by trichoderma koningiopsis (NBRI-PR5) under abiotic stress conditions. J King Saud Univ Sci
32(1):791–798
Thiesv FL, König W, König B (2007) Rapid characterization of the normal and disturbed vaginal
microbiota by application of 16S rRNA gene terminal RFLP fingerprinting. J Medical Microbiol
56(6):755–761
Thijs S, Weyens N, Sillen W, Gkorezis P, Carleer R, Vangronsveld J (2014) Potential for plant
growth promotion by a consortium of stress-tolerant 2,4-dinitrotoluene-degrading bacteria:
isolation and characterization of a military soil. Microbial Biotechnol 7:294–306
Tothill IE, Turner APF (1996a) Developments in bioassay methods for toxicity testing in water
treatment. Trac-Trend Anal Chem 15:178–188
Tothill IE, Turner APF (1996b) Developments in bioassay methods for toxicity testing in water
treatment. Trac-Trend Anal Chem 15:178–188
Tscherko D, Kandeler E, Jones TH (2001) Effect of temperature on below-ground N-dynamics in a
weedy model ecosystem at ambient and elevated atmospheric CO2 levels. Soil Biol Biochem 33
(4–5):491–501
Upadhay N, Kumar V, Kumar V (2013) Extraction, UV-visible, FTIR, NMR spectroscopic study of
Acephate and effect of pH. Int J Environ Sci 3:1849–1856
Upadhyay S, Rao GR, Sharma MK, Bhattacharya BK, Rao VK, Vijayaraghavan R (2009) Immo-
bilization of acetylcholine esterase–choline oxidase on a gold–platinum bimetallic nano
particles modified glassy carbon electrode for the sensitive detection of organophosphate
pesticides, carbamates and nerve agents. Biosens Bioelectron 25:832–883
Vaishnav A, Choudhary DK (2019) Regulation of drought-responsive gene expression in Glycine
max l. Merrill is mediated through Pseudomonas simiae strain AU. J Plant Growth Reg 38
(1):333–342
Van den Berg F, Kubiak R, Benjey WG, Majewski MS, Yates SR, Reeves GL, Smelt JH, Van der
Linden AMA (1999) Emission of pesticides into the air, in fate of pesticides in the atmosphere,
implications for environmental risk assessment. Springer, Dordrecht, pp 195–218
Vance ED, Brookes PC, Jenkinson DS (1987) Microbial biomass measurements in forest soils: the
use of the chloroform fumigation-incubation method in strongly acid soils. Soil Biolog Biochem
19(6):697–702
Venugopal NVS, Sumalatha B, Bonthula S (2012) Spectrophotometric determination of malathion
(an organo phosphorous insecticide) with potassium bromate. Eurasian J Anal Chem 8:131–135
Verma JP, Yadav J, Tiwari KN, Jaiswal DK (2014) Evaluation of plant growth promoting activities
of microbial strains and their effect on growth and yield of chickpea (Cicer arietinum L.) in
India. Soil Biol Biochem 70:33–37
Verma JP, Jaiswal DK, Krishna R, Prakash S, Yadav J, Singh V (2018) Characterization and
screening of thermophilic Bacillus strains for developing plant growth promoting consortium
from hot spring of Leh and Ladakh region of India. Front Microbiol 9:1293
Vigneshwaran S, Preethi J, Meenakshi S (2019) Removal of chlorpyrifos, an insecticide using metal
free heterogeneous graphitic carbon nitride (g-C3N4) incorporated chitosan as catalyst:
photocatalytic and adsorption studies. Int J Biol Macromol 132:289–299
Viruel E, Erazzú LE, Martínez Calsina L, Ferrero MA, Lucca ME, Siñeriz F (2014) Inoculation of
maize with phosphate solubilizing bacteria: effect on plant growth and yield. J Soil Sci Plant
Nutrit 14(4):819–831
Vurukonda SSKP, Vardharajula S, Shrivastava M, Sk ZA (2016) Enhancement of drought stress
tolerance in crops by plant growth promoting rhizobacteria. Microbiol Res 184:13–24
Vyas P, Gulati A (2009) Organic acid production in vitro and plant growth promotion in maize
under controlled environment by phosphate-solubilizing fluorescent pseudomonas. BMC
Microbiol 9(1):174
Wang X, Piao X, Chen J, Hu J, Xu F, Tao S (2006) Organochlorine pesticides in soil profiles from
Tianjin, China. Chemosphere 64(9):1514–1520
Wang G, Liu Y (2016) Diazinon degradation by a novel strain Ralstonia sp. DI-3 and X-ray crystal
structure determination of the metabolite of diazinon. J Biosci 41:359–366
Wainwright M (1978) A review of the effects of pesticides on microbial activity in soils. J Soil Sci
29:287–298
Wallenstein MD, Weintraub MN (2008) Emerging tools for measuring and modeling the in-situ
activity of soil extracellular enzymes. Soil Biol Biochem 40:2098–2106
Woo OG, Kim H, Kim JS, Keum HL, Lee KC, Sul WJ, Lee JH (2020) Bacillus subtilis strain GOT9
confers enhanced tolerance to drought and salt stresses in Arabidopsis thaliana and Brassica
campestris. Plant Physiol Biochem 148:359–367
Yang Y, Campbell CD, Clark L, Cameron CM, Paterson E (2006) Microbial indicators of heavy
metal contamination in urban and rural soils. Chemosphere 63:1942–1952
Yasouri FN (2003) Plasmid mediated degradation of diazinon by three bacterial strains Pseudomo-
nas sp., Flavobacterium sp. and Agrobacterium sp. Asian J Chem 18:2437
Yu X, Ai C, Xin L, Zhou G (2011) The siderophore-producing bacterium, Bacillus subtilis CAS15,
has a biocontrol effect on Fusarium wilt and promotes the growth of pepper. Eur J Soil Biol
47:138–145
Zalat OA, Elsayed MA, Fayed MS, Abd El Megid MK (2014) Validation of UV spectrophotometric
and HPLC methods for quantitative determination of chlorpyrifos. Int Lett Chem Phys Astron
2:58–63
Zantua MI, Bremner JM (1977) Stability of urease in soils. Soil Biol Biochem 9:135–140
Zhang R, Jiang J, Gu JD, Li S (2006) Long term effect of methyl parathion contamination on soil
microbial community diversity estimated by 16S rRNA gene cloning. Ecotoxicology
15:523–530
‘Cu-Chi-Tri’, a New Generation Combination
for Knowledge-Based Management 13
of Oomycete Pathogen, Phytophthora
infestans
Nandani Shukla, P. Lemke, B. M. Moerschbacher, and J. Kumar
Abstract
Globally, an estimated 35% of the potential crop is lost annually to diseases, pest
and weeds, while decreases in arable land and increases in world population,
global climate change and increased production of energy crops continue to
enhance the pressure. Chemical plant protection is too expensive for resource
poor farmers, and it is potentially unsafe to both environment and consumer.
However, as an alternative to chemical plant protection, biological plant protec-
tion is less consistently reliable. Nevertheless, a combination of different
strategies could make biological plant protection more reliable. The present
chapter focuses on developing a novel combination product for economically
viable and ecologically safe plant protection, with emphasis on devastating
diseases caused by oomycete plant pathogens, particularly Phytophthora
infestans, the dreaded late blight causative in potato. Late blight disease manage-
ment has largely relied on the use of chemical fungicides, with the above-
mentioned problems and the added threat of the development of chemical resis-
tant strains of the pathogen. Therefore, the need of alternative approaches for late
blight management without compromising benefits as attained by the use of
chemicals has been variously flagged. To this end, a new generation fungicide
involving a low-dose fungicide (Cu(OH)2), a biocontrol agent (Trichoderma) and
a plant defence activator (chitosan) has been developed and tested under field
conditions for the management of potato late blight. The ‘triple combination’
evokes newer avenues of application of biocontrol agents for safer and sustain-
able management of oomycete plant pathogens.
N. Shukla · J. Kumar (*)

Department of Plant Pathology, GBPUA&T, Pantnagar, Uttarakhand, India
P. Lemke · B. M. Moerschbacher
Institute for Biology and Biotechnology of Plants, University of Münster, Münster, Germany

https://doi.org/10.1007/978-981-15-6275-4_13
298 N. Shukla et al.
Keywords
Copper hydroxide · Chitosan · Trichoderma · Triple combination · Phytophthora

infestans
13.1 Introduction
Extreme and unrestricted use of synthetic pesticides and chemical fertilizers in

agriculture to prevent crop yield losses or product damage leads to alarming
consequences of toxicity. Green revolution technologies have inadvertently
increased dependence on harmful chemicals, pesticides and fertilizers, not only
damaging the ecosystems but also threatening human health. Further, climate change
and faulty practices have aggravated the problem, making the situation even worse.
Pests and diseases cause about 35% losses to crop plants, and their management
contributes to a huge quantum of pesticide use (Flood 2010).
Since the beginning of recorded history, plant disease outbreaks have had a
significant impact on human society. Among these devastating outbreaks, the ‘late
blight disease’ that caused the Irish potato famine of the 1840s triggered the official
beginning of the science of plant pathology and was the first plant disease for which
a microorganism was proved to be the causal agent (Schumann and D’Arcy 2000).
Late blight caused by the oomycete Phytophthora infestans (Mont.) de Bary is
affecting cultivation of potatoes and tomatoes. The disease poses very serious
economic threats to the vast majority of potato production systems as well as
many tomato production systems worldwide (Madden 1983) and has been estimated
in 2012 to be responsible for causing $6.7 billion yield losses annually (Nowicki
et al. 2012).
Management of the late blight pathogen under field conditions is a challenge
since the pathogen has the ability to develop new races and is able to adapt to a wide
range of environmental conditions (Njoroge 2019). The control of the disease has
traditionally relied on foliar applications of fungicides. These fungicides include
copper salts, dithiocarbamates, bis-dithiocarbamates, cyanoacetamide oxime and
metalaxyl. Excessive use of these chemicals poses great threats at a global level
(Ragunanthan and Divakar 1996) and also risks several components of agro-
ecosystem (Ghorbani et al. 2004).
The only effective fungicides currently permitted for blight management even in
organic agriculture are copper-based products (Mizubuti et al. 2007). FRAC (Fungi-
cide Resistance Action Committee) has grouped copper-based fungicides in the
group of ‘low resistance risk’ (Clark and Yamaguchi 2002). Nevertheless, appear-
ance of fungicide-resistant pathogen strains and negative environmental impacts
associated with use of chemicals has intensified the need for reducing chemical
use and for alternative disease management methods.
Biocontrol agents (BCAs) could be one of the most promising and effective
means of alternative disease management strategies, and plant defence inducers,
such as chitosan, could also be utilized in plant disease management resulting in less
13 ‘Cu-Chi-Tri’, a New Generation Combination for Knowledge-Based Management. . . 299
impact of the chemicals on the environment. Trichoderma is a widely used

biological control agent in organic and integrated nutrient management approaches;
however, it lacks efficacy in field condition (Monte 2001). Chitosan is a naturally
occurring compound that has a potential in agriculture with regard to controlling
plant diseases (Hadwiger et al. 1986). This biopolymer has antimicrobial activities
against plant pathogens (viruses, bacteria and other pests) and is well known to have
resistance eliciting activities in plants, leading to a variety of defence responses in
host plants in response to microbial infections, including the accumulation of
phytoalexins, pathogen-related (PR) proteins and proteinase inhibitors, lignin syn-
thesis and callose formation (Oh et al. 1998). Based on these and other properties
that help strengthen host plant defences, chitosan is widely used as an antimicrobial
agent either alone or blended with other natural polymers (No and Meyers 1997).
However, these management methods do not prevent late blight infection alone and
therefore might profit from the integration of copper fungicides that already play an
essential role in the control of oomycete pathogens, particularly under organic
production systems.
13.2 Role of Copper in Management of Late Blight Disease

of Potato
Copper is a vital element for all living organisms as it acts as a cofactor for a range of
enzymes, though excessive use of copper can generate highly reactive oxygen
radicals (Zapotoczny et al. 2007) and can lead to mutation (Anand et al. 2006). In
recent years, due to widespread and long-term use of copper-containing pesticides in
agriculture against phytopathogenic microorganisms, its toxicity has become an
agricultural and environmental concern (Cornejo et al. 2013). Use of copper
fungicides has been restricted in Europe due to increasing concerns about long-
term build-up in soil in areas that have a history of high application rates (EU 2018).
Repeated application of copper leads to Cu accumulation in the soil, affects soil
biology and causes damage to non-target organisms such as earthworms (Lumbricus
terrestris) (Homa et al. 2003).
Based on these concerns, use of Cu fungicides had been restricted in Europe to
6 kg Cu ha1 (Anonymous 2008). In Denmark and the Netherlands, copper
fungicides were completely banned, while in Germany, most of the organic farmer
associations allowed up to 3 kg Cu ha1 in potato production. Copper hydroxide is a
frequently used formulation permitted in Germany and thus is authorized for use in
plant protection products (Anonymous 2009). Though application of Cu fungicides
was allowed until 2016, yet research groups and farmers’ associations imposed clear
Cu input reductions in the EU. Recent EU regulations restrict the use of plant
protection products containing copper compounds to a maximum application rate
of 28 kg/ha of copper over a period of 7 years (i.e. on average 4 kg/ha/year) in order
to minimize the potential accumulation in soil and the exposure for non-target
organisms while taking into account agro-climatic conditions occurring periodically
in Member States leading to an increase of the fungal pressure (EC 2018).
Accordingly, alternative, copper-free or copper-reduced management practices

including treatments based on biocontrol agents (BCAs) for controlling late blight
in potato have been intensively investigated (Tamm et al. 2004). Several alternative
preparations tested in recent years showed potential against the late blight pathogen
in vitro but had only insignificant effects on infections of potato crops in the field.
For example, sage essence (Salvia officinalis) reduced zoospore release of
P. infestans (Blaeser et al. 1998) but revealed no reproducible effects on late blight
severity or tuber yield in vitro (Neuhoff et al. 2006). Beneficial microorganisms,
namely, fungi, bacteria or compost extracts, were found less effective in reducing
foliar development of late blight on detached leaves (Ghorbani et al. 2005). Twenty-
seven copper-free preparations in field trials had been tested; however, only 17%
efficacy was recorded in control of the late blight pathogen. Apparently, stability
under existing environmental conditions remained the main reason for the failure
(Dorn et al. 2007a, b). Finally, recent findings using a 4% extract of Frangula cortex
reduced disease progress similar to 3 kg Cu ha1, but with no significant yield effects
(Krebs et al. 2013).
Today, Cu-based fungicides appear to be the best treatment in organic potato
production (Zarb et al. 2002), but efficient application is required, and it should be
applied only after the failure of alternative, non-chemical methods. Reducing the
rates of copper per application or the frequencies of applications may significantly
reduce the annual Cu input. However, insufficient information is available about
field performance of formulations with reduced Cu doses (e.g. Krebs et al. 2013).
Therefore, development of alternative or combination products that can naturally
induce resistance in the plants and reduce the doses of copper is quite desirable.
13.3 Role of Trichoderma in the Management of Late Blight

Disease of Potato
Trichoderma spp. are ubiquitous and often predominant components of the micro-
flora as saprophytes in soil, litter, organic matter and rhizospheric ecosystems of all
climatic zones. Recent discoveries show that they are opportunistic, avirulent plant
symbionts, as well as being parasites of other fungi (Vinale et al., 2008). Strains of
Trichoderma spp. are endophytes and establish robust and long-lasting colonization
of root surfaces and penetrate into the epidermis. However, the ability of these fungi
to sense, invade and destroy other fungi has been the major driving force behind their
commercial success as biopesticides (Benítez et al. 2004). Trichoderma defend the
plants by their direct and indirect effect on plant-pathogen-soil environment
interactions. These fungi do not only protect plants by killing the pathogens, mainly
other fungi and nematodes, but they also induce resistance against plant pathogens
(Hermosa et al. 2011), impart abiotic stress tolerance and improve plant growth,
vigour and nutrients’ uptake, and they are significantly involved in bioremediation of
soils from heavy metals and environmental pollutants (Tripathi et al. 2013). In
addition, this genus comprises fungi that produce secondary metabolites of clinical
significance and enzymes with widespread industrial application. They produce
and/or release a variety of compounds that induce localized or systemic resistance

responses in plants. These root-microorganism interactions cause significant
changes in the plant metabolism. Plants are protected from numerous pathogens
by responses that are similar to systemic acquired resistance and rhizobacteria-
induced systemic resistance. Root colonization by Trichoderma spp. also frequently
enhances root growth and development, crop productivity, uptake and use of
nutrients and resistance to biotic and abiotic stresses (Harman et al. 2004; Hermosa
et al. 2011).
Various organisms like bacteria, fungi, algae and plants have been utilized for
efficient bioremediation of heavy metals (Vidali 2001). Numerous strains of
Trichoderma have also been utilized in bioremediation, waste management and
biotechnology. Binding of heavy metal ions by cell wall polysaccharides is an
important detoxification mechanism in fungal systems (Leung 2004). Bioremedia-
tion can be defined as the use of organisms to break down harmful environmental
contaminants, to clean the environment to a healthier state. Extensive attention has
been paid to the management of environmental pollution in recent investigations and
to minimize heavy metal contamination (Leung 2004). Heavy metals are part of
human life for thousands of years. Although their ill effects on health have been
known for a long time, exposure to heavy metals continues by different ways and is
even increasing as it is a vital part of chemicals used in fields for plant disease
management (Järup 2003). One such heavy metal is copper, which is a highly
polluting metal, mostly used as fungicide in plant disease management.
It has been reported that Trichoderma inhamatum can significantly tolerate Cr
(VI) and is able to reduce its concentrations. Thus, it may have potential applications
in bioremediation of Cr(VI)-contaminated wastewaters (Morales-Barrera and
Cristiani-Urbina 2008). The ability of immobilized T. viride biomass and cell-free
Ca-alginate beads for biosorption has been investigated (Bishnoi and Garina 2005).
Trichoderma atroviride, T. harzianum and T. pseudokoningii directly link the soil to
plants via solubilization of phosphates and micronutrients while promoting plant
growth and reducing metal toxicity (Pascale et al. 2019; Altomare et al. 1999).
However, research is limited on the utilization of heavy metal-tolerant Trichoderma
spp. in plant disease management either alone or in combination with lower doses of
heavy metal-based fungicides, particularly copper. It is widely recognized that
species of Trichoderma protect plants against pathogens by competition,
mycoparasitism and antibiosis and by inducing systemic acquired resistance (Lorito
and Woo 2015; Druzhinina et al. 2011).
Various studies have reported the potential use of biocontrol fungi against potato
late blight (Jindal et al. 1988; Marrone 2002), either preventing the germination of
sporangia or inhibiting the development of late blight. Chitosan is a biopolymer
consisting of β-1,4-linked glycopyranoses, namely, glucosamine and N-
acetylglucosamine. Enzymes that hydrolyse the β-1,4-glycosylic bonds of this
biopolymer (chitinases and chitosanases) have been found in many microorganisms,
such as bacteria and fungi. Trichoderma spp. also possess a battery of lytic enzymes
and have the ability to degrade chitosan, producing chito-oligomers which are active
elicitors of defence reactions in plants (Lin et al. 2005; Vander et al. 1998).
Trichoderma harzianum produces fungal cell wall degrading enzymes that are
strong inhibitors of spore germination and hyphal elongation of a number of
phytopathogenic fungi. Chitin – together with β-(1,3)-glucan – is the major constit-
uent of the cell walls of asco- and basidiomycetes, whereas cellulose is mainly found
in the cell wall of oomycete pathogens (Peberdy 1990). Its enzymatic hydrolysis is
catalysed by the action of exo- and endochitinases, N-acetyl-β-D-glucosaminidases
and hexosaminidase (Carmen et al. 1995; Peterbauer et al. 1996). Several enzymes
degrading chitinous material (endochitinase, exochitinase, exo-β-D-N-
acetylglucosaminidase) have been reported from T. harzianum, and some of those
have also been cloned (Harman et al. 1993; Garcia et al. 1994; Carmen et al. 1995;
Peterbauer et al. 1996). NAG and HEXO are important genes responsible for the
production of N-acetyl-β-D-glucosaminidase and hexosaminidase enzymes, respec-
tively, which may degrade chito-oligosaccharides to N-acetylglucosamine and glu-
cosamine (Peterbauer et al. 1996). Chitosanases have been reported from a number
of organisms including fungi and bacteria (Alfonso et.al. 1992; Sakai et.al. 1991;
Shimosaka et al. 1993).
Exposure of fungi and yeasts to elevated copper concentrations can lead to a rapid
decline in membrane integrity, which is generally manifested as leakage of mobile
cellular solute (e.g. K+). Trichoderma strains can persist in ecosystems with high
concentrations of heavy metals. Copper tolerance in fungi has also been ascribed to
diverse mechanisms involving trapping of the metal by cell wall components, altered
uptake of copper, extracellular chelation or precipitation by secreted metabolites and
intracellular complexing by sulphur compound, namely, metallothioneins and
phytochelatins (Cervents and Corona 1994; Scheck 1996). Cu++ can bind to the
cell wall surface of T. asperellum, a mechanism of metal tolerance making it less
available in the medium (Erayya 2014).
Extensive metal-induced disruption of membrane integrity inevitably leads to loss
of cell viability. However, even relatively small alterations in the physical properties
of biological membranes can elicit marked changes in the activities of essential
membrane-dependent functions, including transport protein activity, ion imperme-
ability (Keenan et al. 1982; Borel 1996) and phagocytosis (Avery et al. 1996). The
physical properties of a membrane are largely determined by its lipid composition.
The fatty acid composition of microbial membranes is highly variable and is
influenced by both environmental and intrinsic factors. For example, the unsaturated
fatty acid contents of microorganisms generally increase at low temperatures. The
low melting temperatures and large physical volumes occupied by unsaturated fatty
acids are thought to partially compensate for the lipid-ordering effect of chilling
(Cossins 1994). Also, the fatty acid composition of membranes differs between
microbial groups. Indeed, microbial fatty acid profiles have proven to be useful
taxonomic criteria (Thompson et al. 1993) and can be indirectly correlated with other
phenotypic characteristics, including pathogenicity (Harbige and Sharief 2007).
Singh and Erayya (2018) confirmed that a copper-tolerant T. asperellum isolate
had the ability to modulate the fatty acid composition of its plasma membrane which
prevented the penetration/absorption of copper ions inside the cell, so that copper
accumulated only outside of the cell membrane. Copper tolerance of T. asperellum
increased considerably with increased plasma membrane fatty acid. Fatty acids such
as octadecenoic acid derivatives (9.67%), octadecadienoic acid derivatives
(17.46%), hexadecanoic acid (4.37%) and petroselinic acid (2.51%) were found to
be significantly higher in copper-treated Trichoderma as compared to untreated
(control) in fatty acid profiling study (Singh and Erayya 2018). Petroselinic acid
peak was found in CuOH-amended T. asperellum culture only. Phospholipids which
play an important role in metal ions movement across the plasma membrane were
observed in less amount (1%) in CuOH-500 (ppm)-treated T. asperellum as com-
pared to control (3.73%). Therefore, CuOH-tolerant T. asperellum modify total fatty
acid content in the presence of CuOH which made the plasma membrane stable in
copper-amended media (Singh and Erayya 2018). Nischwitz et al. (2007) evlauted
differences in fatty acid methyl ester (FAME) profiles among copper tolerant and
copper sensitive strains of Pantoea ananatis and reported that higher concentrations
of myristic acid and oleic acid were found in copper tolerant starins.
Two unsaturated fatty acids (linoleic acid and oleic acid) were markedly
increased at high copper concentration in copper-tolerant fungi (Abboud and
Alawlaqi 2011). However, due to the aggressive nature of the pathogen and the
typically explosive disease development, management of P. infestans is rather
difficult with biocontrol agents when they are applied alone (Li et al. 1995; Dorn
et al. 2007a, b). Therefore, a new generation of combinations of biopesticides are
required for safe management of oomycete plant pathogens.
13.4 Antifungal Activity of Chitosan Against Phytophthora

infestans and Activation of Defence Mechanisms in Late
Blight Management
Chitosan is a linear amino polysaccharide consisting of glucosamine and N-

acetylglucosamine units. It is obtained commercially by chemical deacetylation of
chitin. It exhibits a range of biological activities that are potentially useful in
agriculture. Chitosan can promote plant growth and development (Malerba and
Cerana, 2016), or it can inhibit the growth of fungi (Oliveira Junior et al. 2012)
and bacteria (Benhabiles et al. 2012), and it has been reported to activate genes
involved in defence responses in plants (Hadwiger et al. 1986; Oh et al. 1998;
Povero et al. 2011). Moreover, chitosan can bind heavy metals due to the free
electron pair of the nitrogen atom in the amino group of glucosamine at neutral or
alkaline pH, and this ability may be useful in combination with copper fungicides
(Verma et al. 2004).
Chitosan also proved to be an effective inhibitor of spore germination and germ
tube elongation and caused morphological changes in hyphal growth of several
phytopathogenic fungi including P. infestans, the causal agent of potato late blight
(Oh et al. 1998). Foliar or root applications of chitosan before inoculation can
enhance protection against pathogenic fungi (Muzzarelli et al. 1990; Vasyukova
et al. 2005). Vasyukova et al. (2005) also reported 50% reduction in late blight
severity with application of low molecular mass (5 kDa) chitosan and, thus, induced
systemic resistance in potato tubers due to increased rishitin synthesis. The level of
rishitin accumulated was directly related to the chitosan concentrations applied. In
chitosan-treated leaves, an increase in endogenous salicylic acid (SA) as well as of
intercellular chitinase and glucanase activities was observed. In general, chitosan
treatment can induce various biochemical changes in plants, such as DNA damage,
chromatin alterations (Hartney et al. 2007; Hadwiger 2008), activation of MAP
kinases, oxidative bursts and callose apposition (Paulert et al. 2010), pathogenesis-
related protein synthesis (Loschke et al. 1983), phytoalexin accumulation, hypersen-
sitive response (Hadwiger and Beckman 1980), synthesis of jasmonic acid and
abscisic acid and accumulation of hydrogen peroxide (Iriti and Faoro 2009) and
increase in cytosolic Ca2+ (Zuppini et al. 2003). Chitosan has been shown to exhibit
elicitor activity and to induce both local and systemic resistance. Spraying and soil
drench with chitosan suppressed late blight (P. infestans) and Fusarium wilt (Fusar-
ium oxysporum f. sp. lycopersici) of tomato (Oh et al. 1998). It has been reported that
chitosan also induced an increase in the activity of phenylalanine ammonia lyase
(PAL), the formation of phenolic compounds and lignification, which may play a
significant role in induction of resistance mechanisms (Loschke et al. 1983;
Moerschbacher et al. 1990; Vander et al. 1998; Menden et al. 2007).
Chitosan as an antifungal agent is extremely successful. Soil amendment with
chitosan has been shown to control fungal diseases in numerous crops, especially
Fusarium wilts and grey mould (Rabea et al. 2003; Laflamme et al. 1998). It is also
important to note that these studies show chitosan to be fungistatic against both
biotrophic and necrotrophic pathogens. The control of oomycete pathogens has also
been achieved with chitosan treatment, with Phytophthora capsici controlled on
peppers (Xu et al. 2007) and P. infestans in potato (O’Herlihy et al. 2003).
Stimulation of antagonistic biocontrol agents is also an important activity of
disease control performed by chitosan. Antagonistic microbes employ a number of
methods to attack plant pests and pathogens. This includes, but is not limited to, the
production of chitinases (Maksimov et al. 2011), the production of toxins
(e.g. antibiotics and toxins), direct parasitism, competition for nutriment, and the
induction of defence responses in the plant. Therefore, adding chitin-based products
to the growing environment may aid beneficial antagonists by stimulating the
production and activation of chitinases that can then be used to attack pests and
pathogens or be used as a stable nitrogen-rich polysaccharide food source that boosts
the population to the level where other mechanisms control the plant pathogens. El
Mohammadi et al. (2014) studied the effect of individual or combined application of
Trichoderma harzianum and chitosan against Fusarium oxysporum f. sp. radicis-
lycopersici (FORL) causing Fusarium crown and root rot (FCRR) under in vitro and
in vivo conditions. They found that T. harzianum significantly reduced the mycelial
growth of the five FORL tested isolates. Chitosan applied at different concentrations
(from 0.5 to 4 g/l) also significantly decreased the mycelial growth of the pathogen,
and a total inhibition was obtained at a concentration of 4 g/l. Under greenhouse
conditions, application of T. harzianum and chitosan (1 g/l) as root dipping treatment
combined with chitosan (0.5 g/l) as foliar spray has reduced FCRR incidence and
severity by 66.6 and 47.6%, respectively. Treatments based on T. harzianum alone
or in combination with chitosan led to an increase in the total phenols and to an

enhancement of chitinase and β-1,3-glucanase activities in leaves of treated tomato
plants compared with the untreated ones.
Yu et al. (2012) evaluated the antifungal activity of chitosan in combination with
the biocontrol yeast Candida laurentii against the Penicillium expansum of pear. The
results showed that the most effective concentration of chitosan able to enhance blue
mould control was 5 g/l when combined with C. laurentii. Infections of fruit by
fungal pathogens often occur in the field, prior to harvest; preharvest treatment with
BCAs would therefore be advantageous in order to reduce initial infection and to
suppress pathogens in storage (Tiexido et al. 1998). However, the combination of
preharvest treatment with C. laurentii and chitosan coating of table grapes enhanced
the control of fruit decay to a greater extent than the preharvest treatment alone
(Meng et al. 2010). The bacterium Bacillus subtilis is a pathogen of fungi and is one
of the most widely used biopesticides in agriculture. Chitosan addition also
improved the action of B. subtilis against powdery mildew in strawberry (Lowe
et al. 2012). The beneficial effect of chitin-based treatments to antagonistic bacteria
is not restricted to B. subtilis, with both chitin and chitosan improving the control of
Fusarium wilt in both tomato and cucumber (Singh et al. 1999) when applied to the
soil with a range of different species of chitinolytic microbes. The use of chitin/
chitosan to encapsulate microbes also assists with the practicalities of storing and
applying microbes on farms and nurseries, which has been one of the major
restrictions to the use of biopesticides in recent times (John et al. 2011).
Chitosan oligosaccharide (COS), a deacetylated and depolymerized derivative of
chitin, has higher antimicrobial properties than chitosan and is presumed to act by
disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi (Jaime
et al. 2012). The gene targets of COS identified in this study indicate that COS’s
mechanism of action is different from other commonly studied fungicides that target
membranes, suggesting that COS may be an effective fungicide for drug-resistant
fungal pathogens.
Chitosan synergy with other biocontrol agent is well known for plant disease
management. Rahman et al. (2014) compared the antifungal activity of chitosan with
DPn (average degree of polymerization) and FA (fraction of acetylation) and of
enzymatically produced chito-oligosaccharides (CHOS) of different DPn alone and
in combination with commercially available synthetic fungicides, Teldor
(fenhexamid), Switch (cyprodinil + fludioxonil), Amistar (azoxystrobin), Signum
(pyraclostrobin) and Delan (dithianon), against Botrytis cinerea, the causative agent
of grey mould in numerous fruit and vegetable crops. CHOS with DPn in the range
of 15–40 had the greatest anti-fungal activity. The combination of CHOS and low
dosages of synthetic fungicides showed synergistic effects on antifungal activity in
both in vitro and in vivo assays. Their study shows that degree of polymerization
(DP) is an important factor on the antifungal activity and CHOS enhance the activity
of commercially available fungicides. According to them, the mechanisms for the
synergism in inhibition of fungal growth are not known, but most likely the
synergism is due to the compounds’ different modes of action. Therefore, synergistic
effect of chitosan with BCAs and chemicals could be utilized for development of
safe and effective strategy for management of late blight of potato caused by
P. infestans. To this end, two-fold and three-fold combinations of copper, chitosan
and Trichoderma were designed and tested for late blight inhibition.
13.5 Combination of Copper/Chitosan and Copper/Trichoderma

Applications Provide Protection Against Late Blight
of Potato
Potato late blight, caused by P. infestans, can be managed with multiple applications
of commercially available synthetic chemical fungicides, even in favourable envi-
ronmental conditions. An extent of late blight suppression has been realized due to
the development of new potato varieties, tolerant for disease and through cultural
practices such as planting disease-free seed, volunteer control, cull pile and irrigation
management (Dorn et al. 2007a, b; Nowicki et al. 2012). With the uncertainties that
accompany epidemics, disease management measures that directly kill the pathogen
are often essential. The organic grower, however, is restricted to cultural controls,
the use of some copper-containing fungicides or other methods that utilize natural
ingredients (Dorn et al. 2007a, b). Therefore, several alternative strategies including
dual combination of copper and chitosan or copper and Trichoderma have been
reported for the management of late blight of potato (Medeiros et al. 2010).
Hadwiger (2008) described a strategy for late blight management using lower
levels of copper sulphate pentahydrate in combination with a chitosan sticker and
complexing agent in the excised leaf greenhouse experiments. In exciced leaf assays,
processed copper sulphate pentahydrate (CT-100) alone, chitosan alone and CT-100
+ chitosan combination were compared with the two commercial fungicides,
chlorothalonil (Bravo) and copper hydroxide (Kocide 2000). It revealed that combi-
nation of CT-100 + chitosan provided moderate control of late blight and protection
agaianst copper-related leaf yellowing at approximately 40-fold lower copper levels
than those recommended for a commercial fungicide.
In large-scale investigations, the use of BCAs often has to be combined with low
doses of chemicals to attain a level of disease control equivalent to synthetic
fungicides for several diseases (Droby et al. 2003). However, the existing literature
reveals few references to the use of copper and Trichoderma dual combination for
management of late blight disease caused by P. infestans.
The combination of Trichoderma asperellum and a chemical agent, copper, is
intrinsically difficult due to the antimicrobial activities of the latter. Erayya et al.
(2020) developed a dual combination product involving a lower dose of chemicals
(copper) and biocontrol agent (Trichoderma) in which the preferred Trichoderma
strain was copper tolerant. Trichoderma asperellum accumulated copper in/on the
surface of cell wall as a mechanism of metal tolerance. Trichoderma sp. promises
great potential as a natural copper removal agent as it is naturally tolerant to high
concentrations of copper. The developed combination product was found effective in
potato late blight disease management as obtained with higher doses of copper
fungicides.
The literature suggests that dual combination of copper + chitosan and copper +
Trichoderma was effective against reducing disease severity but lacks consistent
field performance. Data is though available for commercial and experimental dual
combination preparations for management of late blight disease. Nevertheless,
innovative and superior control measures could be developed by designing a new
generation of plant protectants, like combination products involving plant defence
inducers along with threshold level of copper and BCAs.
13.6 Combination Product of Biopesticides as Alternative or

Copper Reducing Strategy for Late Blight (Phytophthora
infestans) Management
Due to environmental and health concerns, there is immediate interest in finding

alternatives to chemical pesticides for management of plant pathogens that are not
only safe but also highly efficacious with the objective of reducing the pesticide load
below safe limits. One alternative approach could be strategic designing of a novel
formulation involving lower doses of fungicide, plant strengtheners and biological
plant protectant to manage this destructive disease. Such a combination could be an
effective, low-cost, environment-friendly and consumer-safe plant disease manage-
ment strategy for P. infestans. Very high multiplication rate of P. infestans, rapid
establishment of infection and explosive disease development pose major barrier for
its management with BCAs, and plant defence inducers, if applied alone. Therefore,
the challenge is to design a copper-chitosan-Trichoderma combination for efficient
and safe management of oomycete pathogen, P. infestans, and propose a process of
eco-friendly formulation.
Accordingly, a triple combination natural product involving low dose (safe limit)
of a fungicide, a biocontrol agent and a plant defence activator compound was
conceptualized and developed. The first component of the combination includes
copper hydroxide (CuOH), an effective and well-known fungicide against
P. infestans. The second component includes copper-tolerant strain of Trichoderma
asperellum with cellulase, β-1,3-glucanase and chitosanase activities. Thus, the
copper-tolerant strain of T. asperellum theoretically has capability of decomposing
chitosan into its oligomers to enhance its efficacy in combination. The third compo-
nent, chitosan, a biopolymer and plant strengthener, is a natural substrate that is
compatible for growth and sporulation of Trichoderma and/or stimulates the pro-
duction of chitosanase. The specific concentration of all the three components in the
combination product was as effective as double the dose of standard recommended
fungicides to minimize the growth of P. infestans (Erayya 2014).
Copper fungicides are protectant/preventative products. The copper product must
be applied onto the plant surface prior to appearance of the disease. Copper
fungicides inhibit fungal spore germination and mycelial growth, and control is
provided by the free copper ions (Cu2+) released from the applied copper.
Chitosan is a polysaccharide consisting predominantly of repeating units of
D-glucosamine and obtained by deacetylation of chitin. It is ensured that the level
Fig. 13.1 Synergistic action of ‘Cu-Chi-Tri’ combination
of deacetylation is sufficient to render the chitosan water soluble at acidic pH or else

liquid formulation of chitosan is utilized.
The BCA Trichoderma asperellum having copper tolerance and chitosanase
activity could be applied as tuber treatment and/or foliar application. Trichoderma
asperellum produced high concentration of chitosanase and cellulase. Chitosanase is
an enzyme that selectively targets the chitosan in the ‘Cu-Chi-Tri’ combination and
breaks chitosan into smaller units (oligomers) and enhances the efficacy of the triple
combination in disease management, while cellulose is a fundamental component of
fungal cell wall of oomycete pathogens (Mélida et al. 2013; Grenville-Briggs et al.
2008). Therefore, it could be inferred that Trichoderma plays a key role in the ‘Cu-
Chi-Tri’ combination (Bhardwaj 2016). Once T. asperellum secretes chitosanase and
cellulase, chitosanase utilizes/breaks chitosan into its oligomers and cellulase will
degrade cellulose complex in its vicinity, which will thus be reduced to its simpler
components, thereby lysing the cell wall and exposing its intracellular components
to copper, leading to minimized growth and further spread of P. infestans (Bohra
2018). Based on the findings, a schematic diagram on the synergetic action of ‘Cu-
Chi-Tri’ has been hypothesized (Fig. 13.1).
Efficacy of different copper compounds, namely, CuOH (technical grade), CuOH
(Kocide) and copper sulphate pentahydrate (CuSO4.5H2O), in combination with
chitosan and T. asperellum (Tri) to find out the most effective copper source in the
combination for late blight disease management in potato has also been studied.
Among the three copper compounds, CuOH (TG) was found to be the most effective
copper source in the combination (Erraya 2014; Rautella et al. 2018). Findings of
Rautella et al. (2018) proved that the ‘Cu-Chi-Tri’ combination was similarly
effective for management of late blight disease of tomato.
Chitosan/alginate microcapsules simultaneously loaded with copper cations and
Trichoderma viride have also been developed (Marko et al. 2016). It has been
reported that chitosan/alginate microcapsules could simultaneously incorporate
T. viride spores and chemical bioactive agent without inhibiting their activities and
can be suitable for plant nutrition and protection. Therefore, integration of induced
resistance with the management of disease using chemical fungicides is a desirable
component of integrated disease management.
13.7 Conclusion
Improved plant protection strategies are urgently needed that are less burdening to
the environment and safer for the customer than existing chemical technologies, but
that at the same time secure the same high quality and quantity of harvest known
today. Biological treatments alone cannot fulfil these demands, but synergistic
combinations of chemicals and biologics including biostimulants promise to allow
significant reductions of chemical inputs. A synergistic combination of a biochemi-
cal biostimulant, further reinforced with a potential chemical plant protectant,
justifies exploitation to promote growth, development and strengthening of plants
to increase tolerance of abiotic and resistance against biotic stresses. Among the
most widely used biostimulants are selected strains of the fungal genus
Trichoderma. These biocontrol agents are difficult to combine with chemical plant
protectants such as copper-based fungicides as these inhibit their growth. Previous
attempts have shown that chitosan tolerance in Trichoderma can be increased, but
not to the point that a combination with copper dosages required for effective plant
protection would become possible. On the other hand, chitosan as a plant strength-
ener can lower the effective copper dosage, and Trichoderma are known to possess
good chitosanolytic abilities so that they can be combined with potentially antimi-
crobial, plant strengthening chitosans. It has been shown that the synergistic combi-
nation of copper-chitosan-Trichoderma can give good plant protection against
oomycete pathogen, P. infestans, at significantly lowered copper dosages. Thus,
the ‘Cu-Chi-Tri’ combination promises sustainable, healthy and safe plant protection
and necessitates development of a stable formulation product.
Acknowledgement The information given on the ‘triple combination’ was generated through an
Indo-German ‘2 + 2’ grant funded by the Department of Biotechnology, Govt. of India, and the
Federal Ministry of Education and Research, Germany, with contributions of the project partners.
References
Abboud M, Alawlaki M (2011) Biouptake of copper and their impact on fungal fatty acids. Aust J
Basic Appl Sci 5:283–290
Alfonso C, Martinez MJ, Reyes F (1992) Purification and properties of two endochitosanases from
Mucor rouxii implicated in its cell wall degradation. FEMS Microbiol Lett 95:187–194
Altomare C, Norvell WA, Bjorkman T, Harman GE (1999) Solubilization of phosphates and
micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum
Rifai 1295-22. Appl Environ Microbiol 65:2926–2933
Anand P, Isar J, Saran S, Saxena RK (2006) Bioaccumulation of copper by Trichoderma viride.
Bioresour Technol 97:1018–1025
Anonymous (2008) Commission Regulation (EC) No 889/2008 of 5 September 2008 laying down
detailed rules for the implementation of Council Regulation (EC) No 834/2007 on organic
production and labelling of organic products with regard to organic production,
labellingandcontrol (http://ec.europa.eu/agriculture/organic/organicfarming/what-organic en,
09.01.2010)
Anonymous (2009)Commission Directive 2009/37/EC.(http://eurlex.europa.eu/LexUriServ/
LexUriServ.do? uri ¼ OJ:L:2009:104:0023:0032:EN:PDF, 09.01.2010)
Avery SV, Howlett NG, Radice S (1996) Copper toxicity towards Saccharomyces cerevisiae:
dependence on plasma membrane fatty acid composition. Appl Environ Microb 62:3960–3966
Benhabiles MS, Salah R, Lounici H, Drouiche N, Goosen MFA, Mameri N (2012) Antibacterial
activity of chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrocoll
29:48–56
Benítez T, Rincón AM, Limón MC, Codón AC (2004) Biocontrol mechanisms of Trichoderma
strains. Int Microbil 7:249–260
Bhardwaj NR (2016) Elucidating the role of Trichoderma in the triple combination ‘Copper-
Chitosan-Trichoderma’ for the management of late blight disease of potato (Solanum tuberosum
L.). Ph.D. Thesis, G.B Pant University of Agriculture and Technology, Pantnagar, Uttarakhand
(India)
Bishnoi NR, Garima A (2005) Fungus an alternative for bioremediation of heavy metal containing
waste water, a review. J Sci Ind Res. 64:93
Blaeser P, Steiner U, Dehne H-W (1998) Fungizide Wirkstoffeaus Pflanzenextrakten Mitteilungen
der Biologischen Bundesanstaltfu¨r Land- und Forstwirtschaft Berlin-Dahlem, 357:99
Bohra Y (2018) Elucidating cu-Trichoderma interaction and Trichoderma-chitosan interaction in
“Cu-Chi-Tri”, a novel consortium for potato late blight management. Ph.D Thesis, GB Pant
University of Agriculture and Technology, Pantnagar, Uttarakhand (India)
Borel S (1996) Biogenesis of polytopic membrane proteins: membrane segments assemble within
translocation channels prior to membrane integration. Cell 185:379–389
Carmen-Limon M, Lora JM, Garcia I, de la Cruz J, Llobell A, Benitez T, Pintor-Toro JA (1995)
Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic
fungus Trichoderma harzianum. Curr Genet 28:478–483
Cervantes C, Gutierrez-Corona F (1994) Copper resistance mechanisms in bacteria and fungi.
Microbiol Rev 14:121–137
Clark JM, Yamaguchi I. (2002) Scope and status of pesticide resistance. In. Agrochemical resis-
tance. ACS Symposium Series; American Chemical Society: Washington, DC, 2001. See
https://pubs.acs.org/sharingguidelines for options Downloaded via 117.254.37.95 on March
30, 2020
Cossins AR (1994) Homo viscous adaptation and its functional significance. In: Cossins AR
(ed) Temperature adaptation of biological membranes. Portland Press, London, pp 63–75
Cornejo P, Pérez-Tienda J, Meier S, Valderas A, Borie F, Azcón-Aguilar C, Ferrol N (2013) Copper
compartmentalization in spores as a survival strategy of mycorrhizal fungi in cupolluted
environments. Soil Biol Biochem 57:925–928. https://doi.org/10.1016/j.soilbio.2012.10.031
Dorn B, Musa T, Krebs H, Fried PM, Forrer HR (2007a) Control of late blight in organic potato
production: evaluation of copper-free preparations under field, growth chamber and laboratory
conditions. Eur J Plant Pathol 119:217–240
Dorn B, Nusa-Steenblock T, Forrer H (2007b) Control of late blight in organic potato production.
Evaluation of copper free preparations under field, growth chamber and laboratory conditions.
Eur J Plant Path 119:217–240
Droby S, Wisniewski M, El-Ghaouth A, Wilson C (2003) Influence of food additives on the control
of postharvest rots of apple and peach and efficacy of the yeast based biocontrol product Aspire.
Postharvest Biol Technol 27:127–135
Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM, Monte E,
Mukherjee PK, Zeilinger S, Grigoriev IV, Kubicek CP (2011) Trichoderma: the genomics of
opportunistic success. Nat Microbiol Rev 16:749–759
EC (2018) COMMISSION IMPLEMENTING REGULATION (EU) 2018/1981 of 13 December
2018 renewing the approval of the active substances copper compounds, as candidates for
substitution, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and
of the Council concerning the placing of plant protection products on the market, and amending
the Annex to Commission Implementing Regulation (EU) No 540/2011 https://eur-lex.europa.
eu/eli/reg_impl/2018/1981/oj
El-Mohamedy RSR, Abdel-Kareem F, Jabnoun-Khiareddine H, Daami-Remadi M (2014) Chitosan
and Trichoderma harzianum as fungicide alternatives for controlling Fusarium crown and root
rot of tomato. Tunis J Plant Prot 9:31–43
Erayya (2014) Designing a triple combination of copper, Trichoderma and chitosan and its
evaluation against late blight of potato caused by Phytophthora infestans (Mont.) de Bary.
Ph.D Thesis, GB Pant University of Agriculture and Technology, Pantnagar, Uttarakhand
(India)
Erayya SN, Bohra Y, Tiwari AK, Kumar J (2020) Copper tolerant Trichoderma asperellum
increases bio-efficacy of copper against Phytophthora infestans in dual combination.
Phyparasitica, (In Press)
EU (2018). European Parliamentary Research Service of 6 November 2017 on Directive 2009/128/
EC on the sustainable use of pesticides, QA-06-18-079-EN-N http://www.europarl.europa.eu/
thinktank
Flood J (2010) The importance of plant health to food security. Food Secur 2:215–231
Garcia I, Lora JM, De la Cruz J, Benitez T, Llobell A, Pintor-Toro JA (1994) Cloning and
characterization of a chitinase (CHIT42)cDNA from the mycoparasitic fungus Trichoderma
harzianum. Curr Genet 27:83–89
Ghorbani R, Wilcockson SJ, Glotis C, Leifert C (2004) Potato late blight management in organic
agriculture. Outlooks Pest Manag 15:176–280
Ghorbani R, Wilcockson S, Leifert C (2005) Alternative treatments for late blight control in organic
potato: antagonistic micro-organisms and compost extracts for activity against Phytophthora
infestans. Potato Res 48:181–189
Grenville-Briggs LJ, Anderson VA, Fugelstad J, Avrova AO, Bouzenzana J, Williams A, Wawra S,
Whisson SC, Birch PRJ, Bulone V, van Westa P (2008) Cellulose synthesis in Phytophthora
infestans is required for normal appressorium formation and successful infection of potato. Plant
Cell 20:720–738
Hadwiger LA (2008) Pea–Fusarium solani interactions contributions of a system toward under-
standing disease resistance. Phytopathology 98:372–379
Hadwiger LA, Beckman JM (1980) Chitosan as a component of Pea-Fusarium solani interactions.
Plant Physiol 66:205–211
Hadwiger LA, Kendra DF, Fristensky BW, Wagoner W (1986) Chitosan both activates genes in
plants and inhibits RNA synthesis in fungi. In: Muzzarelli R, Jeuniaux C, Gooday GW (eds)
Chitin in nature and Technol. Springer, Boston
Harbige LS, Sharief MK (2007) Polyunsaturated fatty acids in the pathogenesis and treatment of
multiple sclerosis. Br J Nutr 1:S46–S53
Harman GE, Hayes CK, Lorito M, Broadway RM, Di Pietro A, Peterbauer CK, Tronsmo A (1993)
Chitinolytic enzymes of Trichoderma harzianum: purification of chitobiosidase and
endochitinase. Phytopathology 83:313–318
Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species opportunistic,
avirulent plant symbionts. Nat Rev Microbiol 2:43–56
Hartney S, Carson J, Hadwiger LA (2007) The use of chemical genomics to detect functional
systems affecting the non-host disease resistance of pea to Fusarium solani f. sp. phaseoli. Plant
Sci 172:45–56
Hermosa R, Botella L, Keck M, Jimenez JA, Montero Barrientos M, Arbona V, Gomez Cadenas A,
Monte E, Nicolas C (2011) The overexpression in Arabidopsis thaliana of a Trichoderma
harzianum gene that modulates glucosidase activity, and enhances tolerance to salt and osmotic
stresses. J Plant Physiol 168:1295–1302
Homa J, Niklinska M, Plytycz B (2003) Effect of heavy metals on coelomocytes of the earthworm
Allolobophora chlorotica. Pedobiologia 47:640–645
Iriti M, Faoro F (2009) Chemical diversity and defence metabolism: how plants cope with
pathogens and ozone pollution. Int J Mol Sci 10:3371–3399
Jaime MD, Lopez-Llorca LV, Conesa A, Lee AY, Proctor M, Heisler LE, Gebbia M, Giaever G,
Westwood JT, Nislow C (2012) Identification of yeast genes that confer resistance to chitosan
oligosaccharide (COS) using chemogenomics. BMC Genomics 22(13):267. https://doi.org/10.
1186/1471-2164-13-267
Jarup L (2003) Hazards of heavy metal contamination. Br Med Bull 68:167–182
Jindal KK, Singh H, Meeta M (1988) Biological control of Phytophthora infestans on potato. Indian
J Plant Pathol 6:59–62
John RP, Tyagi RD, Bra SK, Surampalli RY, Prévost D (2011) Bio-encapsulation of microbial cells
for targeted agricultural delivery. Crit Rev Biotechnol 31:211–226
Keenan MHJ, Rose AH, Silverman BW (1982) Effect of plasma membrane phospholipid
unsaturation on solute transport into Saccharomyces cerevisiae NCYC366. J Gen Microbiol
128:2547–2556
Krebs H, Vogelsang S & Forrer HR (2013) Phosphonat zur Bekämpfung der Phytophthora
infestans bei Kartoffeln – eine mögliche Alternative zu Kupfer? In: Neuhoff D, Stumm C,
Ziegler G, Rahmann G, Hamm U, Köpke U (Eds.), Ideal und Wirklichkeit Perspektiven
ökologischer Landbewirtschaftung. Beiträge zur 12. Wissenschaftstagung kologischen
Landbau, 5–8 March 2013, Bonn, Germany. Verlag Dr. Köster, Berlin. 270–273
Laflamme P, Benhamou N, Bussiéres G, Dessureault M (1998) Differential effect of chitosan on
root rot fungal pathogens in forest nurseries. Can J Bot 77:1460–1468
Li JX, Chen GH, Webster JM, Czyzewska E (1995) Antimicrobial metabolites from a bacterial
symbiont. J Nat Products 58:1081–1086
Lin W, Hu X, Zhang W, Rogers WJ, Cai W (2005) Hydrogen peroxide mediates defence responses
induced by chitosans of different molecular weights in rice. J Plant Physiol 162:937–944
Lorito M, Woo SL. (2015) Chapter 36: Trichoderma: a multipurpose tool for IPM. In:
B. Lugtenberg (ed.) Principles of Plant Microbe Interactions. pp. 345–353
Loschke DC, Hadwiger LA, Wagoner W (1983) Comparison of mRNA populations coding for
phenylalanine ammonia lyase and other peptides from pea tissue treated with biotic and abiotic
phytoalexin inducers. Physiol Mol Plant Pathol 23:163–173
Lowe A, Rafferty-McArdle SM, Cassells AC (2012) Effects of AMF- and PGPR-root inoculation
and a foliar chitosan spray in single and combined treatments on powdery mildew disease in
strawberry. Agric Food Sci 21:28–38
Leung M (2004) Bioremediation: techniques for cleaning up a mess. J Biotechnol 2:18–22
Vidali M (2001) Bioremediation. An overview. Pure Appl Chem 73:1163–1172
Madden LV (1983) Measuring and modelling crop losses at the field level. Phytopathology
73:1591–1596. 2
Maksimov IV, Abizgildin RR, Pusenkova LI (2011) Plant growth promoting rhizobacteria as
alternative to chemical crop protectors from pathogens (review). Appl Biochem Microbiol
47:333–345
Marko V, Nenad J, Snježana TP, Edyta Đ, Marija B, Slaven J (2016) Encapsulation of biological
and chemical agents for plant nutrition and protection: chitosan/alginate microcapsules loaded
with copper cations and Trichoderma viride. J Agric Food Chem 64:8073–8083
Marrone P (2002) An effective biofungicide with novel mode of action. Pestic Outlook 1:193–194
Malerba M, Cerana R (2016) Chitosan effects on plant systems. Int J Mol Sci 23:17. pii: E996.
https://doi.org/10.3390/ijms17070996
Medeiros FHV, Pomella AWV, de Souza JT, Niella GR, Valle R, Bateman RP, Fravel D,
Vinyard B, Hebbar PK (2010) A novel, integrated method for management of witches' broom
disease in Cacao in Bahia, Brazil. Crop Pro 29:704–711
Mélida H, Sandoval-Sierra JV, Diéguez-Uribeondo J, Bulonea V (2013) Analyses of extracellular
carbohydrates in Oomycetes unveil the existence of three different cell wall types. Eukaryot Cell
12:194–203
Menden B, Kohlhoff M, Moerschbacher BM (2007) Wheat cells accumulate a syringyl-rich lignin
during the hypersensitive resistance response. Phytochemistry 68:513–520
Meng XH, Qin GZ, Tian SP (2010) Influences of preharvest spraying Cryptococcus laurentii
combined with postharvest chitosan coating on postharvest diseases and quality of table grapes
in storage. LWT – Food Sci Technol 43:596–601
Mizubuti ESG, Junior VL, Forbes GA (2007) Management of late blight of potato with alternative
products. Pest Technol 1:106–116
Monte E (2001) Understanding trichoderma: between biotechnology and microbial ecology. Int
Microbiol 4(1):1–4. https://doi.org/10.1007/s101230100001
Moerschbacher BM, Noll U, Gorrichon L, Reisener HJ (1990) Specific inhibition of lignification
breaks hypersensitive resistance of wheat to stem rust. Plant Physiol 93:465–470
Morales –Barrera L, Cristiani Urbina E (2008) Hexavalent chromium removal by a Trichoderma
inhamatum fungal strain isolated from tannery effluent. Water Air Soil Pollut 187:327–336
Muzzarelli RAA, Tarsi R, Filippini O, Giovanetti E, Biagini G, Varaldo PE (1990) Antimicrobial
properties of N-carboxybutyl chitosan. Antimicrob Agents Chemother 34:2019–2023
Neuhoff D, Tadesse M, Köpke U. (2006). Nutzung von Braunalgenextrakten
(Ascophyllumnodosum) zur Kontrolle der Krautfäule (Phytophthorainfestans) im ökologischen
Kartoffel-und Tomatenanbau. Landwirtschaftliche Fakultät der Universität Bonn,
Schriftenreihe des
Njoroge A (2019) Population structure and pathogenicity evolution of Phytophthora infestans
affects epidemiology and management of late blight disease. Diss. (sammanfattning/summary)
Uppsala: Sverigeslantbruksuniv. Acta Universitatis Agric Sueciae 14:1652–6880
Nischwitz C, Gitaitis R, Sanders H, Langston D, Mullinix B, Torrance R (2007) Use of fatty acid
methyl ester profiles to compare copper tolerant and copper-sensitive strains of Pantoea
ananatis. Phytopathology 97:1298–1304
No HK, Meyers SP (1997) Preparation of chitin and chitosan. In: Muzzarelli RAA, Peter MG (eds)
Chitin Handbook, pp 475–489
Nowicki M, Follad MR, Nowakowska M, Kozik EU (2012) Potato and tomato late blight caused by
Phytophthora infestans: an overview of pathology and resistance breeding. Plant Dis 96:4–17
O’Herlihy EA, Duffy EM, Cassells AC (2003) The effects of arbuscular mycorrhizal fungi and
chitosan sprays on yield and late blight resistance in potato crops from microplants. Folia
Geobot 38:201–207. https://doi.org/10.1007/BF02803152
Oliveira Junior, E.N., , Gueddari, N.E., Moerschbacher, B.M., and Franco, T.T. (2012). Growth rate
inhibition of phytopathogenic fungi by characterized chitosans. Braz J Microbiol 43:800–809.
doi: https://doi.org/10.1590/S1517-83822012000200046
Oh SK, Cho D, Yu SH (1998) Development of integrated pest management techniques using
biomass for organic farming (I). Suppression of late blight and Fusarium wilt of tomato by
chitosan involving both antifungal and plant activating activities. Korean J Plant Pathol
14:278–285
Paulert R, Ebbinghaus D, Urlass C, Moerschbacher BM (2010) Priming of the oxidative burst in
rice and wheat cell cultures by ulvan a polysaccharide from green macroalgae and enhanced
resistance against powdery mildew in wheat and barley plants. Plant Pathol 59:634–642
Povero G, Loreti E, Pucciariello C, Santaniello A, Di Tommaso D, Di Tommaso G, Kapetis D,
Zolezzi F, Piaggesi A, Perata P (2011) Transcript profiling of chitosan-treated Arabidopsis
seedlings. J Plant Res 124:619–629
Peberdy JF (1990) Fungal Cell Walls — A Review. In: Kuhn PJ, Trinci APJ, Jung MJ, Goosey
MW, Copping LG (eds) Biochemistry of Cell Walls and Membranes in Fungi. Springer,
Heidelberg. https://doi.org/10.1007/978-3-642-74215-6_2
Peterbauer C, Lorito M, Hayes C et al (1996) Molecular cloning and expression of the nag1 gene
(N-acetyl-β-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1. Curr Genet
30:325–331. https://doi.org/10.1007/s002940050140
Pascale A, Proietti S, Pantelides IS, Stringlis IA (2019) Modulation of the root microbiome by plant
molecules: the basis for targeted disease suppression and plant growth promotion. Front Plant
Sci 10:1741
Verma AJ, Deshpande SD, Knnedy JF (2004) Metal complexation by chitosan and its derivatives: a
review. Carbohydr Polym 55:77–93
Rabea EI, El Badawy MT, Stevens CV, Smagghe G, Steurbaut W (2003) Chitosan as antimicrobial
agent: applications and mode of action. Biomacromolecules 4:1457–1465
Ragunathan V, Divakar BJ (1996) Integrated pest management strategies. In: Gunasekaran M,
Weber DJ (eds) Molecular biology of the biological control of pests and disease of plants. CRC
Press, Florida, pp 191–194
Rahman MH, Shovan LR, Hjeljord LG, Aam BB, Eijsink VGH, Sørlie M et al (2014) Inhibition of
fungal plant pathogens by synergistic action of chito-oligosaccharides and commercially avail-
able fungicides. PLoS One 9:e93192. https://doi.org/10.1371/journal.pone.0093192
Rautela A, Shukla N, Ghatak A, Tewari AK, Kumar J (2018) Field evaluation of different copper
sources in a consortium of ‘Copper-Chitosan-Trichoderma’ for management of late blight
disease of tomato. J Pharmacogn Phytochem 7:1260–1266
Sakai K, Katsumi R, Isobe A, Nanjo F (1991) Purification and hydrolytic action of a chitosanase
from Nocardia orientals. Biochem Biophys Acta 1079:65–72
Scheck JH (1996) Effect of copper bactericides on copper resistant and -sensitive strains of
Pseudomonas syringae pv. syringae. Phytopathol 82:397–406
Shimosaka M, Nogawa M, Ohno Y, Okazaki M (1993) Chitosanase from the plant pathogenic
fungus Fusarium solani f. sp. phaseoli—purification and some properties. Biosci Biotechnol
Biochem 57:231–235
Schumann GL, D’Arcy CJ (2000) Late blight of potato and tomato. The Plant Health Instructor
https://doi.org/10.1094/PHI-I-2000-0724-01. Updated 2018
Singh A, Erayya KJ (2018) Mechanism of copper tolerance in Trichoderma asperellum
(MH593785): a key component in the consortium of Copper-Chitosan- Trichoderma. In J
Chem Stud 6:2285–2293
Singh PP, Shin YC, Park CS, Chung YR (1999) Biological control of Fusarium wilt of cucumber
by chitinolytic bacteria. Phytopathology 89:92–99
Yu T, Chen Y, Fangxia C, Kuang S, Tao Z, Mahbuba Z et al (2012) Integrated control of blue mold
in pear fruit by combined application of chitosan, a biocontrol yeast and calcium chloride.
Postharvest Biol Technol 69:49–53
Tamm L et al (2004) Assessment of the socio-economic impact of late blight and state of the art of
management in European organic potato production systems. In: FiBl Report, vol 106. Research
Institute of Organic Agriculture FiBL, Frick, pp 5–8
Teixido N, Vinas I, Usall J, Magan N (1998) Control of blue mold of apples by preharvest
application of Candida sake grown in media with different water activity. Phytopathology
88:960–964
Thompson IP, Bailey MJ, Ellis JR, Purdy KJ (1993) Subgrouping of bacterial populations by
cellular fatty acid composition. FEMS Microbiol Lett 102:75–84
Tripathi P, Singh PC, Mishra A et al (2013) Trichoderma: a potential bioremediator for environ-
mental clean up. Clean Techn Environ Policy 15:541–550. https://doi.org/10.1007/s10098-012-
0553-7
Vander P, Varum KM, Domard A, El Gueddari N, Moerschbacher BM (1998) Comparison of the
ability of partially N-Acetylated chitosans and chitooligosaccharides to elicit resistance
reactions in wheat leaves. Plant Physiol 118:1353–1359
Vasyukova NI, Chalenko GI, Gerasimova NG, Perekhod EA, Ozeretskovskaya OL, Irina AV,
Varlamov VP, Albulov AI (2005) Chitin and chitosan derivatives as elicitors of potato resis-
tance to late blight. Appl Biochem Microbiol 372–376:36. (translated from Prik. Biokhim.
Mikrobiol. 36: 433–438)
Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Wo SL, Lorito M (2008) Trichoderma–
plant–pathogen interactions. Soil Biol Biochem 40:1–10
Xu J, Zhao X, Han X, Du Y (2007) Antifungal activity of oligochitosan against Phytophthora
capsici and other plant pathogenic fungi in vitro. Pesticide Biochem Physiol 87:220–228
Zapotoczny S, Jurkiewicz A, Tylko G, Anielska T, Turnau K (2007) Accumulation of copper by
Acremonium pinkertoniae, a fungus isolated from industrial wastes. Microbiol Res 26:198–298
Zarb J, Ghorbani R, Juntharathep P, Shotton P, Santos J, Wilcockson S, Leifert C. (2002) Control
strategies for late blight in organic potato production. In: Powell J (Ed.) UK organic research
2002: proceedings of the COR conference, 26–28 March 2002, Aberystwyth, UK, 221–222.
Zellner M, 2004. Zur Epidemiologie und Bekämpfung
Zuppini A, Baldan B, Millioni R, Favaron F, Navazio L, Mariani P (2003) Chitosan induces Ca2+-
mediated programmed cell death in soybean cells. New Phytol 161:557–568
Seaweed and Associated Products: Natural
Biostimulant for Improvement of Plant 14
Health
Jai Singh Patel and Arpan Mukherjee
Abstract
Seaweeds are macroalgae fit into the class Phyophyceae and best known as brown
algae. They are mainly composed of polysaccharides such as laminarin, fucoidan,
and alginates. Several products based on seaweeds are known to be useful for
humans and plants. Extracts obtained from seaweeds contain several bioactive
compounds. Such bioactive compounds induce resistance in plants against dif-
ferent biotic and abiotic stresses. Seaweed extracts can also contain countless
plant-bioactive inorganic and organic compounds such as mannitol,
polysaccharides, oligosaccharides, phytohormones (auxins, cytokinins,
gibberellins, betaine), antioxidants, and vitamins. It also contains a low concen-
tration of minerals (calcium, boron, zinc, potassium, phosphorus, magnesium,
and several other trace elements). Seaweed extract can stimulate plant growth and
enhance the rate of photosynthesis. Seaweed extracts boosted rates of seed
germination, crop growth, yields, and shelf life of produce in post-harvest
conditions. It can reduce the effect of diseases due to fungal, viral, and bacterial
pathogens. The present chapter describes the impact of seaweed and their
products in the agricultural system.
Keywords
Seaweed extract · Biostimulants · Plant defense · Plant growth promotion · Plant-

microbe interactions
J. S. Patel (*)
Department of Plant, Food and Environmental Sciences, Faculty of Agriculture, Dalhousie
University, Halifax, Canada
e-mail: jaisingh.patel@dal.ca
A. Mukherjee
Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi, Uttar
Pradesh, India

https://doi.org/10.1007/978-981-15-6275-4_14
318 J. S. Patel and A. Mukherjee
14.1 Introduction
A biostimulant could be defined as any beneficial microorganism or any organic

material having the ability to increase plant growth, enhance nutrient uptake,
enhance abiotic and biotic stress tolerance, and increase crop yield. The knowledge
about specific roles of biostimulants and the mechanism(s) behind its mode of action
is the necessary requirement for industries based on the biostimulants. Several
biostimulant products are available in the market. Based on their source,
biostimulants were derived from seaweeds, bacteria, higher plants, fungi, humic
acid, and several other industrially processed materials.
Phaeophycean seaweed, also known as brown seaweeds, are the biggest group
having 2000 species. Their maximum biomass is available on the rocky shores of the
temperate area of different countries. Brown seaweed-based products are mostly
used (Blunden and Gordon 1986) in agriculture, and Ascophyllum nodosum (L.) Le
Jolis is one of those that is thoroughly studied (Ugarte 2011). Several other brown
seaweeds, including Laminaria spp., Turbinaria spp., Fucus spp., and Sargassum
spp., were also known to be used as biofertilizer in agriculture (Hong et al. 2007).
Plenty of reports are available regarding the role of seaweed-based biostimulants for
crop protection and crop production in terms of elevated resistance against stresses,
improved crop yield, early germination of the seeds, etc. (Beckett and van Staden
1989; Hankins and Hockey 1990; Norrie and Keathley 2005).
The use of brown seaweed-based products can enhance the cation exchange
capacity of the soil. It can increase nutrient availability to the plants. It can be the
source of nitrogen-based fertilizers as well as increase solubility of the nutrients
available in soil. Many studies have shown the role of seaweed-based products for
stimulation of root formation and growth (Pacholczak et al. 2016; Vernieri et al.
2006). Another study by Vernieri et al. (2006) showed enhanced root biomass of the
plants grown in hydroponic system. Polysaccharide-rich extracts of seaweed have
shown to have an enhancing effect on plant growth (Hernández-Herrera et al. 2016).
Such activity of extracts suggests the role of oligosaccharides as signaling molecule
for regulation of phytohormone-related genes in the treated plants. However, the
polysaccharide-rich extracts that promoted root growth in mung bean plants also
showed presence of synthetic hormones in the extract. A recent report suggested the
role of Ascophyllum nodosum extract on the reduction of mycotoxin production in
the Fusarium head blight infected wheat plants (Gunupuru et al. 2019).
14.2 Role of Seaweed as a Plant Growth Promoter
The emerging formulations based on the seaweed extract have the ability to improve
plant growth and also enhance tolerance against abiotic stresses, including heat,
drought, and salinity. Several plant metabolic pathways are targeted by the seaweed
extracts to improve plant growth and tolerance against the abiotic stresses
(Fig. 14.1). The use of seaweed in agriculture is a very ancient technology and
still serving as organic fertilizers for the plants (Craigie 2011).
14 Seaweed and Associated Products: Natural Biostimulant for Improvement of Plant. . . 319
Fig. 14.1 Different physiological activities affected by the seaweed-based biostimulants
Presently more than 50 companies worldwide are producing seaweed extracts for
promotion of plant growth, and these seaweed extracts are based on different
seaweeds present in the sea. The products based on Ascophyllum nodosum getting
the highest attention among them (Sharma et al. 2015). Several plant species have
shown growth promotion under the application of the seaweed extract, but the
mechanism behind such activity is not very well explored (Verkleij 1992;
Battacharyya et al. 2015). The complex nature of the seaweed extracts makes their
studies exceedingly challenging in determining the components of seaweeds that are
responsible for such activities. The nature of commercial formulations available in
the marketplace depends on the method of extraction used for isolation, such as
water-based extractions, acid-based extraction, alkaline-based extraction,
microwave-assisted extraction, ultrasound-assisted extraction, and enzyme-based
extraction (Shukla et al. 2019) (Fig. 14.2).
Most of the commercial products are derived from red algae such as
Lithothamnium calcareum as well as brown algae, including Durvillaea potatorum
and Ascophyllum nodosum (Khan et al. 2009). Some of the products are listed in
Table 14.1. Application of the seaweed extracts enhanced the seedling growth of
lettuce (Lactuca sativa L.) (Moller and Smith 1998). The formulation based on
A. nodosum has been reported to increase growth and accumulation of K+ in almond
plants (Prunus dulcis). The commercial products GroZyme and MegaFol showed a
similar effect on foliar application and stimulated plant growth (Saa et al. 2015).
Fig. 14.2 Extraction processes, mode of application, and responses in the plants by seaweed-based
biostimulants
14.3 Role of Seaweed Extract in Chilling Stress Mitigation
Production of several crops is going down due to the chilling stress. It can cause
sterility in pollens and loss of grain setting in wheat (Chakrabarti et al. 2011).
Seaweed extracts are also reported to develop tolerance in plants against the chilling
stress. Several products have been tested for enhancement of cold tolerance in maize
plants, and the extracts rich in Zn and Mn were able to enhance tolerance by
increasing production of the reactive oxygen species (ROS) (Bradáčová et al.
2016). Nutrient deficiency stress via chilling stress can be overcome by the use of
seaweed extract, which imposes its role in improvement of oxidative stress toler-
ance. The model plant Arabidopsis thaliana was also used for demonstration of the
role of seaweed extract. Rayirath et al. (2009) suggested the role of Ascophyllum
nodosum extract in A. thaliana against chilling stress. They used different organic
fractions of the extract to search the component responsible for the activity. The
authors have demonstrated that ethyl acetate fraction of seaweed extract was rich in
the fatty acid content, which was responsible for increasing the tolerance against
chilling stress. The treated plants with ethyl acetate fractions showed faster recovery
of A. thaliana plants after chilling shock as well as increased expression of some key
genes, including CBF3, COR15A, and RD29A. The extracts are responsible for the
increment in total soluble sugar and proline content, which are critical compounds
for the chilling stress (Nair et al. 2012). Not only chilling stress, seaweed extracts
were also reported to mitigate heat stress (Zhang and Ervin 2008).
Table 14.1 Seaweed-based biostimulants and their functions

Nature of
processed
S. N. product Alga used Function References
1. Extract Sargassum polycystum Provide resistance Khompatara
to leaf fall disease et al. (2019)
2. Sulphated Acanthophora spicifera Defense responses Pettongkhao
polysaccharide against et al. (2019)
Phytophthora
palmivora
3. A sulfated Sargassum vulgare Anticoagulant, Dore et al.
polysaccharide antithrombotic, (2013)
antioxidant, and
anti-inflammatory
effect
4. Extract Sargassum fusiforme Induced resistance Sbaihat et al.
in Solanum (2015)
lycopersicum
5. Lipophilic Ascophyllum Enhance freezing Rayirath et al.
components nodosum tolerance in (2009)
Arabidopsis
thaliana
6. Extract Ascophyllum Enhance freezing Nair et al. (2012)
nodosum tolerance in
Arabidopsis
thaliana
7. Extract Ascophyllum Drought tolerance Xu and Leskovar
nodosum in spinach (2015)
8. Seaweed Ascophyllum Enhances seedling Möller and
suspensions nodosum and Laminaria growth in lettuce Smith, (1998)
hyperborea
9. Extract Sargassum muticum and Mitigate salinity Latef et al.
Jania rubens stress in chickpea (2017)
10. Extract Ascophyllum nodosum Increases growth Ali et al. (2016)
and yield of tomato
11. Extract Ulva intestinalis Regulation of Ghaderiardakani
hormone production et al. (2019)
in Arabidopsis
12. Extract Ascophyllum nodosum Clementine Fornes et al.
mandarin and (2002)
Navelina orange
13. Seaweed Ecklonia maxima Hormone regulation Finnie and Van
concentrate in tomato Staden (1985)
14. Extract Ulva lactuca, Caulerpa Seed germination in Hernández-
sertularioides, Padina tomato plant Herrera et al.
gymnospora, and (2014)
Sargassum liebmannii
15. Extract Gracilaria textorii and Seed germination of Rao and
Hypnea musciformis some vegetable Chatterjee
crops (2014)
(continued)

Nature of
processed
S. N. product Alga used Function References
16. Extract Sargassum myriocystum Stimulant of Kalaivanan and
seedling growth of Venkatesalu
Vigna (2012)
mungo
17. Extract Sargassum wightii Increases growth of Kumar et al.
green gram (2012)
18. Extract Ulva reticulata Growth of Vigna Selvam and
mungo Sivakumar
(2013)
19. Extract SUNRED Positive effect on Deng et al.
the pigment (2019)
characteristics of
grape crop
20. Nano-size Ascophyllum nodosum Vine growth, yield, Sabir et al.
fertilizer berry quality of (2014)
grapes
21. Biochar of Saccharina, Undaria, Soil amelioration Roberts et al.
seaweed and Sargassum (2015)
22. Extract Kelp Growth and Zheng et al.
development of (2016)
Brassica chinensis
23. Extract Gracilaria edulis and Plant growth of Vinoth et al.
Sargassum wightii tomato (2012)
24. Extract Ascophyllum nodosum Growth of spinach Fan et al. (2013)
25. Extract Ascophyllum nodosum Growth promotion Jannin et al.
Brassica napus (2013)
26. Extract Sargassum wightii and Growth of Vigna Sivasankari et al.
Caulerpa chemnitzia sinensis (2006)
14.4 Role of Seaweed Extract in Salinity Stress Mitigation
Salt stress is one of the significant threats to crops. Reports have shown that more
than 20% of the croplands and around 50% of the irrigated lands are affected by salt
stress, causing loss of production below the genetic capability of the crop plants (Ren
et al. 2005). Algal extracts are also known to mitigate salinity stress, such as in
turfgrass (Nabati et al. 1994). Several studies showed the role of seaweed extract for
improvement of salinity tolerance in tomato, avocado, Arabidopsis, and passion fruit
(Jithesh et al. 2019; Bonomelli et al. 2018; Jolinda et al. 2018; Di Stasio et al. 2018).
Two different commercial extracts FiftyR and RygexR based on A. nodosum
stimulated accumulation of antioxidants, essential amino acids, and minerals in
tomato plants under salt stress conditions (Di Stasio et al. 2018). Application of
extracts obtained from A. nodosum mitigates the salinity stress in avocado via
improvement in nutrient uptake and plant growth. There was a higher accumulation
of Ca2+ and K+ in the seaweed extract-treated avocado plants compared to the
control (Bonomelli et al. 2018). Seaweed extract based on A. nodosum was also
found to improve growth of turfgrass under salt stress conditions (Elansary et al.
2017).
The organic fraction of A. nodosum extract especially the ethyl acetate extract
induces salt stress tolerance in Arabidopsis plants. Global transcriptomics of the
ethyl acetate fraction-treated plants revealed the pathways induced by the ethyl
acetate fraction (Jithesh et al. 2019). Several genes related to salt stress were found
to be influenced by two different seaweed extracts (Goñi et al. 2016). The mecha-
nism includes decrease in water loss from cellular structures, accumulation of
various ions, and protection of proteins from denaturation due to seaweed applica-
tion (Wise and Tunnacliffe 2004; Goyal et al. 2005). Several genes related to
flavonoid synthesis were shown to be induced by application of seaweed extract,
which protects the cells from ROS-mediated oxidative damage under salt stress
(Jithesh et al. 2019). Not only the regulatory genes but also expression of amino
acids, carbohydrate synthesis, and sugar alcohol synthesis-related genes were also
increased under the control of seaweed extracts (Elansary et al. 2017; Jithesh et al.
2019).
Accumulation of the stress amino acid proline can alleviate salt stress in different
crop plants. The major mechanisms include enhancement of antioxidant activity and
stabilization of intracellular organelles (Ashraf and Harris 2004; Ashraf and Foolad
2007). A recent study by Elansary et al. (2017) has shown enhanced synthesis of
structural carbohydrate after application of seaweed extract on turfgrass. Research
has also shown enhanced expression of genes related to sugar transport under the
effect of seaweed extract in turfgrass.
14.5 Role of Seaweed Extract in Mitigation of Drought Stress
Drought is one of the major abiotic stress-causing losses of crop production. More
than 40% of yield loss was observed in maize as well as more than 21% in the wheat
crop due to 40% reduction in water availability (Daryanto et al. 2016). A yield loss
ranging between 34 and 68% in cowpea was recorded due to drought stress (Farooq
et al. 2017). The extract obtained from the A. nodosum has been reported to enhance
drought tolerance in ornamental plants, including Pittosporum eugenioides and
Spiraea nipponica. The major compounds, such as proline, phenolic compounds,
and flavonoids, were increased after application of seaweed extract under drought
stress (Elansary et al. 2016). Application of A. nodosum-based seaweed extract also
enhances the fresh and dry weight in the leafy vegetable spinach under drought stress
(Xu and Leskovar 2015). Isopropanol fraction of the seaweed extract was reported to
increase the water potential and conductance of stomata in grape plants (Vitis
vinifera L) and the K+ and Ca2+ fluxes under drought stress (Mancuso et al. 2006).
Ionic and osmotic stresses can be overcome by the accumulation of K+. Application
of commercial extract of A. nodosum increases the water use efficiency in the orange
Table 14.2 Some seaweed biostimulant-based products and their producing companies
Name of the Name of the
S. N. Name of the product seaweed used company
1. Aquasap (seaweed extract powder) Kappaphycus SeaNutri LLC
alvarezii
2. Kelpak (liquid seaweed concentrate) Ecklonia maxima Kelp Products
International
3. Asco-root (granular supplement with Ascophyllum Organic Ocean Inc.
controlled release) nodosum
4. Stimulagro (concentrated liquid extract of Ascophyllum Organic Ocean Inc.
cold seaweed) nodosum
5. Tonic (seaweed-based liquid fertilizer) Ascophyllum Organic Ocean Inc.
nodosum
6. Ecklomar (liquid extract) Ecklonia maximum Plymag
7. AlgaFlex (liquid concentrated seaweed Ascophyllum Biotechnica
extract) nodosum Services Ltd.
8. Maxicrop liquid seaweed (liquid seaweed Ascophyllum Maxicrop
extract) nodosum
tree Citrus sinensis under drought stress (Spann and Little 2011). The role of
seaweed extract to increase water use efficiency under the drought stress could be
a beneficial application for the drought stress-prone areas for cultivation of fruit trees
(Table 14.2).
14.6 Role of Seaweed Extract in Plant Defense
Random change in the climatic conditions and unscientific way of agricultural

practices have weakened the defense system and induced diseases in plants and
thereby showed a negative effect directly on agricultural crops (Anderson et al.
2004; Ayliffe and Lagudah 2004). Infectious diseases in plants are caused by some
biological agents including the family of bacteria, fungi, or viruses (Pieterse and
Dicke 2007; Stadnik and Freitas 2014), and infectious diseases directly harm the
plant health and crop productivity. To prevent pathogenic infection, plants have
evolved inducible defense processes (Conrath et al. 2002; Wiesel et al. 2014), and it
can be stimulated by any potential stimulants including seaweed extracts.
Till now, two types of disease defense mechanisms have been reported by
scientists, viz., induced systemic resistance (ISR) and systemic acquired resistance
(SAR). To protect plants from a wide range of pathogens, in induced systemic
resistance (ISR), the defense responses are mediated through jasmonate (JA) and
ethylene (ET), whereas salicylic acid (SA) is important to PR (pathogenesis-related)
gene activation for systemic acquired resistance (SAR) mechanisms (Gaffney et al.
1993; van Loon et al. 1998). In plants, the elicitor molecules from pathogens are
responsible for inducing the defense system (Conrath et al. 2002; Wiesel et al. 2014).
Not only chitin, lipopolysaccharides, and flagella of microbes but also some
chemically synthesized components like 2,6-dichloro-isonicotinic acid,

b-aminobutyric acid, chitosan, benzothiadiazole, and methyl jasmonate have the
ability to induce plant defence mechanisms (SAR/ISR) by working as an elicitor,
against a wide range of pathogens (Dixon 2001; Mercier et al. 2001; Bektas and
Eulgem 2015; Iriti and Varoni 2015).
Over time, different seaweeds have evolved and equipped themselves with
important defense mechanisms to protect themselves from their own pathogens
(Potin et al. 1999; Shukla et al. 2016). Because of the presence of some important
bioactive components such as carrageenans, fucans, ulvans, and fucose containing
polymers (or laminarins) in the seaweeds, they showed resistance against a wide
range of pathogens (Klarzynski et al. 2003; Sangha et al. 2010; Vera et al. 2011).
These bioactive components of seaweeds work as elicitor molecules and play a role
in inducing defense mechanisms against pathogens (Khan et al. 2009; Sharma et al.
2014; Shukla et al. 2016). These elicitors can act as pathogen-associated molecular
patterns (PAMPs) (Sharma et al. 2014). PAMPs bind to the host pattern recognition
receptors (PRRs), which are transmembrane proteins, and protect the plants, through
induction of defense mechanisms ISR and SAR via mediation of a systemic signal
(Eckardt 2008; Zipfel 2009). The primed plants showed higher defense response
than the non-primed plants during pathogen infections.
Bioactive components in A. nodosum extract (ANE) induce defense responses
against different pathogens (Patier et al. 1995; Sharma et al. 2014). The report
showed that commercial extract of A. nodosum, namely, Marmarine (IFTCTM,
Amman, Jordan), induces defense in cucumber plants against Phytophthora melonis
(Abkhoo and Sabbagh 2016). Application of the seaweed extract led to induce
activation of some important disease resistance enzymes like polyphenol oxidase
(PPO), peroxidase (PO), phenylalanine ammonia-lyase (PAL), lipoxygenase, and, β
1,3-glucanase. This report suggested the role of seaweed extracts in inducing
different enzymes and genes that increase the resistance power in cucumber
(Abkhoo and Sabbagh 2016). Another report by Panjehkeh and Abkhoo (2016)
showed that application of A. nodosum extract known as Dalgin was able to induce
resistance (ISR) against Phytophthora capsici, a potential disease-causing agent of
tomato. Another application of A. nodosum extract StimplexR with chlorothalonil
(fungicide) showed decrease in the disease-causing ability by fungal pathogens in
cucumber plants by inducing some of the defense genes and enzymes (Jayaraman
et al. 2011). Stella MarisR, another A. nodosum-derived product, was reported to
induce plant immunity by boosting the concentration of ROS through the synthesis
of hydrogen peroxide. Cook et al. (2018) reported that the immunity response gene
WRKY30 (early phase), CYP71A12 (mid phage), and PR-1 (late phage) were
upregulated by the application of bioactive components. A. thaliana plants treated
with 1 g/L of ANE showed resistance against necrotic fungal pathogen Sclerotinia
sclerotiorum (Subramanian et al. 2011). The report showed that fungal pathogens
A. radicina and B. cinerea did not show any significant disease progression in carrot
plants when ANE extract was sprayed (Jayaraj et al. 2008). It was noticed that the
ANE-primed carrot plants induced defense-related enzymes, including PO, PPO,
PAL, β 1,3-glucanase, and chitinase, and also increased the transcript accumulation
of the PR-1, PR-5, NPR-1, LTP, chalcone syntheses, and PAL. Mukherjee and Patel
(2019) reported that application of seaweeds on agriculture crops enhances plant
growth with respect to seedling, root, and shoot development, improves nutrient
uptake ability and fruit setting, boosts immunity against biotic and abiotic stresses,
and also enhances soil fertility and health.
References
Abkhoo J, Sabbagh SK (2016) Control of Phytophthora melonis damping-off, induction of defense
responses, and gene expression of cucumber treated with commercial extract from Ascophyllum
nodosum. J Appl Phycol 28:1333–1342
Ali N, Farrell A, Ramsubhag A, Jayaraman J (2016) The effect of Ascophyllum nodosum extract on
the growth, yield and fruit quality of tomato grown under tropical conditions. J Appl Phycol
28:1353–1362
Anderson PK, Cunningham AA, Patel NG, Morales FJ, Epstein PR, Daszak P (2004) Emerging
infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers.
Trends Ecol Evol 19:535–544
Ashraf MFMR, Foolad M (2007) Roles of glycine betaine and proline in improving plant abiotic
stress resistance. Environ Exp Bot 59:206–216
Ashraf MPJC, Harris PJC (2004) Potential biochemical indicators of salinity tolerance in plants.
Plant Sci 166:3–16
Ayliffe MA, Lagudah ES (2004) Molecular genetics of disease resistance in cereals. Ann Bot
94:765–773
Battacharyya D, Babgohari MZ, Rathor P, Prithiviraj B (2015) Seaweed extracts as biostimulants in
horticulture. Sci Hortic 196:39–48
Beckett RP, Van Staden J (1989) The effect of seaweed concentrate on the growth and yield of
potassium stressed wheat. Plant Soil 116:29–36
Bektas Y, Eulgem T (2015) Synthetic plant defense elicitors. Front Plant Sci 5:804
Blunden GERALD, Gordon SM (1986) Betaines and their sulphonio analogues in marine algae.
Prog Phycol Res 4:39–80
Bonomelli C, Celis V, Lombardi G, Mártiz J (2018) Salt stress effects on avocado (Persea
americana mill.) plants with and without seaweed extract (Ascophyllum nodosum) application.
Agronomy 8:64
Bradáčová K, Weber NF, Morad-Talab N, Asim M, Imran M, Weinmann M, Neumann G (2016)
Micronutrients (Zn/Mn), seaweed extracts, and plant growth-promoting bacteria as cold-stress
protectants in maize. Chem Biol Technol Agric 3:19
Chakrabarti B, Singh SD, Nagarajan S, Aggarwal PK (2011) Impact of temperature on phenology
and pollen sterility of wheat varieties. Aust J Crop Sci 5:1039
Conrath U, Pieterse CM, Mauch-Mani B (2002) Priming in plant–pathogen interactions. Trends
Cook J, Zhang J, Norrie J, Blal B, Cheng Z (2018) Seaweed extract (Stella Maris®) activates innate
immune responses in Arabidopsis thaliana and protects host against bacterial pathogens. Mar
Drugs 16:221
Craigie JS (2011) Seaweed extracts stimuli in plant science and agriculture. J Appl Phycol
23:371–393
Daryanto S, Wang L, Jacinthe PA (2016) Global synthesis of drought effects on maize and wheat
production. PLoS One 11:e0156362
Deng Q, Xia H, Lin L, Wang J, Yuan L, Li K, Liang D (2019) SUNRED, a natural extract-based
biostimulant, application stimulates anthocyanin production in the skins of grapes. Sci Rep
9:2590
Di Stasio E, Van Oosten MJ, Silletti S, Raimondi G, Dell’Aversana E, Carillo P, Maggio A (2018)
Ascophyllum nodosum-based algal extracts act as enhancers of growth, fruit quality, and
adaptation to stress in salinized tomato plants. J Appl Phycol 30:2675–2686
Dixon RA (2001) Natural products and plant disease resistance. Nature 411:843
Dore CMPG, Alves MGDCF, Will LSEP, Costa TG, Sabry DA, de Souza Rêgo LAR, Leite EL
(2013) A sulfated polysaccharide, fucans, isolated from brown algae Sargassum vulgare with
anticoagulant, antithrombotic, antioxidant and anti-inflammatory effects. Carbohydr Polym
91:467–475
Eckardt NA (2008) Chitin signaling in plants: insights into the perception of fungal pathogens and
rhizobacterial symbionts. Plant Cell 20:241–243
Elansary HO, Skalicka-Woźniak K, King IW (2016) Enhancing stress growth traits as well as
phytochemical and antioxidant contents of Spiraea and Pittosporum under seaweed extract
treatments. Plant Physiol Biochem 105:310–320
Elansary HO, Yessoufou K, Abdel-Hamid AM, El-Esawi MA, Ali HM, Elshikh MS (2017)
Seaweed extracts enhance Salam turfgrass performance during prolonged irrigation intervals
and saline shock. Front Plant Sci 8:830
Fan D, Hodges DM, Critchley AT, Prithiviraj B (2013) A commercial extract of brown macroalga
(Ascophyllum nodosum) affects yield and the nutritional quality of spinach in vitro. Commun
Soil Sci Plant 44:1873–1884
Farooq M, Gogoi N, Barthakur S, Baroowa B, Bharadwaj N, Alghamdi SS, Siddique KHM (2017)
Drought stress in grain legumes during reproduction and grain filling. J Agron Crop Sci
203:81–102
Finnie JF, Van Staden J (1985) Effect of seaweed concentrate and applied hormones on in vitro
cultured tomato roots. J Plant Physiol 120:215–222
Fornes F, Sanchez-Perales M, Guardiola JL (2002) Effect of a seaweed extract on the productivity
of ‘de Nules’ clementine mandarin and navelina orange. Bot Mar 45:486–489
Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ryals J (1993) Requirement of
salicylic acid for the induction of systemic acquired resistance. Science 261:754–756
Ghaderiardakani F, Collas E, Damiano DK, Tagg K, Graham NS, Coates JC (2019) Effects of green
seaweed extract on Arabidopsis early development suggest roles for hormone signalling in plant
responses to algal fertilisers. Sci Rep 9:1983
Goñi O, Fort A, Quille P, McKeown PC, Spillane C, O’Connell S (2016) Comparative
transcriptome analysis of two Ascophyllum nodosum extract biostimulants: same seaweed but
different. J Agric Food Chem 64:2980–2989
Goyal K, Walton LJ, Tunnacliffe A (2005) LEA proteins prevent protein aggregation due to water
stress. Biochem J 388:151–157
Gunupuru LR, Patel JS, Sumarah MW, Renaud JB, Mantin EG, Prithiviraj B (2019) A plant
biostimulant made from the marine brown algae Ascophyllum nodosum and chitosan reduce
Fusarium head blight and mycotoxin contamination in wheat. PLoS One 14
Hankins SD, Hockey HP (1990) The effect of a liquid seaweed extract from Ascophyllum nodosum
(Fucales, Phaeophyta) on the two-spotted red spider mite Tetranychus urticae. In: Thirteenth
international seaweed symposium. Springer, Dordrecht, pp 555–559
Hernández-Herrera RM, Santacruz-Ruvalcaba F, Ruiz-López MA, Norrie J, Hernández-Carmona G
(2014) Effect of liquid seaweed extracts on growth of tomato seedlings (Solanum lycopersicum
L.). J Appl Phycol 26:619–628
Hernández-Herrera RM, Santacruz-Ruvalcaba F, Zañudo- Hernández J, Hernández-Carmona G
(2016) Activity of seaweed extracts and polysaccharide-enriched extracts from Ulva lactuca and
Padina gymnospora as growth promoters of tomato and mung bean plants. J Appl Phycol
28:2549–2560
Hong DD, Hien HM, Son PN (2007) Seaweeds from Vietnam used for functional food, medicine
and biofertilizer. J Appl Phycol 19:817–826
Iriti M, Varoni EM (2015) Chitosan-induced antiviral activity and innate immunity in plants.
Environ Sci Pollut Res 22:2935–2944
Jannin L, Arkoun M, Etienne P, Laîné P, Goux D, Garnica M, Houdusse F (2013) Brassica napus
growth is promoted by Ascophyllum nodosum (L.) Le Jol. Seaweed extract: microarray analysis
and physiological characterization of N, C, and S metabolisms. J Plant Growth Regul 32:31–52
Jayaraj J, Wan A, Rahman M, Punja ZK (2008) Seaweed extract reduces foliar fungal diseases on
carrot. Crop Prot 27:1360–1366
Jayaraman J, Norrie J, Punja ZK (2011) Commercial extract from the brown seaweed Ascophyllum
nodosum reduces fungal diseases in greenhouse cucumber. J Appl Phycol 23:353–361
Jithesh MN, Shukla PS, Kant P, Joshi J, Critchley AT, Prithiviraj B (2019) Physiological and
transcriptomics analyses reveal that Ascophyllum nodosum extracts induce salinity tolerance in
Arabidopsis by regulating the expression of stress responsive genes. J Plant Growth Regul
38:463–478
Jolinda M, Leitão ET, Gomes CD, Rodrigues MH, Valéria FDO, dos Santos GL, Santos ADS
(2018) The initial growth of passion fruit plant irrigated with saline water and the application of
biostimulants. J Agric Sci 10:357–363
Kalaivanan C, Venkatesalu V (2012) Utilization of seaweed Sargassum myriocystum extracts as a
stimulant of seedlings of Vigna mungo (L.) Hepper. Span J Agric Res 10:466–470
Khan W, Rayirath UP, Subramanian S, Jithesh MN, Rayorath P, Hodges DM, Prithiviraj B (2009)
Seaweed extracts as biostimulants of plant growth and development. J Plant Growth Regul
28:386–399
Khompatara K, Pettongkhao S, Kuyyogsuy A, Deenamo N, Churngchow N (2019) Enhanced
resistance to leaf fall disease caused by Phytophthora palmivora in rubber tree seedling by
Sargassum polycystum extract. Plan Theory 8:168
Klarzynski O, Descamps V, Plesse B, Yvin JC, Kloareg B, Fritig B (2003) Sulfated fucan
oligosaccharides elicit defense responses in tobacco and local and systemic resistance against
tobacco mosaic virus. Mol Plant-Microbe Interact 16:115–122
Kumar NA, Vanlalzarzova B, Sridhar S, Baluswami M (2012) Effect of liquid seaweed fertilizer of
Sargassum wightii Grev. On the growth and biochemical content of green gram (Vigna radiata
(L.) R. Wilczek). Recent Res Sci Technol 4
Latef AAHA, Srivastava AK, Saber H, Alwaleed EA, Tran LSP (2017) Sargassum muticum and
Jania rubens regulate amino acid metabolism to improve growth and alleviate salinity in
chickpea. Sci Rep 7:10537
Mancuso S, Azzarello E, Mugnai S, Briand X (2006) Marine bioactive substances (IPA extract)
improve foliar ion uptake and water stress tolerance in potted Vitis vinifera plants. Adv Hortic
Sci:156–161
Mercier L, Lafitte C, Borderies G, Briand X, Esquerré-Tugayé MT, Fournier J (2001) The algal
polysaccharide carrageenans can act as an elicitor of plant defence. New Phytol 149:43–51
Moller M, Smith ML (1998) The significance of the mineral component of seaweed suspensions on
lettuce (Lactuca sativa L.) seedling growth. J Plant Physiol 153:658–663
Mukherjee A, Patel JS (2019) Seaweed extract: biostimulator of plant defense and plant productiv-
ity. Int J Environ Sci Technol:1–6
Nabati DA, Schmidt RE, Parrish DJ (1994) Alleviation of salinity stress in Kentucky bluegrass by
plant growth regulators and iron. Crop Sci 34:198–202
Nair P, Kandasamy S, Zhang J, Ji X, Kirby C, Benkel B, Prithiviraj B (2012) Transcriptional and
metabolomic analysis of Ascophyllum nodosum mediated freezing tolerance in Arabidopsis
thaliana. BMC Genomics 13:643
Norrie J, Keathley JP (2005) Benefits of Ascophyllum nodosum marine-plant extract applications to
Thompson seedless grape production. In: X international symposium on plant bioregulators in
fruit production, vol 727, pp 243–248
Pacholczak A, Nowakowska K, Pietkiewicz S (2016) The effects of synthetic auxin and a seaweed-
based biostimulator on physiological aspects of rhizogenesis in ninebark stem cuttings. Notulae
Botanicae Horti Agrobotanici Cluj-Napoca 44:85–91
Panjehkeh N, Abkhoo J (2016) Influence of marine brown alga extract (Dalgin) on damping-off
tolerance of tomato. J Mater Environ Sci 7:2369–2374
Patier P, Potin P, Rochas C, Kloareg B, Yvin JC, Liénart Y (1995) Free or silica-bound oligokappa-
carrageenans elicit laminarinase activity in Rubus cells and protoplasts. Plant Sci 110:27–35
Pettongkhao S, Bilanglod A, Khompatara K, Churngchow N (2019) Sulphated polysaccharide from
Acanthophora spicifera induced Hevea brasiliensis Defense responses against Phytophthora
palmivora infection. Plan Theory 8:73
Pieterse CM, Dicke M (2007) Plant interactions with microbes and insects: from molecular
mechanisms to ecology. Trends Plant Sci 12:564–569
Potin P, Bouarab K, Küpper F, Kloareg B (1999) Oligosaccharide recognition signals and defence
reactions in marine plant-microbe interactions. Curr Opin Microbiol 2:276–283
Rao GN, Chatterjee R (2014) Effect of seaweed liquid fertilizer from Gracilaria textorii and
Hypnea musciformis on seed germination and productivity of some vegetable crops. Univ J
Rayirath P, Benkel B, Hodges DM, Allan-Wojtas P, MacKinnon S, Critchley AT, Prithiviraj B
(2009) Lipophilic components of the brown seaweed, Ascophyllum nodosum, enhance freezing
tolerance in Arabidopsis thaliana. Planta 230:135–147
Ren ZH, Gao JP, Li LG, Cai XL, Huang W, Chao DY, Lin HX (2005) A rice quantitative trait locus
for salt tolerance encodes a sodium transporter. Nat Genet 37:1141
Roberts DA, Paul NA, Dworjanyn SA, Bird MI, De Nys R (2015) Biochar from commercially
cultivated seaweed for soil amelioration. Sci Rep 5:9665
Saa S, Rio OD, Castro S, Brown PH (2015) Foliar application of microbial and plant based
biostimulants increases growth and potassium uptake in almond (Prunus dulcis [mill.] DA
Webb). Front Plant Sci 6:87
Sabir A, Yazar K, Sabir F, Kara Z, Yazici MA, Goksu N (2014) Vine growth, yield, berry quality
attributes and leaf nutrient content of grapevines as influenced by seaweed extract (Ascophyllum
nodosum) and nano-size fertilizer pulverizations. Sci Hortic 175:1–8
Sangha JS, Ravichandran S, Prithiviraj K, Critchley AT, Prithiviraj B (2010) Sulfated macroalgal
polysaccharides λ-carrageenan and ι-carrageenan differentially alter Arabidopsis thaliana resis-
tance to Sclerotinia sclerotiorum. Physiol Mol Plant Pathol 75:38–45
Sbaihat L, Takeyama K, Koga T, Takemoto D, Kawakita K (2015) Induced resistance in Solanum
lycopersicum by algal elicitor extracted from Sargassum fusiforme. Sci World J
Selvam GG, Sivakumar K (2013) Effect of foliar spray from seaweed liquid fertilizer of Ulva
reticulata (Forsk.) on Vigna mungo L. and their elemental composition using SEM–energy
dispersive spectroscopic analysis. Asian Pac J Reprod 2:119–125
Sharma HS, Fleming C, Selby C, Rao JR, Martin T (2014) Plant biostimulants: a review on the
processing of macroalgae and use of extracts for crop management to reduce abiotic and biotic
stresses. J Appl Phycol 26:465–490
Shukla PS, Borza T, Critchley AT, Prithiviraj B (2016) Carrageenans from red seaweeds as
promoters of growth and elicitors of defense response in plants. Front Mar Sci 3:81
Shukla PS, Mantin EG, Adil M, Bajpai S, Critchley AT, Prithiviraj B (2019) Ascophyllum
nodosum-based biostimulants: sustainable applications in agriculture for the stimulation of
plant growth, stress tolerance, and disease management. Front Plant Sci 10
Sivasankari S, Venkatesalu V, Anantharaj M, Chandrasekaran M (2006) Effect of seaweed extracts
on the growth and biochemical constituents of Vigna sinensis. Bioresour Technol 97:1745–1751
Spann TM, Little HA (2011) Applications of a commercial extract of the brown seaweed
Ascophyllum nodosum increases drought tolerance in container-grown ‘Hamlin’sweet orange
nursery trees. HortScience 46:577–582
Stadnik MJ, Freitas MBD (2014) Algal polysaccharides as source of plant resistance inducers. Trop
Subramanian S, Sangha JS, Gray BA, Singh RP, Hiltz D, Critchley AT, Prithiviraj B (2011)
Extracts of the marine brown macroalga, Ascophyllum nodosum, induce jasmonic acid depen-
dent systemic resistance in Arabidopsis thaliana against Pseudomonas syringae pv. Tomato
DC3000 and Sclerotinia sclerotiorum. Eur J Plant Pathol 131:237–248
Ugarte RA (2011) An evaluation of the mortality of the brown seaweed Ascophyllum nodosum (L.)
Le Jol. Produced by cutter rake harvests in southern New Brunswick, Canada. J Appl Phycol
23:401–407
Van Loon LC, Bakker PHM, Pieterse CMJ (1998) Systemic resistance induced by rhizosphere
bacteria. Annu Rev Phytopathol 36:453–483
Verkleij FN (1992) Seaweed extracts in agriculture and horticulture: a review. Biol Agric Hortic
8:309–324
Vernieri P, Borghesi E, Tognoni F, Serra G, Ferrante A, Piagessi A (2006) Use of biostimulants for
reducing nutrient solution concentration in floating system. Acta Hortic 718:477–484
Vinoth S, Gurusaravanan P, Jayabalan N (2012) Effect of seaweed extracts and plant growth
regulators on high-frequency in vitro mass propagation of Lycopersicon esculentum L (tomato)
through double cotyledonary nodal explant. J Appl Phycol 24:1329–1337
Wiesel L, Newton AC, Elliott I, Booty D, Gilroy EM, Birch PR, Hein I (2014) Molecular effects of
resistance elicitors from biological origin and their potential for crop protection. Front Plant Sci
5:655
Wise MJ, Tunnacliffe A (2004) POPP the question: what do LEA proteins do. Trends Plant Sci
9:13–17
Xu C, Leskovar DI (2015) Effects of A. nodosum seaweed extracts on spinach growth, physiology
and nutrition value under drought stress. Sci Hortic 183:39–47
Zhang X, Ervin EH (2008) Impact of seaweed extract-based cytokinins and zeatin riboside on
creeping bentgrass heat tolerance. Crop Sci 48:364–370
Zheng S, Jiang J, He M, Zou S, Wang C (2016) Effect of kelp waste extracts on the growth and
development of Pakchoi (Brassica chinensis L.). Sci Rep 6:38683
Zipfel C (2009) Early molecular events in PAMP-triggered immunity. Curr Opin Plant Biol
12:414–420
Secondary Metabolites from Microbes
for Plant Disease Management 15
U. V. A. Buddhika and S. Abeysinghe
Abstract
Microorganisms inhabiting soil/plant systems produce antimicrobial compounds

including secondary metabolites (SMs). They are low-molecular-weight structur-
ally diverse and complex compounds that are not essential in their life unless they
meet undesirable conditions. SMs such as antibiotics, toxins, ribosomal peptides
(RPs), low-molecular-weight volatile organic compounds (VOCs), polyketides
(PKs), non-ribosomal peptides (NRPs) and hybrids between PKs and NRPs have
shown a diverse performance in antagonistic activity against plant pathogens.
These compounds are involved immensely with combatting pathogens following
interactions with pathogens inhabiting in soil/plant systems, particularly by
developing a disease-suppressive soil. SMs produced by certain microorganisms
induce plant defence reactions leading to a systemic resistance to pathogen
infection. Bacterial endophytes, particularly inhabiting inside plant tissues, are
also source of SMs, which may act as elicitors of plant defences. Biochemical
techniques and genomic-based studies have uncovered that several genes
encoding SMs associated with biocontrol activity are located in gene clusters.
Despite the fact that metabolites have been developed as biopesticides,
bio-weedicides, the in-cooperation of new techniques is important for finding
new resources from disease suppressive soil, which do not represent when the
cultures are tested in vitro. However, this review finds isolation of active
compounds is one of the major challenges in biotechnology as many biosynthetic
genes are not expressed under standard culture conditions, thus proposing a
possible solution to overcome the issue.
U. V. A. Buddhika
School of Agricultural and Wine Sciences, Charles Sturt University, Wagga, NSW, Australia
S. Abeysinghe (*)
Department of Botany, University of Ruhuna, Matara, Sri Lanka
e-mail: saman@bot.ruh.ac.lk

https://doi.org/10.1007/978-981-15-6275-4_15
332 U. V. A. Buddhika and S. Abeysinghe
Keywords
Plant disease · Plant pathogens · Biocontrol agents · Secondary metabolites ·

Disease suppressive soil · Co-cultivation of microbes
15.1 Introduction
Plant diseases caused by pathogenic microorganisms such as bacteria, fungi and

virus can be controlled so as to protect plants from being infected with the help of
certain microorganisms inhabiting rhizosphere, endosphere and phyllosphere
(Mathivanan et al. 2008; Ryu 2013). These microorganisms are different species
from bacteria (Caulier et al. 2019), fungi (Gawai 2018) and actinomycetes (Chen
et al. 2018b; Olanrewaju and Babalola 2019) and are considered as biological
control agents. They owe to several mechanisms involving hyperparasitism, compe-
tition, production of cell wall degrading enzymes and induced resistance (Khokhar
et al. 2012). The production of antimicrobial secondary metabolites (SMs) and other
factors such as siderophores and microbial cyanide and lytic enzymes also play an
important role in plant protection against pathogens (Keswani et al. 2020; Andrić
et al. 2020; Cipollone et al. 2008). The involvement of SMs produced by
microorganisms inhabiting soil/plant systems as to this phenomenal process has
been reported (Raaijmakers et al. 2002).
SMs of microorganisms are low-molecular-weight, structurally diverse and com-
plex compounds that are not essential in their life unless they meet undesirable
conditions. It has been reported that soil/plant-associated bacteria, fungi and
actinomycetes produced a plethora of antimicrobial SMs contributing plant protec-
tion. For instance, fungal species that have shown antagonism against plant
pathogens produce an array of SMs such as antibiotics and toxins (Mathivanan
et al. 2008; Pusztahelyi et al. 2015). Bacillus spp. have also been reported to produce
a range of bioactive metabolites such as ribosomal peptides (RPs), volatile
compounds, polyketides (PKs), non-ribosomal peptides (NRPs) and hybrids
between PKs and NRPs (Caulier et al. 2019). Actinomycetes that represent high
proportion of the soil microbial biomass have the capacity to produce an array of
SMs with the function of antibacterial, antifungal, antibiotic, antiparasitic, insecti-
cide and herbicide (Aggarwal et al. 2016).
Microorganisms producing SMs have been shown to be able to use as biocontrol
agents because of the antimicrobial effects those bioactive compounds hold
(Mathivanan et al. 2008; Sansinenea et al. 2016). There is a growing importance
of performing genomic analysis, which enables researchers to identify biosynthetic
gene clusters encoding SMs that are associated with biocontrol activity (Chowdhury
et al. 2015). The expression of these genes in liquid cultures and the ability of
harvesting secondary metabolites in sufficient quantities are imperative when it
comes to their application in disease management in agriculture. Therefore, it is
important to discover new methods of finding how to improve the yield of SMs in
liquid cultures.
15 Secondary Metabolites from Microbes for Plant Disease Management 333
The aim of this book chapter is to summarize current knowledge about SMs
produced by microorganisms and their application in plant disease management. In
addition, we highlight setbacks, when it comes to the expression of SMs in liquid
cultures that limit the yield, and possibilities of enhancing the expression of genes in
a way to get the sufficient quantity.
15.2 Secondary Metabolites from Microbes and Plant Disease

Suppression
Microorganisms are a gifted source of a spectrum of natural products, which

immensely contribute to the existence of all life forms and the planet. Natural
compounds of microorganisms include primary metabolites such as amino acids,
enzymes, vitamins, organic acids and alcohol, which are useful for human and
planetary health, agriculture, forestry, etc. They are used as nutritional supplements
as well as in the production of industrial commodities through biotransformation.
Whereas microorganisms produce some other compounds such as pigments,
alkaloids, toxins, antibiotics, gibberellins, carotenoids, etc. with no exact function
for their life and derived from the primary metabolism, which are known as SMs
(Malik 1980).
The production of SMs is not widespread among microbes from several genera,
and the formation is generally repressed during logarithmic growth and is
derepressed during the stationary growth phases (Malik 1980). These bioactive
compounds have been studied widely due to the ecological significance, the envi-
ronmental health and agricultural purposes (Behie et al. 2017; Gloer 2007) and for
the purpose of enhancing human and animal health (Singh et al. 2017b). Plant-
associated microorganisms, especially endophytic and rhizosphere bacteria,
actinomycetes and fungi, have been shown to produce a series of bioactive small
molecule/natural products (Gunatilaka 2006; Singh et al. 2017a) and produce SMs
for interacting with other soil microorganisms to suppress the growth of disease-
causing micrograms (Meisner and de Boer 2018) subsequently develop the disease
suppressive soil in ecosystems.
A range of SMs produced by plant-associated microorganisms have shown to be
involved immensely with eradications of pathogens. The soil application of plant
probiotic Bacillus spp. has resulted in early colonization on root system and elimi-
nation of the potential of pathogen colonization and infection triggering the induc-
tion of a range of beneficial SMs and disease resistance (Rahman et al. 2018). In
some cases, low-molecular-weight volatile organic compounds (VOCs) among the
range of microbial SMs have been uncovered (Bailly and Weisskopf 2017; Giorgio
et al. 2015; Hua et al. 2014; Kanchiswamy et al. 2015). Streptomyces spp. have
showed to produce compounds including morphinan, 7,8-didehydro-4,5-epoxy-17-
methyl-3,6-bis[(trimethylsilyl)oxy]-, (5.alpha. 6.alpha)-(C23H35NO3Si2),
cyclononasiloxane, octadecamethyl-(C18H54O9Si9) and benzoic acid, 2,5-bis
(trimethylsiloxy) (C16H30O4Si3) with good antifungal activity against Rhizoctonia
solani (Ahsan et al. 2017).
15.2.1 Fungal Secondary Metabolites
Fungi are found to be the most prolific producer of SM including non-ribosomal

peptides (NRPs), peptaibols, polyketides, pyrones, siderophores and volatile and
non-volatile terpenes (Mukherjee et al. 2012; Crutcher et al. 2013) as they are the
fundamental element to the health and prosperity of every terrestrial ecosystem (Bills
and Gloer 2016). Despite the fact that fungal SMs promote vitality and plant growth
and enhance the resilience against abiotic stress factors, they are known to trigger
plant-induced systemic resistance (ISR) for the protection of plants from pathogen
infections (Mathivanan et al. 2008; O’Brien 2017). A record number of
microparasitic fungal species were reported to produce a diverse range of SMs
with biological activity. Hundreds of SMs produced by beneficial fungi have been
isolated and characterized, especially from biocontrol strains of the genus,
Trichoderma.
Trichoderma spp., the common rhizosphere inhabitants, have been widely stud-
ied due to their capacity to parasitize other fungi (mycoparasitism) and to compete
with deleterious microorganisms in the soil/plant system. The production of SMs in
Trichoderma spp., as tested in in vitro conditions, is strain dependent and varies on i)
the compound considered, ii) the phytopathogen used for elicitation, iii) the viability
of elicitors and iv) the balance between elicited biosynthesis and biotransformation
rate, and also the biocontrol agent seems to modulate the production according to the
presence or the absence of the target pathogen (Vinale et al. 2009). However, it has
been reported that their culture filtrates comprise SMs with biocontrol potential
against severe pathogens, when the culture filtrates were examined. 6-Pentyl-α-
pyrone (6PP) has been found as the major compound that can protect pruning
wounds of grapevines from trunk disease pathogens (Mutawila et al. 2016).
The analysis of culture filtrates of fungal strains is known to be effective in
identifying and characterizing SMs produced against severe plant pathogens. For
instance, the culture filtrate of Clitocybe nuda strain LA82 when tested against
Phytophthora blight of pepper caused by Phytophthora capsici and the leaf spot
on pepper caused by Xanthomonas axonopodis pv. vesicatoria has resulted in an
inhibitory substance. This substance was stable at low and high pH and high
temperature, has a molecular weight between 1000 and 500 and negatively charged
and is a hydrophilic compound, but not a protein (Chen and Huang 2009). From a
similar kind of study, a variety of antimicrobial SMs, including polyketides and
alkanes, has also been found from a new Trichoderma asperellum strain, GDFS1009
(Wu et al. 2017), enabling researchers to pursue investigation into the genetic
analysis of SMs and their widespread application in plant disease management.
As shown by genomic analysis, genes responsible for the biosynthesis of SMs
that have antagonistic activity against microbial plant pathogens are often clustered
in the genome (Fanelli et al. 2018). Ascomycetes have more genes in SMs coding
gene clusters than basidiomycetes, archeo-ascomycetes and chytridiomycetes,
whereas hemi-ascomycetes and zygomycetes have none (Pusztahelyi et al. 2015).
Ascomycete genomes code for an average 16 polyketide synthases (PKS), ten
non-ribosomal protein synthases (NRPS), two tryptophan synthetases (TS) and two
dimethylallyl tryptophan synthetases (DMATS) with crucial importance in SM
synthesis (Pusztahelyi et al. 2016). Despite the fact that NRPS is vital for the
synthesization of siderophores facilitating virulence in several fungi such as
Cochliobolus heterostrophus, C. miyabeanus, F. graminearum and A. brassicicola
(Oide et al. 2006), other types of SM biosynthetic gene clusters (BGC) including
destruxin, NG39x and ferricrocin, together with putative helvolic acid and pseurotin
and tropolone/citrinin-related compound clusters, have been reported to be involved
with the suppression of plant pathogens (Sbaraini et al. 2016).
15.2.2 Bacterial Secondary Metabolites
The common knowledge is that a range of bacterial species such as Pseudomonas

and Bacillus have been used as biological control agents in agriculture due to the
production of an array of antimicrobial SMs with antagonistic potential against
disease causing microbial pathogens. It has been reported that soil bacterium Pseu-
domonas fluorescens BBc6R8 produced three siderophores such as enantio-
pyochelin, pyoverdine and biosurfactant viscosin, which are mainly responsible
for the antagonistic activity of the bacterium under iron-limited conditions apart
from stimulation of the growth of ectomycorrhizal fungus Laccaria bicolor S238N
that kills fungal pathogen (Palin et al. 2016). Seventy-one (71) strains of Bacillus
spp. isolated from different Mexican sites have shown antagonism against several
phytopathogenic fungi, Fusarium oxysporum, Fusarium equiseti, Fusarium
avenaceum, Bipolaris spp. and Alternaria spp., and a range of SMs leading to the
antagonism, which trigger morphological changes on reproductive structures of
pathogenic microbes (Sansinenea et al. 2016). SMs such as bacillomycin D can
cause severe injury to both cell wall and cell membrane of fungal spores and hypha
as observed in the killing of Aspergillus flavus (Gong et al. 2014). In addition, they
have the ability to produce compounds that belong to multiple classes of antibiotics,
which is why a large number of Bacillus strains have been developed for an effective
control of a broad range of plant diseases (Shafi et al. 2017).
In terms of the function, bacterial SMs seemed to be volatile apart from being
soluble (Tyc et al. 2017). VOCs produced by a range of bacterial species including
many species of Pseudomonas and Bacillus have a significant potential of enhancing
antagonistic activity against disease-causing microbial pathogens (Rajer et al. 2017;
Raza et al. 2016; Tahir et al. 2017; Xie et al. 2018). However, their production seems
to be dependent on the inoculation strategy and the size of the inoculum. For
instance, Bacillus spp. produce four VOCs such as benzaldehyde, nonanal,
benzothiazole and acetophenone, when tested against Clavibacter michiganensis
ssp. sepedonicus, the causative agent of bacterial ring rot (Rajer et al. 2017), and
benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene with a robust antag-
onistic activity, when tested against Ralstonia solanacearum, the causal organism of
bacterial wilt disease (Tahir et al. 2017). Pseudomonas spp. also produce volatilome
with a robust chemical resources that could help plant for efficient control of
pathogens. Besides HCN, NH3 and H2S, a blend of other potential VOCs majoring
1-undecene and dimethyl disulphide (DMDS) has also been found, when Pseudo-
monas spp. tested against Phytophthora infestans (Bailly and Weisskopf 2017).
Some BVCs, such as DMDS and 2-methylpentanoate, are highly toxic to plant
pathogens (Ossowicki et al. 2017; Raza et al. 2016; Sharifi and Ryu 2018), and
mechanism of disease suppression primarily seems to be ISR (Sharifi and Ryu
2018). VOCs alter the transcriptional expression levels of genes involved in induced
systemic resistance (ISR) in plants, leading to an inhibition of disease suppression.
For instance, acetoin, 2,3-butanediol and tridecane from Paenibacillus polymyxa
induce ISR genes such as salicylic acid, jasmonic acid and ethylene signaling marker
genes PR1, ChiB and VSP2 in Arabidopsis plants (Lee et al. 2012). In addition,
VOCs have a direct inhibitory effect on conidia germination and the growth of plant
pathogens, such as Botrytis cinerea (Sharifi and Ryu 2016). Carvacrol and trans-2-
hexenal have reported to be effective in hampering in vitro growth and germination
of Monilinia laxa, the agent of brown rot of stone fruit (Neri et al. 2007).
Most SMs from bacterial species are also mainly produced by gene clusters such
as NRPSs and PKSs. Genomic analysis showed 13 biosynthetic gene clusters
(BGCs) encoding SMs associated with biocontrol activity were identified in Bacillus
spp. (Chen et al. 2018a). BGCs in Bacillus spp. included five NRPS clusters
encoding three lipopeptides (surfactin, iturin and fengycin), a siderophore
(bacillibactin) and the antibiotic dipeptide bacilysin. Three PKS clusters were
identified which encoded for the antibacterials: bacillaene, difficidin and
macrolactin. In addition, a ribosomally originated biosynthetic cluster, which
encodes antibiotic plantazolicin, has been found from Bacillus amyloliquefaciens.
Genomic analysis coupled with LC-MS/MS has confirmed the presence of nine
metabolites or their derivatives and eight completed genomes in
B. amyloliquefaciens (Dunlap et al. 2013). Bioactive metabolites of microorganisms
identified by biochemical techniques and genomic-based studies enable researchers
a rapid identification of bioactive metabolites and assemble extra information for
applying them in plant disease management.
15.2.3 Secondary Metabolites from Actinomycetes
Actinomycetes represent a part of microbiome in the soil and soil/plant systems.

Their potential of using in plant protection has been studied widely (El-Tarabily and
Sivasithamparam 2006; Goudjal et al. 2014; Sharma and Salwan 2018; Shrivastava
and Kumar 2018) as they display several desirable characteristics such as production
of SMs, including a range of antibiotics and anti-infection agents against several
phytopathogenic fungi in nature, when it comes to the suppression of plant diseases
(Chen et al. 2018b; Viaene et al. 2016). Some of the SMs from actinomycetes have
been found as blasticidin S, kasugamycin, streptomycin, oxytetracycline,
validamycin, polyoxins, mildiomycin, natamycin, etc. Several SMs produced by a
range of species act as fungicide and bactericides (Aggarwal et al. 2016).
Among diverse range of actinobacteria, the genus Streptomyces belong to the

rhizosphere microbial communities are efficiently colonize rhizosphere and plant
tissues inside, act as a promising resource of SMs (Aggarwal et al. 2016; Olanrewaju
and Babalola 2019; Vurukonda et al. 2018). These compounds have two inhibitory
effects such as fungicidal and fungistatic. The production of growth inhibitory
secondary metabolites such as antibiotics, toxins, biosurfactants, volatiles and others
can suppress or kill microbial rivals through interference competition (Vurukonda
et al. 2018). The liquid culture of Streptomyces strain TKA-5 when tested against
Phytophthora capsici, phytophthora blight of bell pepper, and Alternaria
brassicicola, black leaf spot of spoon cabbage, showed antagonism due to the
low-molecular-weight, non-protein chemical constituents, which have been found
as SMs (Ko et al. 2010). These compounds enable them to stimulate morphological
deformations such as shrinkage, collapse and tortuosity and debilitate spore germi-
nation (Chen et al. 2018b) and inhibit protein biosynthesis in different ways, toxic to
the nervous system making irreversible paralysis, disrupt neuronal activity and
inhibit neurotransmission of pathogenic microorganisms (Aggarwal et al. 2016).
They eventually contribute to develop a disease suppressive soil (Viaene et al. 2016).
Disease suppression in soil appeared to be due to the concerted activities of
multiple microbial genera working together at specific sites or operating at different
stages of the infection process of the pathogen. Next-generation sequencing and
other ‘omics’ technologies have provided new insights into microbial and chemical
ecology that the suppressive soils contain a valuable source of biocontrol agents,
which do not represent when the cultures are tested in vitro (Gómez Expósito et al.
2017). Therefore, practical solutions are important for developing new technologies
to isolate effective biocontrol compounds for formulations as fungicide and
bactericides.
15.2.4 Use of Secondary Metabolites for Controlling Plant Diseases
Eco-friendly chemical resources could help select for efficient biocontrol strategies
and lead to a greener chemical disease management in the field. Therefore, it is
important to use cultures of microorganisms for controlling weeds, pest and
pathogens as their metabolites such as antibiotics, volatile compounds, enzymes
and other toxic substances are the key factors involved in biocontrol potential. These
metabolites have been developed as biopesticides, bio-weedicides and biofertilizers.
It has been found that metabolites from Trichoderma spp. were more effective as
biopesticides and biofertilizers than using living microbes (Vinale et al. 2009). Singh
et al. (2016) have also shown that SMs of Trichoderma and Streptomyces have more
antagonistic actions than their live culture treatments. As such use of SMs provides
significant beneficial impact over the live culture application on the management of
diseases in crop plants. Table 15.1 lists secondary metabolites produced by
microorganisms that have been used in current agricultural practices for the purposes
of controlling plant diseases.
Table 15.1 The summary of the aforementioned secondary metabolites produced by microbes that
have been used in plant disease management at present agriculture
Secondary metabolites Source Biological activity
6-Pentyl-α-pyrone (6PP) Fungi Protect pruning wounds of grapevines from
trunk disease pathogens
Polyketides Bacteria, fungi Antibacterials, antifungals, antivirals and
antiparasitics (Han et al. 2018)
Alkanase
Bacillomycin D Bacteria Antifungal, antibacterial
Benzaldehyde, nonanal, Bacteria Antagonism against Ralstonia solanacearum
benzothiazole and (Bacillus spp.)
acetophenone
1-Undecene Bacteria Antibacteral against against Phytophthora
(Pseudomonas infestans
spp.)
DMDS and Bacteria Inducing ISR in plants
2-methylpentanoate
Carvacrol and trans-2- Bacteria Hampering conidia germination
hexenal
Acetoin, 2,3-butanediol Bacteria Induce ISR genes in plants
and tridecane (Paenibacillus
polymyxa)
Blasticidin S Actinomycetes Antifungal against Pyricularia oryzae
(Streptomyces
spp.)
Kasugamycin Actinomycetes Antifungal against Phytophthora sojae
(Streptomyces
spp.)
Streptomycin Actinomycetes Antibacterial and antifungal against
(Streptomyces Xanthomonas oryzae, Xanthomonas citri and
spp.) Pseudomonas tabaci
Oxytetracycline Actinomycetes
(Streptomyces
spp.)
Validamycin Actinomycetes Antifungal against Rhizoctonia solani and
(Streptomyces other Rhizoctonia in rice, potatoes,
spp.) vegetables, strawberries, tobacco, ginger,
cotton, rice, sugar beet, etc.
Polyoxins, mildiomycin, Actinomycetes Antifungal, antibacterial
natamycin (Streptomyces
spp.)
Identification of new molecular effectors from a range of metabolites supports

biotechnological advancement of the use of such molecules in plant disease man-
agement. However, isolation of active compounds is one of the major challenges in
biotechnology as many biosynthetic genes are not expressed under standard culture
conditions, thus resulting in a limited diversity of microbial compounds that can be
obtained through fermentation. Genetic analysis has shown that gene clusters
encoding SMs are not expressed when microbes cultured alone, but in co-culture
with other microorganisms (Lugtenberg 2018; Wakefield et al. 2017). Among
114, 16 bacterial isolates have resulted in compounds with strong activity such as
1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester; 9,12-octadecadienoic acid
(Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid,
bis(2-ethylhexyl) ester, when cultivated together with other microbes (Mohamad
et al. 2018), although broad antimicrobial activities have shown against common
fungal pathogens. All in all, co-cultivation seems to be an effective strategy to
produce bioactive metabolites, especially novel compounds from plant beneficial
fungi (Vinale et al. 2017).
15.2.5 Conclusion and Future Perspectives
SMs from bacterial antagonists have served as important sources of antimicrobial

agents, which are of great use in the field of medicine and agriculture. Although new
scientific approaches such as metagenomics have deployed to find new genes, the
success of achieving the production in vitro is not as simple as it seems, due to the
debilitated performance of microbes isolated from their original source and in vitro
cultures. However, co-cultivation has provided a possible method of yielding strong
and effective SMs from microorganisms. Thus, cultivation of microbes isolated from
disease suppressive soil together with other microbes might be the possible solution
to optimize and enhance yield of target compounds in a way that can be applied to
protect plant from being affected by several diseases.
References
Aggarwal N, Thind SK, Sharma S (2016) Role of SMs of Actinomycetes in crop protection. In:
Subramaniam G, Arumugam S, Rajendran V (eds) Plant growth promoting Actinobacteria: a
new avenue for enhancing the productivity and soil fertility of grain legumes. Springer
Singapore, Singapore, pp 99–121
Ahsan T, Chen J, Zhao X, Irfan M, Wu Y (2017) Extraction and identification of bioactive
compounds (eicosane and dibutyl phthalate) produced by Streptomyces strain KX852460 for
the biological control of Rhizoctonia solani AG-3 strain KX852461 to control target spot disease
in tobacco leaf. AMB Express 7:54
Andrić S, Meyer T, Ongena M (2020) Bacillus responses to plant-associated fungal and bacterial
communities. Front Microbiol 11:1350. https://doi.org/10.3389/fmicb.2020.01350
Bailly A, Weisskopf L (2017) Mining the Volatilomes of plant-associated microbiota for new
biocontrol solutions. Front Microbiol 8:1638–1638
Behie SW, Bonet B, Zacharia VM, McClung DJ, Traxler MF (2017) Molecules to ecosystems:
actinomycete natural products in situ. Front Microbiol 7(2149)
Bills GF, Gloer JB (2016) Biologically active SMstes from the Fungi. Microbiol Spectrum 4(6)
Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J (2019) Overview of the
antimicrobial compounds produced by members of the Bacillus subtilis group. Front Microbiol
10(302)
Chen JT, Huang JW (2009) Control of plant diseases with SMste of Clitocybe nuda. New
Chen L, Heng J, Qin S, Bian K (2018a) A comprehensive understanding of the biocontrol potential
of Bacillus velezensis LM2303 against Fusarium head blight. PLoS One 13:e0198560–
e0198560
Chen Y, Zhou D, Qi D, Gao Z, Xie J, Luo Y (2018b) Growth promotion and disease suppression
ability of a Streptomyces sp. CB-75 from Banana rhizosphere soil. Front Microbiol
8:2704–2704
Chowdhury SP, Hartmann A, Gao X, Borriss R (2015) Biocontrol mechanism by root-associated
Bacillus amyloliquefaciens FZB42 – a review. Front Microbiol. https://doi.org/10.3389/fmicb.
2015.00780
Cipollone R, Ascenzi P, Tomao P, Imperi F, Visca P (2008) Enzymatic detoxification of cyanide:
clues from Pseudomonas aeruginosa Rhodanese. J Mol Microbiol Biotechnol 15(2–3):199–211.
https://doi.org/10.1159/000121331
Crutcher FK, Parich A, Schuhmacher R, Mukherjee PK, Zeilinger S, Kenerley CM (2013) A
putative terpene cyclase, vir4, is responsible for the biosynthesis of volatile terpene compounds
in the biocontrol fungus Trichoderma virens. Fungal Genet Biol 56:67–77. https://doi.org/10.
1016/j.fgb.2013.05.003
Dunlap CA, Bowman MJ, Schisler DA (2013) Genomic analysis and SMs production in Bacillus
amyloliquefaciens AS 43.3: a biocontrol antagonist of Fusarium head blight. Biol Control
64:166–175
El-Tarabily KA, Sivasithamparam K (2006) Non-streptomycete actinomycetes as biocontrol agents
of soil-borne fungal plant pathogens and as plant growth promoters. Soil Biol Biochem
38:1505–1520
Fanelli F, Liuzzi VC, Logrieco AF, Altomare C (2018) Genomic characterization of Trichoderma
atrobrunneum (T. harzianum species complex) ITEM 908: insight into the genetic endowment
of a multi-target biocontrol strain. BMC Genomics 19:662–662
Gawai DU (2018) Role of Fungi as biocontrol agents for the control of plant diseases in sustainable
agriculture. In: Gehlot P, Singh J (eds) Fungi and their role in sustainable development: current
perspectives. Springer, Singapore
Giorgio A, De Stradis A, Lo Cantore P, Iacobellis NS (2015) Biocide effects of volatile organic
compounds produced by potential biocontrol rhizobacteria on Sclerotinia sclerotiorum. Front
Microbiol 6:1056
Gloer JB (2007) Applications of fungal ecology in the search for new bioactive natural products. In:
Kubicek CP, Druzhinina IS (eds) Environmental and microbial relationships. Springer, Berlin
Heidelberg, Berlin, Heidelberg, pp 257–283
Gómez Expósito R, de Bruijn I, Postma J, Raaijmakers JM (2017) Current insights into the role of
rhizosphere bacteria in disease suppressive soils. Front Microbiol 8:2529–2529
Gong Q, Zhang C, Lu F, Zhao H, Bie X, Lu Z (2014) Identification of bacillomycin D from Bacillus
subtilis fmbJ and its inhibition effects against Aspergillus flavus. Food Control 36:8–14
Goudjal Y, Toumatia O, Yekkour A, Sabaou N, Mathieu F, Zitouni A (2014) Biocontrol of
Rhizoctonia solani damping-off and promotion of tomato plant growth by endophytic
actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res 169:59–65
Gunatilaka AAL (2006) Natural products from plant-associated microorganisms: distribution,
structural diversity, bioactivity, and implications of their occurrence. J Nat Prod 69:509–526
Hua SS, Beck JJ, Sarreal SB, Gee W (2014) The major volatile compound 2-phenylethanol from the
biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes
of Aspergillus flavus. Mycotoxin Res 30:71–78
Kanchiswamy CN, Malnoy M, Maffei ME (2015) Chemical diversity of microbial volatiles and
their potential for plant growth and productivity. Front Plant Sci 6:151
Keswani C, Singh HB, García-Estrada C et al (2020) Antimicrobial secondary metabolites from
agriculturally important bacteria as next-generation pesticides. Appl Microbiol Biotechnol
104:1013–1034. https://doi.org/10.1007/s00253-019-10300-8
Khokhar MK, Gupta R, Sharma R (2012) Biological control of plant pathogens using biotechno-
logical aspects: a review. Sci Rep 1:277
Ko WH, Tsou YJ, Lin MJ, Chern LL (2010) Activity and characterization of SMs produced by a
new microorganism for control of plant diseases. New Biotechnol 27:397–402
Lee B, Farag MA, Park HB, Kloepper JW, Lee SH, Ryu CM (2012) Induced resistance by a long-
chain bacterial volatile: elicitation of plant systemic Defense by a C13 volatile produced by
Paenibacillus polymyxa. PLoS One 7:e48744
Lugtenberg B (2018) Putting concerns for caution into perspective: microbial plant protection
products are safe to use in agriculture. J Plant Dis Prot 125:127–129
Malik VS (1980) Microbial SMssm. Trends Biochem Sci 5:68–72
Mathivanan N, Prabavathy VR, Vijayanandraj VR (2008) The effect of fungal SMs on bacterial and
fungal pathogens. In: Karlovsky P (ed) SMs in soil ecology. Springer, Berlin\Heidelberg, pp
129–140
Meisner A, de Boer W (2018) Strategies to maintain natural biocontrol of soil-borne crop diseases
during severe drought and rainfall events. Front Microbiol 9:2279–2279
Mohamad OAA, Li L, Ma JB, Hatab S, Xu L, Guo JW, Rasulov BA, Liu YH, Hedlund BP, Li W-J
(2018) Evaluation of the antimicrobial activity of Endophytic bacterial populations from
Chinese traditional medicinal plant Licorice and characterization of the bioactive SMs produced
by Bacillus atrophaeus against Verticillium dahliae. Front Microbiol 9:924–924
Mukherjee PK, Horwitz BA, Kenerley CM (2012) Secondary metabolism in Trichoderma – a
genomic perspective. Microbiology 158:35–45. https://doi.org/10.1099/mic.0.053629-0
Mutawila C, Vinale F, Halleen F, Lorito M, Mostert L (2016) Isolation, production and in vitro
effects of the major SMs produced by Trichoderma species used for the control of grapevine
trunk diseases. Plant Pathol 65:104–113
Neri SV, Bradley EH, Groce NE (2007) Frequency of HIV testing among persons with disabilities:
results from the National Health Interview Survey, 2002. AIDS Educ Prev 19:545–554
O’Brien PA (2017) Biological control of plant diseases. Australia Plant Pathol 46(4):293–304
Oide S, Moeder W, Krasnoff S, Gibson D, Haas H, Yoshioka K, Turgeon BG (2006) NPS6,
encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism,
is a conserved virulence determinant of plant pathogenic ascomycetes. Plant Cell 18:2836–2853
Olanrewaju OS, Babalola OO (2019) Streptomyces: implications and interactions in plant growth
promotion. Appl Microbiol Biotechnol 103:1179–1188
Ossowicki A, Jafra S, Garbeva P (2017) The antimicrobial volatile power of the rhizospheric isolate
Pseudomonas donghuensis P482. PLoS One 12:e0174362
Palin B, Deveau A, Gross H, Schnepf M, Mehnaz S, Aigle B, Leblond P, Dorrestein PC (2016) Role
of SMs in the interaction between Pseudomonas fluorescens and soil microorganisms under
iron-limited conditions. FEMS Microbiol Ecol:92
Pusztahelyi T, Holb I, Pócsi I (2015) SMs in fungus-plant interactions. Front Plant Sci 6(573)
Pusztahelyi T, Holb IJ, Pócsi I (2016) Plant-fungal interactions: special SMstes of the biotrophic,
necrotrophic, and other specific interactions. In: Mérillon J-M, Ramawat KG (eds) Fungal
metabolites. Springer International Publishing, Cham, pp 1–58
Raaijmakers JM, Vlami M, de Souza JT (2002) Antibiotic production by bacterial biocontrol
agents. Antonie Van Leeuwenhoek 81:537–547
Rahman M, Sabir AA, Mukta JA, Khan MMA, Mohi-Ud-Din M, Miah MG, Rahman M, Islam MT
(2018) Plant probiotic bacteria Bacillus and Paraburkholderia improve growth, yield and
content of antioxidants in strawberry fruit. Sci Rep 8:2504
Rajer FU, Wu H, Xie Y, Xie S, Raza W, Tahir HAS, Gao X (2017) Volatile organic compounds
produced by a soil-isolate, Bacillus subtilis FA26 induce adverse ultra-structural changes to the
cells of Clavibacter michiganensis ssp. sepedonicus, the causal agent of bacterial ring rot of
potato. Microbiology (Reading, England) 163:523–530
Raza W, Ling N, Liu D, Wei Z, Huang Q, Shen Q (2016) Volatile organic compounds produced by
Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia
solanacearum. Microbiol Res 192:103–113
Ryu C-M (2013) Promoting plant protection by root-associated microbes. Plant Pathol J
29:123–124
Sansinenea E, Almaraz M, Ramírez MD, Ortiz A (2016) Cellular damage of plant pathogenic fungi
by antifungal compounds produced by Bacillus spp. isolates. Chem Ecol 32:722–732
Sbaraini N, Guedes RLM, Andreis FC, Junges Â, de Morais GL, Vainstein MH, de Vasconcelos
ATR, Schrank A (2016) SMs gene clusters in the entomopathogen fungus Metarhizium
anisopliae: genome identification and patterns of expression in a cuticle infection model.
BMC Genomics 17:736
Shafi J, Tian H, Ji M (2017) Bacillus species as versatile weapons for plant pathogens: a review.
Biotechnol Biotec Equip 31:446–459
Sharifi R, Ryu C-M (2016) Are bacterial volatile compounds poisonous odors to a fungal pathogen
Botrytis cinerea, alarm signals to Arabidopsis seedlings for eliciting induced resistance, or both?
Front Microbiol 7(196)
Sharifi R, Ryu C-M (2018) Revisiting bacterial volatile-mediated plant growth promotion: lessons
from the past and objectives for the future. Ann Bot 122:349–358
Sharma V, Salwan R (2018) Chapter 6: Biocontrol potential and applications of actinobacteria in
agriculture. In: Singh BP, Gupta VK, Passari AK (eds) New and future developments in
microbial biotechnology and bioengineering. Elsevier, pp 93–108
Shrivastava P, Kumar R (2018) Chapter 5: Actinobacteria: eco-friendly candidates for control of
plant diseases in a sustainable manner. In: Singh BP, Gupta VK, Passari AK (eds) New and
future developments in microbial biotechnology and bioengineering. Elsevier, pp 79–91
Singh A, Gupta R, Srivastava M, Gupta MM, Pandey R (2016) Microbial SMs ameliorate growth,
in planta contents and lignification in Withania somnifera (L.) Dunal. Physiol Mol Biol Plants
22:253–260
Singh M, Kumar A, Singh R, Pandey KD (2017a) Endophytic bacteria: a new source of bioactive
compounds. 3 Biotech 7:315–315
Singh R, Kumar M, Mittal A, Mehta PK (2017b) Microbial metabolites in nutrition, healthcare and
agriculture. 3 Biotech 7:15–15
Tahir HAS, Gu Q, Wu H, Niu Y, Huo R, Gao X (2017) Bacillus volatiles adversely affect the
physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in
tobacco against bacterial wilt. Sci Rep 7:40481–40481
Tyc O, Song C, Dickschat JS, Vos M, Garbeva P (2017) The ecological role of volatile and soluble
SMs produced by soil Bacteria. Trends Microbiol 25:280–292
Viaene T, Langendries S, Beirinckx S, Maes M, Goormachtig S (2016) Streptomyces as a plant’s
best friend? FEMS Microbiol Ecol 92(8)
Vinale F, Ghisalberti EL, Sivasithamparam K, Marra R, Ritieni A, Ferracane R, Woo S, Lorito M
(2009) Factors affecting the production of Trichoderma harzianum SMs during the interaction
with different plant pathogens. Lett Appl Microbiol 48:705–711
Vinale F, Nicoletti R, Borrelli F, Mangoni A, Parisi OA, Marra R, Lombardi N, Lacatena F,
Grauso L, Finizio S, Lorito M, Woo SL (2017) Co-culture of plant beneficial microbes as source
of bioactive metabolites. Sci Rep 7:14330
Vurukonda SSKP, Giovanardi D, Stefani E. (2018) Plant growth promoting and biocontrol activity
of Streptomyces spp. as Endophytes. Intern J Mol Sci. 19:952
Wakefield J, Hassan HM, Jaspars M, Ebel R, Rateb ME (2017) Dual induction of new microbial
SMs by fungal bacterial co-cultivation. Front Microbiol 8:1284
Wu Q, Sun R, Ni M, Yu J, Li Y, Yu C, Dou K, Ren J, Chen J (2017) Identification of a novel
fungus, Trichoderma asperellum GDFS1009, and comprehensive evaluation of its biocontrol
efficacy. PLoS One 12:e0179957
Xie S, Zang H, Wu H, Uddin Rajer F, Gao X (2018) Antibacterial effects of volatiles produced by
Bacillus strain D13 against Xanthomonas oryzae pv. oryzae. Mol Plant Pathol 19:49–58
Beneficial Root Microbiota: Transmogrifiers
of Secondary Metabolism in Plants 16
Akanksha Singh, Rupesh Chaubey, Stuti Srivastava,
Sumit Kushwaha, and Rakesh Pandey
Abstract
All plants in the ecosystem are found in close association with complex group of
microbes both belowground and aboveground surfaces. Reports suggest that the
association can be harmful, neutral, or beneficial to the plants depending upon the
category of colonizing microbes. It is among them that certain microorganisms
bring about modification in the plant metabolome, maneuvering to modifications
in the biosynthetic pathway of plant metabolites of known and unknown origin.
Plant secondary metabolites are exceptional group of chemicals released as an
end product of biosynthetic pathways which have numerous secondary roles in
survival and growth of the plants. Among the multifarious roles played by the
metabolites, some of the important traits include repulsion of pathogens and
attraction of beneficial group of microbes. The present chapter thus summarizes
the till-date understanding of the role of root microbiome on the secondary
metabolic status of plants, how the remodeling affects the health and defense
status of the concerned plants, and finally the knowledge hiatus that needs to be
fulfilled for harnessing the full potential of microbes.
Keywords
Root microbiome · Secondary metabolites · Plant defense · Cross kingdom
communication
A. Singh · R. Chaubey · S. Srivastava · S. Kushwaha · R. Pandey (*)

Department of Microbial Technology and Nematology, CSIR-Central Institute of Medicinal and
Aromatic Plants, Lucknow, India
e-mail: r.pandey@cimap.res.in

https://doi.org/10.1007/978-981-15-6275-4_16
344 A. Singh et al.
16.1 Introduction
Host-associated microbial populations are reported to be engaged in elementary

roles like nutrition status, different developmental phases, and immunity of both
animal and plant kingdom. The different factors which help in architecturing the
host–microbiome interactions are inadequately understood, which hold an important
place in evolutionary and ecological sciences (Fitzpatrick et al. 2018). Talking about
the plants, the roots bring together two different microbial sections namely rhizo-
sphere and the endosphere. The colonization at the rhizospheric surface by microbes
can either be beneficial, neutral, or harmful associations, depending upon the
relationship they share with the host plant. With the advancement in technologies,
especially pertaining to sequencing, the picture of different root-associated
microbiomes is getting clearer day by day (Rout and Southworth 2013). The most
recent information which is coming out from the experimental evidences is that the
role of microbiome differs not only with plant tissues but also with the change in
environmental conditions too (Yu et al. 2019). The Next Generation Sequencing
(NGS) data clearly demonstrates that amazing number of taxonomically dissimilar
microbes colonize the plant system, whose density can be sometimes much higher
than the plant cells figures (Mendes et al. 2013; Panke-Buisse et al. 2015). The
colonization affects the plant system either directly or indirectly either by facilitating
nutrient uptake, phytohormone production, induction of systemic resistance, forma-
tion of physical barriers, and changes in secondary metabolite status of concerned
plants (Etalo et al. 2018). The most recent area of current research in plant–microbe
interaction is changes in metabolomic status of plants leading to alteration in some
key metabolites of agricultural and medical importance (Etalo et al. 2018). Hence, it
is hypothesized that exploring plant–microbe communication will pave way for not
only boosting production of metabolites of pharmaceutical importance but other
unknown secondary metabolites too. The current chapter has thus been written with
the aim to provide exhaustive information about the key players involved in alter-
ation of secondary metabolites in plants with special emphasis on beneficial
microbes, root exudates, and bioactive metabolites.
16.2 Root Microbes
Microorganisms have been defined as smallest organisms that cannot be seen with
the naked eye and can only be seen with a special equipment called microscope.
Among the diverse range of microbes, we in this chapter have specifically discussed
about the microbes colonizing the root zone of plants. Microorganisms are mostly
found as free-living microbes and when they stick around the plant roots and root
hairs, they are called root microbes.
Root microbes are classified into two types:
16 Beneficial Root Microbiota: Transmogrifiers of Secondary Metabolism in Plants 345
1. Beneficial microbes are those microbes which work toward enhancing the yield
and overall well-being of plants and which can easily perform plant growth
promotion, for example Pseudomonas, Bacillus, etc.
2. Harmful microbes are the category of root microbes that inhibits the growth of
plants by destroying the plant cells, making the plants nutrients deficient, and
killing the beneficial microbes.
16.2.1 Beneficial Root Microbes
In the early 1904, Lorenz Hiltner observed and stated that there are numerous
microorganisms which live in the soil near the rhizospheric region than the distant
part of soil (Hiltner 1904). Soil is been widely accepted as the home for array of
microbial species, fungi, invertebrates, archaea, and mostly bacteria (Tringe et al.
2005). Hiltner gave the term Rhizosphere for that region where microbial popula-
tion was the highest near plant roots. It has also been derived that some region of soil
which is conventionally benefitted by root secretion and associated with microbes of
soil is referred to as root microbiome. Moreover, plant root system always expands
through the soil and penetrates it, resulting in release of water-soluble materials such
as amino acids, organic molecules, certain sugars, and carbohydrate derivatives
which are essential for microorganism to survive.
Surprisingly, plant physiologists noted that soil plays a role in providing nutrients
to plants, but they forgot to add that soil is a different complex ecological system
having a huge species like protists, animals, bacteria, and fungi specially
(Bonkowski et al. 2009; Müller et al. 2016). The microorganisms’ living in soil
are the basic invisible mangers of soil fertility, and it doesn’t matter if the soil
condition or crop species favour them or not, because it is the nature which promotes
microbes to become root symbionts. These symbionts promote plant growth and
increase yield by different actions like nutrient uptake and nitrogen metabolism
resulting in nitrogen fixation, and these particular activities help plants to counter
pests, diseases, and biotic and abiotic stresses (Fig. 16.1). Collectively, by the
enhancement of plant capacity in photosynthesis and production of organic acids,
plants derive their health and the microbes which helped throughout this process are
referred as “beneficial root microbes.”
16.2.1.1 Types of Beneficial Root Microbes

The beneficial root microbes have been basically categorized into five different
types, namely actinomycetes, bacteria, fungi, protozoans, and nematodes. The
detailed information about these microbes has been discussed in the following
sections.
Bacteria
Bacteria are the smallest living organisms and major key player of soil in bringing
together the simpler forms around the root system so that the plants can firmly take
up all the nutrients important to their growth and development, for examples
346 A. Singh et al.
Fig. 16.1 Role of rhizospheric microbial community in mitigating biotic and abiotic stressors
macronutrients like nitrogen, phosphorus, potassium, etc. Phosphorus is usually not

found in available form for the plants in soil, but some of the beneficial bacteria turns
the nonavailable phosphorus into available form which a plant can easily utilize. In
soil system there are huge number of bacteria which enhance plant growth and have
thus been referred to as plant growth–promoting rhizobacteria (PGPR) (Bonkowski
et al. 2009). Moreover, in obligate symbionts, PGPRs can easily interact with the
host plants and enhance growth of the plant either by direct benefit via nitrogen
fixation (Müller et al. 2016; Kloepper and Schroth 1987) or indirectly by secreting
certain enzymes and hormones which can suppress other pathogens’ activity
(Soyano et al. 2014; Ferguson and Mathesius 2014). The root hierarchy is also
dependent upon PGPRs, as the structural modification in root results in better
conduction of molecules into plant parts which is inversely proportional to better
crop yield (Pérez-Montaño et al. 2014; Lugtenberg and Kamilova 2009; Uga et al.
2013). The projectile PGPR activity and plant growth promotion attributes are also
reviewed in some articles (Ogawa et al. 2014; Ning et al. 2014).
Example – Indole-3-acetic acid production by the Rhizobium leguminosarum has
been discussed in literature for playing a key role in promoting certain effects on rice
seedlings (Biswas et al. 2000). In the same way, Azotobacter has been reported to do
the job for maize seedlings (Zahir et al. 2000).
Actinomycetes
Actinomycetes are spore-forming, gram-positive aerobic bacteria which form
thread-like structures called filaments, and work in cycling or turning up the organic
matters, mainly by decomposition of complex mixtures found from decomposed
plants, animals, or fungal sheets over rocks. Somehow these enzymes and hormones
also help in suppressing certain plant pathogens which pose threat to plants, for
example Streptomyces sp. have been found responsible for nutrient uptake and plant
growth in rice and chickpea plants (Gopalakrishnan et al. 2014, 2015). Likewise,
Frankia has been found to be responsible for nitrogen fixation in Alnus plant
(Simonet et al. 1990).
Fungi
Fungi are multicellular, eukaryotic, heterotrophic organisms that have absorptive
mode of nutrition. They live in the root zone of plants and act like natural recycling
bins, help in reabsorbing soil nutrients from dead organic matter, and redistributing
them back to plants roots. In addition, they also help in making nutrients available to
plants through formation of siderophores. For example, mycorrhizal association is a
mutual relationship which exists between roots of plant and fungus for sharing the
benefits. The association is usually two ways – ectomycorrhizal when the fungus
resides outside of root, or endomycorrhizal when the fungus penetrates inside of the
root. It is well reported in literature that most of the rhizospheric fungus produces
metabolites for the inhibition of plant pathogens (Ali et al. 2015; Saraf et al. 2014).
Example – Plant defense mechanisms can directly or indirectly be controlled by
arbuscular mycorrhizal fungi (AMF) (Di Benedetto et al. 2017). Trichoderma
harzianum are involved in active colonization of tomato root and induced systemic
resistance-like defense in Arabidopsis. (Engelberth et al. 2001). Likewise,
Trichoderma viride has been found to be responsible for elicitation of jasmonic
acid and salicylic acid biosynthesis in lima bean (Morán-Diez et al. 2009).
Protozoa
Protozoa are single-celled, microscopic, eukaryotic, and heterotrophic organisms
(using organic carbon as a source of energy). They are non-filamentous and
restricted to moist or aquatic habitats. Protozoans play important roles in the fertility
of soils by eating soil bacteria and maintaining bacterial populations. Protozoans
sometime help in promoting plant health by the mineralization of nutrients and
alteration in the hierarchy or activity of plant root–associated families (Bonkowski
2004). It was also stated and reported that predation of some of the different plant
pathogenic species has an inverse effect on the plant growth hormone production
(Krome et al. 2010) or sometimes they support the beneficial microbes to survive
(Jousset et al. 2010; Müller et al. 2013). Protozoans also excrete nitrogen in the form
of ammonium and phosphorus as products of their metabolism, and it is because of
this reason that the presence of protozoans in soil has been reported to enhance plant
growth and development.
Example – Acanthamoeba castellanii grazing has been reported to maintain the
bacterial population in the rhizospheric soil by consumption etc.
348 A. Singh et al.
Nematodes
Nematodes are microscopic worms which live around or inside the plant and
periodically rely and feed over bacteria, fungus, and other soil microbes. Nematodes
can easily carry live microbes over their bodies and also inside their digestive
systems, and by this activity wherever they go nematodes deliver microbes over
the roots of plant or in soil. Few nematodes are also disease causing, while others
feed over disease-causing organisms which can be identified as potential biocontrol
agents.
Example – Steinernema, Risbravis, Rhabditis, etc., are the useful nematodes
responsible for decomposing the organic matter and managing attack on insects
and other pests.
16.3 How Useful Root Microbes Boost Crop Productivity?
Beneficial root microbes present in the rhizospheric soil near plant roots ameliorate
plant productivity and its performance in a variety of ways like deterioration of
pathogens, providing resistance against any infection, and help in plant growth
promotion. The major mode action involves following steps:
16.3.1 Nutrient Availability
Rhizospheric microorganisms always take part in obtaining trace elements which are
found in insoluble forms, where microbes turn this into soluble form and make them
available to plants. By the use of certain molecules, like siderophore, iron chelation
and conversion of complex to simpler form takes place (Aznar and Dellagi 2015).
Most of the bacterial community works as key component to unlock the nutrients
which are locked in the form of hydrocarbons essential for the plants. Some of the
saprotrophs and fungi have been reported as nutrient extractors through solubiliza-
tion or reabsorption processes, among which actinomycetes play a significant role in
decaying organic matter to make it in available form (Aznar and Dellagi 2015).
16.3.2 Plant Growth Promotion
In a different manner we have seen PGPRs playing essential role in plant growth
promotion where they produce metabolites which eventually trigger the release of
plant hormones reported to play beneficial role for plants. Apart from working as
PGPRs, some microbes work as bio-remediators. As a biocontrol trait, microbes
effect plant pathogens through the different synthesis like regulation of ethylene
level in plant, siderophore activity, acquired systemic resistance, antibiosis, quorum
sensing, etc. (Babalola 2010; Olanrewaju et al. 2017). In addition, the beneficial
microbes are reported to increase photosynthesis and production of hormones and
enzymes as a result of improvement in crop growth. They also control various
insects and plant diseases as a consequence improvement in crop quality. The use of
such kinds of microorganisms leads to reduction in the usage of chemical fertilizers.
16.4 Root Exudates: Role in Shaping Root Microbiome
In natural environment, plants health status mainly depends on complex and active
microbial community present in the rhizospheric soil. In plants, root system is the
essential part for nutrient and water conduction, which is inhabited and encircled by
a major microbial community called root microbiota or rhizomicrobiome (Del
Carmen Orozco-Mosqueda et al. 2018; Hacquard et al. 2015). Complex microbial
community present in the root microbiome is referred to as plant’s second genomic
part which consists of total rhizosphere community’s interactions present in relation
to plant health (Berendsen et al. 2012). Crop growth and yield inside natural
environment depends on microbial interactions, that is, bacteria and fungi,
actinomycetes, etc. (Schmidt et al. 2016). Attachment of microbial diversities was
preferred to be connected in two steps:
16.4.1 Rhizosphere
Rhizosphere as a term was first coined by Lorentz Hiltner (Hiltner 1904) and
reconsidered by Pinton as the zone around the plant roots in the soil which is
colonized by microbial community (Morgan et al. 2005; Pinton et al. 2007).
Example – Azotobacter, Nitrobacter, Proteobacteria, Rhizobacteria,
Actinobacteria, Pseudomonas are some of the ruling populations of bacteria over
rhizosphere (Sylvia and Prévost 2005).
16.4.2 Rhizoplane
Region of surface of the plant roots with epidermis and mucilage which is direct
contact with the soil and colonized by microbial community.
Example – Burkholderia, Acidobacterium, Dyella, and Edaphobacter are the
major genera abundant in the rhizoplane.
The soil–microbe interactions are usually specific and depend upon coevolution-
ary dilemma (Dobbelaere et al. 2003; Duffy et al. 2004); (Morgan et al. 2005). In the
underground world, the specific plant–microbe interactions hold a very important
place in various processes governing ecosystem, just like carbon metabolism,
sequestration, and nutrient cycling (Singh et al. 2004).
For the export and secretion of molecules into the rhizospheric soil, plants use a
hierarchical transport technique where plant roots along with root hairs and adventi-
tious part release root exudates either by passive or active diffusion/secretion
mechanism (Badri et al. 2009; Weston et al. 2012).
350 A. Singh et al.
Table 16.1 Different types of root exudates released by the plants

Classes of
compounds Components identified
Amino acids All 20 proteinic amino acids, homoserine, aminobutyric acid, mugineic
acid, l-hydroxyproline
Lignins Coumaric acid, sinapoyl aldehyde, benzoic acid, pyroglutamic acid
phloroglucinol, gallic acid, nicotinic acid, chlorogenic acid, vanillin,
catechol, sinapyl alcohol, quinic acid
Proteins and Peroxidases, PR proteins, proteases, lipase, acid phosphatases, lectins,
enzymes hydrolases
Phenolics and Umbelliferone, Caffeic acid, cinnamic acid, coumarin, ferulic acid,
Coumarins salicylic acid, syringic acid, vanillic acid
Indole compounds Indole-3-acetic acid, brassilexin, sinalexin, methyl indole carboxylate,
camalexin glucoside, brassitin
Flavonols Strigolactone, kaempferol, quercitin, genistein, myricetin, naringin, rutin,
naringenin, and their substitutes with sugars
Sugars Galactose, mannitol, fructose, pentose, rhamnose, arabinose, raffinose,
ribose, sucrose, xylose and glucose
Sterols Stigmasterol, campestrol, sitosterol
Allomones Juglone, 5,7,40 -trihydroxy-30 , 50 -dimethoxyflavone, sorgoleone, DIBOA
DIMBOA
Anthocyanins Pelargonidin, delphinidin, cyanidin and their substitutes with sugar
molecules
Organic acids Succinic acid, l-aspartic acid, l-glutamic acid, salicylic acid, oxalic acid,
shikimic acid, chorismic acid, acetic acid, sinapic acid,, isocitric acid, citric
acid, p-hydroxybenzoic acid, tartaric acid, gallic acid, malic acid,
protocatechuic acid, p-coumaric acid, mugineic acid, piscidic acid
Aurones Sinapoyl choline, benzyl aurones synapates
Glucosinolates Cyclobrassinone, desulphoglucoalyssin, desulphoprogoitrin,
desulphonapoleiferin, desulphoguconapin
Fatty acids Palmitic acid, linoleic acid, stearic acid, oleic acid
Root exudates are usually referred to as a group of chemical molecules in

rhizosphere which are secreted by plant root systems. They are a mixture of complex
substances like sugars, organic acids, enzymes, amino acids, etc., which act as major
source of organic carbon specifically obtained from rhizospheric soil (Hütsch et al.
2002; Nguyen 2003). Usually quality and quantity of root exudates depends upon
plant species and is variable in different plants, individual plant’s age, and some
external factors like biotic and abiotic stresses. Knudson (1920) and Lyon and
Wilson (1921) were the first who had provided indication regarding root exudation
and microbe abundance in rhizosphere of the plants. Some of the important exudates
usually found in the rhizosphere have been mentioned in Table 16.1.
16.5 Requirement of Root Exudates in Plant–Microbe

Interactions
Phytochemicals secreted by plant roots mediate certain number of interactions like
• Plant–plant interaction
• Plant–microbe interaction
• Microbe–microbe interaction
Microorganisms live in the rhizospheric soil where they interact with roots and
their components to enhance the plant health (Berendsen et al. 2012; Panke-Buisse
et al. 2015). The interaction might be neutral in some ways and either advantageous
or harmful in others (Mercado-Blanco and Bakker 2007; Raaijmakers et al. 2009).
Most probably, depending on the environment, microbes also turn the table from
pathogenesis to symbiotic association (Newton et al. 2010). In different examples,
Rhizobia includes Bradyrhizobium, Azorhizobium, symbiotic nitrogen, and
nitrogen-fixing bacteria like Sinorhizobium and Mesorhizobium (Davidson and
Robson 1986; Zahran 1999). In nitrogen-limiting conditions, attraction and intima-
tion of legume–rhizobia symbiosis result in secretion of flavones and flavonols by
legumes (Coronado et al. 1995; Zhang et al. 2009). In the same way equal exchange
of plant nutrients benefit both the partners like the mycorrhizal associations which is
a common association found in alomost 80 percent of the plant species (Kiers et al.
2011).
16.6 Effect of Microbe–Microbe Interactions on the Soil

Microbial Communities
For plants, rhizospheric zone is a kind of nutrient-rich site where the competition for
food among microbes always takes place. Secondary metabolites produced by
microbes are released in the environment to overcome other competitors which
fight to occupy similar zone for establishing firmly itself outside or within the
roots (Thomashow and Weller 1988; van Loon and Bakker 2005; Pierson and
Pierson 2010; Kim et al. 2011). The metabolites released in environment consist
of siderophore, lytic enzymes, toxic elements, and antibiotics (Bais et al. 2006).
Some rhizospheric microbes hold a variety of genes for the production of
siderophores and other antibiotics like Bacillus amyloliquefaciens (Chen et al.
2007) and few species of Pseudomonas (Paulsen et al. 2005). Antibiotics like
2,4-diacetylphloroglucinol (DAPG) and oomycin are also products of microbes
(van Loon and Bakker 2005). The referred antibiotics play a significant role in
restraining the pathogenic microbes (Aminov 2009; Pierson and Pierson 2010;
Thomashow and Weller 1988; Kim et al. 2011).
Besides antibiotics, plant secondary metabolites also work toward altering sig-
naling pathway and metabolic activity of plants (Přikryl et al. 1985; Brazelton et al.
2008; Costacurta and Vanderleyden 1995; Kim et al. 2011). These kinds of
352 A. Singh et al.
microbial attributes sometime change the root exudates’ composition, leading to the
selective enhancement of any particular microbial partner in the rhizosphere (Přikryl
et al. 1985; Bulgarelli et al. 2013). The whole scenario of communication between
two bacterial communities results in release of signaling molecules which are
relatively recognized by other communities via inter- and intra-species communica-
tion (An et al. 2014). In bacteria this scenario comprises of biofilm formation,
motility, and cell adhesion (Sperandio et al. 2002; Chu et al. 2011); production of
the virulence-associated factors; and cell proliferation. This kind of density-
dependent stimulus and exchange of signals is referred to as quorum sensing
(Fuqua et al. 1994; Miller and Bassler 2001; Atkinson and Williams 2009; An
et al. 2014) (Yajima 2014).
In fungi, two important molecules namely farnesol and tyrosol have been
reported for regulating quorum sensing–controlled traits like biofilm formation,
resistance to drugs, and morphogenesis (Chen et al. 2007; Enjalbert and Whiteway
2005; Albuquerque and Casadevall 2012). Likewise, tryptophol has been reported to
control morphogenetic behavior in Saccharomyces cerevisiae through both density-
dependent approach as well via nutritional trigger (Chen and Fink 2006).
16.7 Coevolutionary Relationship of Root Exudates

with the Rhizosphere
Microbial communities present in the soil are involved in multilevel intercommuni-

cation which are known to influence vital environmental activities, like biogeochem-
ical cycling of nutrients, soil quality, and plant well-being (Barea et al. 2005; Giri
2005).
The age of the plants, crop species, and types of soil determine the variation in
microbial communities present in the rhizospheric soil (Wieland et al. 2001; Buyer
et al. 2002); (Kowalchuk et al. 2002). In some recent evidences it was observed that
specific plant species cultivate their own soil fungal community and diversity
composition, and this “culture” is mediated by root exudates (Broeckling et al.
2008).
Example – In native soil, when Arabidopsis thaliana and Medicago truncatula
were grown at different places, it was observed that Arabidopsis and Medicago
maintained its own fungal community in their resident soil. When the plants were
grown in other soil different from the native soil that did not promote Arabidopsis or
Medicago plants, the microbial communities in those soils decreased considerably.
Similarly, when root exudates were added to the soil, the same response was
observed, thus showing that plants secrete root exudates to drive these responses
and this interaction has a coevolutionary component.
16.8 Bioactive Metabolites
Plants play a variety of roles either in metabolism or metabolites, which are required
for the sustainability of plant system. These plant metabolites could be made up of
proteins, lipids, carbohydrates, or nucleic acids which are then known as primary
metabolites. Metabolites are primarily known as helping hand for plant system
which directly intervenes in the growth and development (Ballhorn et al. 2009).
The metabolites produced by plants have been broadly categorized into two groups
namely:
16.8.1 Primary Metabolites
Primary metabolites are certain compounds which directly benefitted the plants for
their overall growth. They have been classified as carbohydrates, lipids, proteins,
etc., which are likely used by the plants directly for different works (Schafer and
Wink 2009).
16.8.2 Secondary Metabolites
Plant secondary metabolites are those compounds which do not having any direct
role in plant metabolism and are often useful in respect to defense-related properties.
They are usually low molecular weight around 3000 dalton (Osbourn et al. 2003).
The production and secretion of secondary metabolite varies from species to species
and somehow difference between natural products and secondary metabolites is hard
to define (Vasconsuelo and Boland 2007).
In so many different ways, secondary metabolites are involved in upregulation of
primary metabolism and act as triggers for signaling any known process. Secondary
metabolites often maintain the balance of plant molecules with the environment
either via adaptation mechanism or by making a complementary framework to
intricate fine balance (Osbourn et al. 2003; Berni et al. 2018; Grayson 1998).
16.9 Principal Groups of Secondary Metabolites
Plant secondary metabolites have been majorly categorized into four major classes
(Goldberg 2003). These four categories include terpenoids, nitrogen-containing
compounds, phenolics, and sulfur-containing compounds (GSH, defensins, and
lectins) (Mazid et al. 2011).
354 A. Singh et al.
16.9.1 Terpenes
Terpenoids are the on the whole most varied class of plant secondary metabolites as
they have approximately 40,000 dissimilar compounds, and thus they stand out as
the biggest class of important plant metabolites (Bohlmann and Keeling 2008).
16.9.2 Phenolics
Phenolics are molecules that have an aromatic ring bound with one or more hydroxyl
groups (Nicholson and Hammerschmidt 1992). By the chemical formula and its
structure, it differs from simple phenols like catechol to catechol melanins through a
long chain polymer. Phenolic compounds are reported to guard plants from different
herbivores and pathogens. Apart from protecting plants from above-mentioned
stressors, phenolics also protect plants from UV radiation, heat shock, and frost
situation (Parr and Bolwell 2000).
16.9.3 Alkaloids
Alkaloids are amino acids–derived nitrogen-containing compounds just like tyrosine

and tryptophan. They also present in huge amount but take 20% of total metabolites
(Hegnauer 1988). Alkaloids occupy a major share in drug industry and are being
mainly used as narcotics or in pharmaceuticals (Hesse 2002; Yao et al. 2004). The
most common alkaloids derived from plant sources are vincristine and vinblastine,
morphine, and codeine (Crozier et al. 2006).
16.9.4 Sulfur-Containing Secondary Metabolites
Sulfur-containing metabolites are derived from two different ways; one group is
formed from hydrolyzation of glucosinolates by myrosinase enzyme. Second group
is made up of allin by alliinase enzyme found basically in onion and garlic. Both of
these groups are in nature for a purpose which we always face off with and help in
guarding plants from the herbivores (Ober et al. 2003).
16.10 Role of Rhizospheric Microbiome on Plant Growth

Promotion
Microbial communities are well acclaimed for playing a crucial part in the overall
development and growth of plants by manipulating diverse physiological processes.
The shaping of rhizospheric microbiome is a mutual process which is largely
influenced by the rhizodeposits (Sharma and Chauhan 2017). Recently, people
have started focusing on studying the microbiome associated with host plants in
order to expand sustainable farming customs via the utilization of microbial

biopesticides and biofertilizers. Within a given set of soil type, the indigenous plants
restructure and reframe the native rhizospheric microbial community by applying a
selective pressure. It is exhaustively reported in literature that within a given set of
soil type, the indigenous plants put forth a selective pressure on this immense
biodiversity pool, thereby reshaping the rhizospheric microbial community
structure.
Manipulation of bacterial microbiome has attracted more attention of researchers
in recent times than the other groups of organisms, as it has helped the scientists in
altering numerous plant beneficial activities, namely enhancement in growth and
yield, as well as suppression of phytopathogens with final effect on the usage of
chemical fertilizers which is considerably reduced (Adesemoye and Kloepper 2009).
Microorganisms living belowground are known to affect composition and total yield
of natural plant communities directly and indirectly (Van Der Heijden et al. 2008;
Turner et al. 2013). It is because of this reason that the soil microbial richness has
been directly linked with the diversity and productivity aboveground plant (Lau and
Lennon 2011; Wagg et al. 2011).
16.11 Role of Rhizospheric Microbiome on Plant Secondary

Metabolite Status
The interconnection between plants and their microbial communities is active

practice in which plants interact to their surrounding environment and accordingly
respond to the changes (Chaparro et al. 2012). Microbes play important role in
agriculture in order to maintain environmental equilibrium (Fig. 16.2). Both the
shoot and root systems of plant are directly or indirectly contact with diverse group
of microorganisms. Due to the presence of infinite number of microbes, various
mechanisms occur around the plant root, and one of them is secretion by root
exudate. The root exudation comprises the secretion of carbon-containing
compounds that are primary and secondary metabolites products and many more
molecules (Uren 2000).
Elicitors are chemical compounds for stress factors which when applied in minute
quantity to a living being enhances the biosynthesis of metabolites, mainly second-
ary metabolites (Radman et al. 2003). In context to the plant system, elicitors play
vital role in defense process against pathogens and environmental stress. The biotic
elicitors include bacteria, fungi, and viruses whereas abiotic elicitors involve metal,
ions, and inorganic molecules. Thus, PGPR can produce elicitors which in turn will
originate the synthesis of secondary metabolites (Sekar and Kandavel 2010)
[Table 16.2]. The herbaceous plant Catharanthus roseus, which is commonly called
rose periwinkle, belonging to family Apocynaceae releases bioactive compound
ajmalicine under drought stress (Jaleel et al. 2009). Likewise, in another study
Pseudomonas fluorescens, a plant growth–promoting rhizobacteria was reported to
increase the production of ajmalicine under drought stress. This bacterium also
increased plant biomass and helped in protecting the plants against stress condition.
356 A. Singh et al.
Fig. 16.2 Schematic representation of the role of rhizospheric microbiome on the growth, second-
ary metabolite, and defense status of host plants
C. roseus is also reported to secrete some metabolites like serpentine, catharanthine,

tabersonine, and vindoline but among all of them ajmalicine content was found to be
maximally increased (Jaleel et al. 2009).
The perennial plant Crocus sativus, commonly called saffron crocus, secretes
crocetin, picrocrocin, and safranal compounds. In a study it was found that the
contents were increased when plants were inoculated with Bacillus subtilis FZB24
(Sharaf-Eldin et al. 2008). Among all the compounds, crocetin was found to be
increased maximally. Trichoderma belonging to fungal genera is usually present in
almost all soil types (Hermosa et al. 2012). It has property to kill other harmful
bacteria and fungi that act as biocontrol agent for the plant (Druzhinina et al. 2011).
Trichoderma acts as a biotic elicitor for oleanolic acid which is secreted by Calen-
dula officinalis plant. Oleanolic acid amount is intensified by application of
Trichoderma viride (Wiktorowska et al. 2010).
Scopolia parviflora is a flowering plant belonging to family Solanaceae, which
produces scopolamine compound whose concentration was found to be increased
along with the amount of tropane alkaloids by different microbes such as Bacillus
cereus and Pseudomonas aeruginosa (Jung et al. 2003). Tropane alkaloids concen-
tration is high in roots as compared to stem and leaves. Tropane has cyclic amine
group which has piperidine and pyrrolidine ring with single nitrogen atom and two
carbon atoms (Hanuš et al. 2005). They are used as anesthetics, bronchodilators, and
mydriatics (Grynkiewicz and Gadzikowska 2008).
Apart from PGPRs, endophytes are those bacterial or fungal microbes that live
their entire life with living cells of plant without causing any disease to the host
(Wilson 1995; Sturz et al. 2000). Nowadays endophytes have been considered as an
important source for secondary metabolites which include phenols, alkaloids, and
Table 16.2 Effects of different beneficial microbes on the status of important secondary
metabolites
Secondary
S. No. Plant name metabolite Microbes Reference
1. Medicago Luteolin Rhizobium meliloti Hartwig et al.
sativa (1990)
2. Capsicum Capsidiol Trichoderma viride Brooks et al.
annum (1986)
3. Catharanthus Ajmalicine Trichoderma viride Namdeo et al.
roseus (2002) and
Namdeo (2004)
4. Catharanthus Ajmalicine Pseudomonas Bais et al. (2002)
roseus fluorescens
5. Catharanthus Serpentine Pseudomonas Jaleel et al. (2009)
roseus fluorescens
6. Salvia Tanshinone IIA Trichoderma Ming et al. (2013)
miltiorrhiza atroviride
7. Gymnema Gymnemic acid Saccharomyces Chodisetti et al.
sylvestre cerevisiae (2013)
8. Gymnema Gymnemic acid Bacillus subtilis Chodisetti et al.
sylvestre (2013)
9. Gymnema Gymnemic acid Escherichia coli Chodisetti et al.
sylvestre (2013)
10. Datura metel Atropine Bacillus cereus Shakeran et al.
(2015)
11. Taverniera Glycyrrhizic acid Rhizobium Awad et al. (2014)
cuneifolia leguminosarum
12. Vicia sativa 7,30-Dihydroxy- Rhizobium Zaat et al. (1989)
40-methoxyflavone
13. Pisum Apigenin and Rhizobium Firmin et al.
sativum eriodictyol (1986)
14. Sesbania 7,40- Azorhizobium Messens et al.
rostrata Dihydroxyflavaone (1991)
15. Glycine max Daidzein and Bradyrhizobium Kosslak et al.
genistein japonium (1987) and
Bassam et al.
(1988)
16. Trifolium 7,40- Rhizobium Redmond et al.
repens dihydroxyflavone (1986)
and geraldone
17. Ocimum Rosmaric acid Aspergillus niger Bais et al. (2002)
basilium
18. Glycine max (i) Iturine Bacillus subtilis Ohno et al. (1995)
19. Hyoscyamus (i) Hyoscyamine Pseudomonas putida Ghorbanpour et al.
niger L. (ii) scopolamine and Pseudomonas (2010)
fluorescens
20. Crocus Picrocrocin, Bacillus subtilis Sharaf-Eldin et al.
sativus L. crocetin and (2008)
safranal compounds
.21. Calendula Oleanolic acid Trichoderma viride Wiktorowska
officinalis L. et al. (2010)
358 A. Singh et al.
terpenoids products. For example, hypericin is a bioactive compound which was

isolated from Hypericum perforatum and whose production was increased upon
inoculation of Thielavia subthermophila (Kusari et al. 2008, 2009).
Plumbago rosea L., commonly called Indian leadwort, is classified under
angiosperms. It is used for medicinal purposes like in curing of certain kinds of
chronic diseases, skin diseases, and used as an anticancer plant (Parimala and
Sachdanandam 1993). It releases useful metabolite compound plumbagin from its
root and Aspergillus niger, Rhizopus oryzae, Bacillus subtilis, and Pseudomonas
aeruginosa have been reported to be its elicitor. Among the above-mentioned
genera, fungal elicitors enhanced the content of plumbagin, whereas bacteria
elicitors were not so effective (Komaraiah et al. 2002) The maize crop (Zea mays)
discharges a compound named benzoxazinoid whose amount changed by
rhizobacterium Pseudomonas putida KT2440, which protects the plant from patho-
genic microorganism (Neal et al. 2012). These compounds function naturally toward
the protection of plants. In cell culture roots of Taverniera cuneifolia (shrub),
glycyrrhizic acid content was intensified when treated with bacteria Rhizobium
leguminosarum as compared to the control roots. Other bacterial origin elicitors
observed in Taverniera cuneifolia are B. aminovorans, B. cereus, and
Agrobacterium rhizogenes which were also found to increase the amount of
glycyrrhizic acid. But when it is treated with Agrobacterium tumefaciens, no signifi-
cant increase in glycyrrhizic acid was found. In another plant, namely Hypericum
perforatum compound hypericin and pseudohypericin is released, whose concentra-
tion is reported to be increased by Rhizobacterium (Mañero et al. 2012).
Alfalfa (Medicago sativa) belonging to family Fabaceae is a medicinal plant,
which is a rich source of vitamins A, B, and C (Rashmi and Sarkar 1997). Luteolin is
a bioactive compound released by alfalfa plant whose production is enhanced by
plant growth rhizobacteria Rhizobium meliloti (Peters et al. 1986). Likewise, in
Datura metel, Bacillus cereus and Staphylococcus aureus were found to increase
the content of atropine, a compound largely used for relieving pain (Shakeran et al.
2015).
16.12 Conclusion and Future Prospects
Owing to the presence of diverse variety and multidimensional role of secondary

metabolites, we can assume that these organic compounds are of immense impor-
tance for the growth, development, defense, and survival of plants. Plants preferably
produce these compounds when they encounter herbivores or pathogen attacks. In
totality, these compounds are also produced when plants face challenges like abiotic
stresses, that is, salinity, drought, UV radiations, heavy metals, and harsh climate. In
addition to the above, the biotic elicitors, namely rhizospheric microbes many times
positively change the status of plant secondary metabolites production. Additionally,
being relatively an unexplored area, the rhizospheric microbiome offers a huge
potential for not only manipulating the plant growth but also the secondary metabo-
lite status of plants too.
Therefore, though significance of the microbiome present in the rhizosphere has

been identified way back, but still tremendous efforts needs to be put in to explore
the potential of organisms which might have good properties for our plants and
surrounding environment. Pairing traditional techniques with high-end, next-gener-
ation sequencing techniques for identifying cues, exudates and other molecules will
really help in understanding the complex underground communication existing
between plants and microbes.
References
Adesemoye AO, Kloepper JW (2009) Plant–microbes interactions in enhanced fertilizer-use effi-
ciency. Appl Microbiol Biotechnol 85:1–12
Albuquerque P, Casadevall A (2012) Quorum sensing in fungi–a review. Med Mycol 50:337–345
Ali GS, Norman D, El-Sayed AS (2015) Soluble and volatile metabolites of plant growth-
promoting rhizobacteria (PGPRs): role and practical applications in inhibiting pathogens and
activating induced systemic resistance (ISR). In: Advances in botanical research, vol 75.
Academic Press, pp 241–284
Aminov RI (2009) The role of antibiotics and antibiotic resistance in nature. Environ Microbiol
11:2970–2988
An JH, Goo E, Kim H, Seo YS, Hwang I (2014) Bacterial quorum sensing and metabolic slowing in
a cooperative population. Proc Natl Acad Sci 111:14912–14917
Atkinson S, Williams P (2009) Quorum sensing and social networking in the microbial world. J R
Soc Interface 6:959–978
Awad V, Kuvalekar A, Harsulkar A (2014) Microbial elicitation in root cultures of Taverniera
cuneifolia (Roth) Arn. for elevated glycyrrhizic acid production. Ind Crop Prod 54:13–16
Aznar A, Dellagi A (2015) New insights into the role of siderophores as triggers of plant immunity:
what can we learn from animals. J Exp Bot 66:3001–3010
Babalola OO (2010) Beneficial bacteria of agricultural importance. Biotechnol Lett 32:1559–1570
Badri DV, Weir TL, Van der Lelie D, Vivanco JM (2009) Rhizosphere chemical dialogues: plant–
microbe interactions. Curr Opin Biotechnol 20:642–650
Bais HP, Walker TS, Schweizer HP, Vivanco JM (2002) Root specific elicitation and antimicrobial
activity of rosmarinic acid in hairy root cultures of Ocimum basilicum. Plant Physiol Biochem
40:983–995
Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM (2006) The role of root exudates in rhizosphere
interactions with plants and other organisms. Annu Rev Plant Biol 57:233–266
Ballhorn DJ, Kautz S, Heil M, Hegeman AD (2009) Cyanogenesis of wild lima bean (Phaseolus
lunatus L.) is an efficient direct defence in nature. Plant Signal Behav 4:735–745
Barea JM, Pozo MJ, Azcon R, Azcon-Aguilar C (2005) Microbial co-operation in the rhizosphere. J
Exp Bot 56:1761–1778
Bassam BJ, Djordjevic MA, Redmond JW, Batley M, Rolfe BG (1988) Identification of a nodD-
dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by
soybeans and other legumes. Mol Plant-Microbe Interact 1:161–168
Berendsen RL, Pieterse CM, Bakker PA (2012) The rhizosphere microbiome and plant health.
Trends Plant Sci 17:478–486
Berni R, Cantini C, Romi M, Hausman JF, Guerriero G, Cai G (2018) Agrobiotechnology goes
wild: Ancient local varieties as sources of bioactives. Int J Mol Sci 19:2248
Biswas JC, Ladha JK, Dazzo FB, Yanni YG, Rolfe BG (2000) Rhizobial inoculation influences
seedling vigor and yield of rice. Agron J 92:880–886
Bohlmann J, Keeling CI (2008) Terpenoid biomaterials. Plant J 54:656–669
360 A. Singh et al.
Bonkowski M (2004) Protozoa and plant growth: the microbial loop in soil revisited. New Phytol
162:617–631
Bonkowski M, Villenave C, Griffiths B (2009) Rhizosphere fauna: the functional and structural
diversity of intimate interactions of soil fauna with plant roots. Plant Soil 321:213–233
Brazelton JN, Pfeufer EE, Sweat TA, Gardener BBM, Coenen C (2008) 2, 4-Diacetylphloroglucinol
alters plant root development. Mol Plant-Microbe Interact 21:1349–1358
Broeckling CD, Broz AK, Bergelson J, Manter DK, Vivanco JM (2008) Root exudates regulate soil
fungal community composition and diversity. Appl Environ Microbiol 74:738–744
Brooks CJ, Watson DG, Freer IM (1986) Elicitation of capsidiol accumulation in suspended callus
cultures of Capsicum annuum. Phytochemistry 25:1089–1092
Bulgarelli D, Schlaeppi K, Spaepen S, Van Themaat EVL, Schulze-Lefert P (2013) Structure and
functions of the bacterial microbiota of plants. Annu Rev Plant Biol 64:807–838
Buyer JS, Roberts DP, Russek-Cohen E (2002) Soil and plant effects on microbial community
structure. Can J Microbiol 48:955–964
Chaparro JM, Sheflin AM, Manter DK, Vivanco JM (2012) Manipulating the soil microbiome to
increase soil health and plant fertility. Biol Fertil Soils 48:489–499
Chen H, Fink GR (2006) Feedback control of morphogenesis in fungi by aromatic alcohols. Genes
Dev 20:1150–1161
Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Junge H (2007)
Comparative analysis of the complete genome sequence of the plant growth–promoting bacte-
rium Bacillus amyloliquefaciens FZB42. Nat Biotechnol 25:1007
Chodisetti B, Rao K, Gandi S, Giri A (2013) Improved gymnemic acid production in the suspension
cultures of Gymnema sylvestre through biotic elicitation. Plant Biotechnol Rep 7:519–525
Chu W, Jiang Y, Yongwang L, Zhu W. (2011) Role of the quorum-sensing system in biofilm
formation and virulence of Aeromonas hydrophila. Afr J Microbiol Res 5: 5819–5825
Coronado C, Zuanazzi JS, Sallaud C, Quirion JC, Esnault R, Husson HP, Ratet P (1995) Alfalfa
root flavonoid production is nitrogen regulated. Plant Physiol 108:533–542
Costacurta A, Vanderleyden J (1995) Synthesis of phytohormones by plant-associated bacteria. Crit
Rev Microbiol 21:1–18
Crozier A, Jaganath IB, Clifford MN (2006) In: Crozier A, Clifford MN, Ashihara H (eds) Plant
secondary metabolites: occurrence, structure and role in the human diet. Blackwell Publishing
Limited. ISBN-13: 978-1-4051-2509-3
Davidson IA, Robson MJ (1986) Effect of contrasting patterns of nitrate application on the nitrate
uptake, N2-fixation, nodulation and growth of white clover. Ann Bot 57:331–338
Del Carmen Orozco-Mosqueda M, del Carmen Rocha-Granados M, Glick BR, Santoyo G (2018)
Microbiome engineering to improve biocontrol and plant growth-promoting mechanisms.
Di Benedetto NA, Corbo MR, Campaniello D, Cataldi MP, Bevilacqua A, Sinigaglia M, Flagella
Z. (2017) The role of plant growth promoting bacteria in improving nitrogen use efficiency for
sustainable crop production: a focus on wheat, 3: 413–434
Dobbelaere S, Vanderleyden J, Okon Y (2003) Plant growth-promoting effects of diazotrophs in the
rhizosphere. Crit Rev Plant Sci 22:107–149
Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM, Monte E, Kubicek
CP (2011) Trichoderma: the genomics of opportunistic success. Nat Rev Microbiol 9:749
Duffy B, Keel C, Défago G (2004) Potential role of pathogen signaling in multitrophic plant-
microbe interactions involved in disease protection. Appl Environ Microbiol 70:1836–1842
Engelberth J, Koch T, Schüler G, Bachmann N, Rechtenbach J, Boland W (2001) Ion channel-
forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling. Cross talk
between jasmonate and salicylate signaling in lima bean. Plant Physiol 125:369–377
Enjalbert B, Whiteway M (2005) Release from quorum-sensing molecules triggers hyphal forma-
tion during Candida albicans resumption of growth. Eukaryot Cell 4:1203–1210
Etalo DW, Jeon JS, Raaijmakers JM (2018) Modulation of plant chemistry by beneficial root
microbiota. Nat Prod Rep 35:398–409
Ferguson BJ, Mathesius U (2014) Phytohormone regulation of legume-rhizobia interactions. J

Chem Ecol 40:770–790
Firmin JL, Wilson KE, Rossen L, Johnston AWB (1986) Flavonoid activation of nodulation genes
in Rhizobium reversed by other compounds present in plants. Nature 324:90
Fitzpatrick CR, Copeland J et al (2018) Assembly and ecological function of the root microbiome
across angiosperm plant species. Proc Natl Acad Sci 115:E1157–E1165
Fuqua WC, Winans SC, Greenberg EP (1994) Quorum sensing in bacteria: the LuxR-LuxI family
of cell density-responsive transcriptional regulators. J Bacteriol 176:269
Ghorbanpour M, Hosseini NM, Rezazadeh S, Omidi M, Khavazi K, Etminn A (2010) Hyoscyamine
and scopolamine production of black henbane (Hyoscyamus niger) infected with Pseudomonas
putida and Pseudomonas. fluorescens strains under water deficit stress. Planta Med 76:P167
Giri BF (2005) In: Varma A (ed) Microorganisms in soils: roles in genesis and functions
(pp. 139–153). Springer, Germany
Goldberg G (2003) Plants: diet and health. The report of a British nutrition foundation task force,
vol 347. Blackwell Publishing Limited, Oxford, U.K.
Gopalakrishnan S, Vadlamudi S, Bandikinda P, Sathya A, Vijayabharathi R, Rupela O, Varshney
RK (2014) Evaluation of Streptomyces strains isolated from herbal vermicompost for their plant
growth-promotion traits in rice. Microbiol Res 169:40–48
Gopalakrishnan S, Srinivas V, Alekhya G, Prakash B, Kudapa H, Varshney RK (2015) Evaluation
of Streptomyces sp. obtained from herbal vermicompost for broad spectrum of plant growth-
promoting activities in chickpea. Org Agric 5:123–133
Grayson DH. (1998) Monoterpenoids. Natl Prod Rep. 5:497–521
Grynkiewicz G, Gadzikowska M (2008) Tropane alkaloids as medicinally useful natural products
and their synthetic derivatives as new drugs. Pharmacol Rep 60:439
Hacquard S, Garrido-Oter R, González A, Spaepen S, Ackermann G, Lebeis S, Schulze-Lefert P
(2015) Microbiota and host nutrition across plant and animal kingdoms. Cell Host Microbe
17:603–616
Hanuš LO, Řezanka T, Spížek J, Dembitsky VM (2005) Substances isolated from Mandragora
species. Phytochemistry 66:2408–2417
Hartwig UA, Maxwell CA, Joseph CM, Phillips DA (1990) Chrysoeriol and luteolin released from
alfalfa seeds induce nod genes in Rhizobium meliloti. Plant Physiol 92:116–122
Hegnauer R (1988) Biochemistry, distribution and taxonomic relevance of higher plant alkaloids.
Phytochemistry 27:2423–2427
Hermosa R, Viterbo A, Chet I, Monte E (2012) Plant-beneficial effects of Trichoderma and of its
genes. Microbiology 158:17–25
Hesse M (2002) Alkaloids: Nature’s Curse or Blessing? Wiley- VCH, New York
Hiltner LT (1904) Uber nevere Erfahrungen und Probleme auf dem Gebiet der Boden Bakteriologie
und unter besonderer Beurchsichtigung der Grundungung und Broche. Arbeit Deut Landw Ges
Berlin 98:59–78
Hütsch BW, Augustin J, Merbach W (2002) Plant rhizodeposition—an important source for carbon
turnover in soils. J Plant Nutr Soil Sci 165:397–407
Jaleel CA, Gopi R, Gomathinayagam M, Panneerselvam R (2009) Traditional and non-traditional
plant growth regulators alters phytochemical constituents in Catharanthus roseus. Process
Biochem 44:205–209
Jousset A, Rochat L, Scheu S, Bonkowski M, Keel C (2010) Predator-prey chemical warfare
determines the expression of biocontrol genes by rhizosphere-associated Pseudomonas
fluorescens. Appl Environ Microbiol 76:5263–5268
Jung HY, Kang SM, Kang YM, Kang MJ, Yun DJ, Bahk JD, Choi MS (2003) Enhanced production
of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora.
Enzyme Microb Tech 33:987–990
Kiers ET, Duhamel M, Beesetty Y, Mensah JA, Franken O, Verbruggen E, Palmer TM (2011)
Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis. Science 333:880–882
362 A. Singh et al.
Kim YC, Leveau J, Gardener BBM, Pierson EA, Pierson LS, Ryu CM (2011) The multifactorial
basis for plant health promotion by plant-associated bacteria. Appl Environ Microbiol
77:1548–1555
Kloepper JW, Schroth MN (1987) Plant growth-promoting rhizobacteria on radishes. Proc 4th Int
Conf Plant Path Bact Angers:879–882
Knudson L (1920) The secretion of invertase by plant roots. Am J Bot 7:371–379
Komaraiah P, Amrutha RN, Kishor PK, Ramakrishna SV (2002) Elicitor enhanced production of
plumbagin in suspension cultures of Plumbagorosea L. Enzyme MicrobTechnol 31:634–639
Kosslak RM, Bookland R, Barkei J, Paaren HE, Appelbaum ER (1987) Induction of
Bradyrhizobium japonicum common nod genes by isoflavones isolated from Glycine max.
Proc Natl Acad Sci 84:7428–7432
Kowalchuk GA, Buma DS, de Boer W, Klinkhamer PG, van Veen JA (2002) Effects of above-
ground plant species composition and diversity on the diversity of soil-borne microorganisms.
Antonie Van Leeuwenhoek 81:509
Krome K, Rosenberg K, Dickler C, Kreuzer K, Ludwig-Müller J, Ullrich-Eberius C, Bonkowski M
(2010) Soil bacteria and protozoa affect root branching via effects on the auxin and cytokinin
balance in plants. Plant Soil 328:191–201
Kusari S, Lamshöft M, Zühlke S, Spiteller M (2008) An endophytic fungus from Hypericum
perforatum that produces hypericin. J Nat Prod 71:159–162
Kusari S, Zühlke S, Kosuth J, Cellarova E, Spiteller M (2009) Light-independent metabolomics of
endophytic Thielavia subthermophila provides insight into microbial hypericin biosynthesis. J
Nat Prod 72:1825–1835
Lau JA, Lennon JT (2011) Evolutionary ecology of plant–microbe interactions: soil microbial
structure alters selection on plant traits. New Phytol 192:215–224
Lugtenberg B, Kamilova F (2009) Plant-Growth-Promoting Rhizobacteria. Annu Rev Microbiol
63:541–556
Lyon TL, Wilson JK (1921) Liberation of organic matter by roots of growing plants, vol 40. Cornell
University
Mañero FJG, Algar E, Gómez MS, Sierra MD, Solano BR (2012) Elicitation of secondary
metabolism in Hypericum perforatum by rhizosphere bacteria and derived elicitors in seedlings
and shoot cultures. Pharm Biol 50:1201–1209
Mazid M, Khan TA, Mohammad F (2011) Role of secondary metabolites in defense mechanisms of
plants. Biol Med 3:232–249
Mendes R, Garbeva P, Raaijmakers JM (2013) The rhizosphere microbiome: significance of plant
beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol Rev
37:634–663
Mercado-Blanco J, Bakker PA (2007) Interactions between plants and beneficial Pseudomonas
spp.: exploiting bacterial traits for crop protection. Antonie Van Leeuwenhoek 92:367–389
Messens E, Geelen D, Van Montagu M, Holsters M (1991) 7, 4-Dihydroxyflavanone is the major
Azorhizobium nod gene-inducing factor present in Sesbania rostrata seedling exudate. Mol
Plant-Microbe Interact 4:262–267
Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annu Rev Microbiol 55:165–199
Ming Q, Su C, Zheng C, Jia M, Zhang Q, Zhang H, Qin L (2013) Elicitors from the endophytic
fungus Trichoderma atroviride promote Salvia miltiorrhiza hairy root growth and tanshinone
biosynthesis. J Exp Bot 64:5687–5694
Morán-Diez E, Hermosa R, Ambrosino P, Cardoza RE, Gutiérrez S, Lorito M, Monte E (2009) The
ThPG1 endopolygalacturonase is required for the Trichoderma harzianum–plant beneficial
interaction. Mol Plant-Microbe Interact 22:1021–1103
Morgan JAW, Bending GD, White PJ (2005) Biological costs and benefits to plant–microbe
interactions in the rhizosphere. J Exp Bot 56:1729–1739
Müller MS, Scheu S, Jousset A (2013) Protozoa drive the dynamics of culturable biocontrol
bacterial communities. PLoS One 8:e66200
Müller DB, Vogel C, Bai Y, Vorholt JA (2016) The plant microbiota: systems-level insights and
perspectives. Annu Rev Genet 50:211–234
Namdeo AG (2004) Investigation on pilot scale bioreactor with reference to the synthesis of
bioactive compounds from cell suspension cultures of Catharanthus roseus Linn. Devi Ahilya
Vishwavidyalaya, Indore
Namdeo A, Patil S, Fulzele DP (2002) Influence of fungal elicitors on production of ajmalicine by
cell cultures of Catharanthus roseus. Biotechnol Prog 18:159–116
Neal AL, Ahmad S, Gordon-Weeks R, Ton J (2012) Benzoxazinoids in root exudates of maize
attract Pseudomonas putida to the rhizosphere. PLoS One 7:e35498
Newton AC, Fitt BD, Atkins SD, Walters DR, Daniell TJ (2010) Pathogenesis, parasitism and
mutualism in the trophic space of microbe–plant interactions. Trends Microbiol 18:365–373
Nguyen C (2003) Rhizodeposition of organic C by plants: Mechanisms and controls. Agronomie
23:375–396
Nicholson RL, Hammerschmidt R (1992) Phenolic compounds and their role in disease resistance.
Ning P, Li S, Li X, Li C (2014) New maize hybrids had larger and deeper post-silking root than old
ones. Field Crop Res 166:66–67
Ober D, Harms R, Witte L et al (2003) Molecular evolution by change of function. Alkaloid specific
homospermidine synthase retained all properties of deoxyhypusine synthase except binding the
eIF5A precursor protein. J Biol Chem 278:12805–12812
Ogawa S, Valencia MO, Ishitani M, Selvaraj MG (2014) Root system architecture variation in
response to different NH4+ concentrations and its association with nitrogen-deficient tolerance
traits in rice. Acta Physiol Plant 36:2361–2372
Ohno A, Ano T, Shoda M (1995) Production of a lipopeptide antibiotic, surfactin, by recombinant
Bacillus subtilis in solid state fermentation. Biotechnol Bioeng 47:209–214
Olanrewaju OS, Glick BR, Babalola OO (2017) Mechanisms of action of plant growth promoting
bacteria. World J Microbiol Biotechnol 33:197
Osbourn AE, Qi X, Townsend B, Qin B (2003) Dissecting plant secondary metabolism- constitutive
chemical defences in cereals. New Phytol 159:101–108
Panke-Buisse K, Poole AC, Goodrich JK, Ley RE, Kao-Kniffin J (2015) Selection on soil
microbiomes reveals reproducible impacts on plant function. Microb Ecol J 9:980
Parimala R, Sachdanandam P (1993) Effect of Plumbagin on some glucose metabolising enzymes
studied in rats in experimental hepatoma. Mol Cell Biochem 125:59–63
Parr AJ, Bolwell GP (2000) Phenols in the plant and in man. The potential for possible nutritional
enhancement of the diet by modifying the phenols content or profile. J Sci Food Agric
80:985–1012
Paulsen IT, Press CM, Ravel J, Kobayashi DY, Myers GS, Mavrodi DV, Dodson RJ (2005)
Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat
Biotechnol 23:873
Pérez-Montaño F, Alías-Villegas C, Bellogín RA, Del Cerro P, Espuny MR, Jiménez-Guerrero I,
Cubo T (2014) Plant growth promotion in cereal and leguminous agricultural important plants:
from microorganism capacities to crop production. Microbiol Res 169:325–336
Peters NK, Frost JW, Long SR (1986) A plant flavone, luteolin, induces expression of Rhizobium
meliloti nodulation genes. Science 233:977–980
Pierson LS, Pierson EA (2010) Metabolism and function of phenazines in bacteria: impacts on the
behavior of bacteria in the environment and biotechnological processes. Appl Microbiol
Pinton R, Varanini Z, Nannipieri P (2007) The rhizosphere: biochemistry and organic substances at
the soil-plant interface. CRC press
Přikryl Z, Vančura V, Wurst M (1985) Auxin formation by rhizosphere bacteria as a factor of root
growth. Biol Plant 27:159–163
364 A. Singh et al.
Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Moënne-Locco Y (2009) The rhizo-
sphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms.
Radman R, Saez T, Bucke C, Keshavarz T (2003) Elicitation of plants and microbial cell systems.
Biotechnol Appl Biochem 37:91–102
Rashmi R, Sarkar MV (1997) Cultivation of alfalfa (Medicago sativa L). Anc Sci Life 17:117
Redmond JW, Batley M, Djordjevic MA, Innes RW, Kuempel PL, Rolfe BG (1986) Flavones
induce expression of nodulation genes in Rhizobium. Nature 323:632
Rout ME, Southworth D (2013) The root microbiome influences scales from molecules to
ecosystems: the unseen majority1. Am J Bot 100:1689–1691
Saraf M, Pandya U, Thakkar A (2014) Role of allelochemicals in plant growth promoting
rhizobacteria for biocontrol of phytopathogens. Microbiol Res 169:18–29
Schafer H, Wink M (2009) Medicinally important secondary metabolites in recombinant
microorganisms or plants: progress in alkaloid biosynthesis. Biotechnol J 4:1684–1703
Schmidt JE, Bowles TM, Gaudin A (2016) Using ancient traits to convert soil health into crop yield:
impact of selection on maize root and rhizosphere function. Front Plant Sci 7:373
Sekar S, Kandavel D (2010) Interaction of plant growth promoting rhizobacteria (PGPR) and
endophytes with medicinal plants “New Avenues for Phytochemicals”. J Phytol 91:100
Shakeran Z, Keyhanfar M, Asghari G, Ghanadian M (2015) Improvement of atropine production by
different biotic and abiotic elicitors in hairy root cultures of Datura metel. Turk J Biol
39:111–118
Sharaf-Eldin M, Elkholy S, Fernández JA, Junge H, Cheetham R, Guardiola J, Weathers P (2008)
Bacillus subtilis FZB24® affects flower quantity and quality of saffron (Crocus sativus). Planta
Med 74:1316–1320
Sharma R, Chauhan A (2017) Rhizosphere microbiome and its role in plant growth promotion. In:
Mining of microbial wealth and metagenomics. Springer, pp 29–56
Simonet P, Normand P, Moiroud A, Bardin R (1990) Identification of Frankia strains in nodules by
hybridization of polymerase chain reaction products with strain-specific oligonucleotide probes.
Arch Microbiol 153:235–240
Singh BK, Millard P, Whiteley AS, Murrell JC (2004) Unravelling rhizosphere–microbial
interactions: opportunities and limitations. Trends Microbiol 12:386–393
Soyano T, Hirakawa H, Sato S, Hayashi M, Kawaguchi M (2014) Nodule inception creates a long-
distance negative feedback loop involved in homeostatic regulation of nodule organ production.
Proc Natl Acad Sci 111:14607–14612
Sperandio V, Torres AG, Kaper JB (2002) Quorum sensing Escherichia coli regulators B and C
(QseBC): a novel two-component regulatory system involved in the regulation of flagella and
motility by quorum sensing in E. coli. Mol Microbiol 43:809–821
Sturz AV, Christie BR, Nowak J (2000) Bacterial endophytes: potential role in developing
sustainable systems of crop production. Crit Rev Plant Sci 19:1–30
Sylvia AH, Prévost D (2005) Ecology of plant growth promoting rhizobacteria. In: PGPR:
Biocontrol and Biofertilization. Springer, Dordrecht, pp 1–38
Thomashow LS, Weller DM (1988) Role of a phenazine antibiotic from Pseudomonas fluorescens
in biological control of Gaeumannomyces graminis var. tritici. J Bacteriol 170:3499–3508
Tringe SG, Von Mering C, Kobayashi A, Salamov AA, Chen K, Chang HW, Bork P (2005)
Comparative metagenomics of microbial communities. Science 308:554–557
Turner TR, James EK et al (2013) The plant microbiome. Genome Biol 14:209
Uga Y, Sugimoto K, Ogawa S, Rane J, Ishitani M, Hara N, Inoue H (2013) Control of root system
architecture by deeper rooting 1 increases rice yield under drought conditions. Nat Genet
45:1097
Uren NC (2000) Types, amounts, and possible functions of compounds released into the rhizo-
sphere by soil-grown plants. In: The Rhizosphere. CRC Press, pp 35–56
Van Der Heijden MG, Bardgett RD et al (2008) The unseen majority: soil microbes as drivers of
plant diversity and productivity in terrestrial ecosystems. Ecol Lett 11:296–310
Van Loon LC, Bakker PAHM (2005) Induced systemic resistance as a mechanism of disease
suppression by rhizobacteria. In: PGPR: Biocontrol and Biofertilization. Springer, Dordrecht,
pp 39–66
Vasconsuelo A, Boland R (2007) Molecular aspects of the early stages of elicitation of secondary
metabolites in plants. Plant Sci 172:861–875
Wagg C, Jansa J et al (2011) Belowground biodiversity effects of plant symbionts support
aboveground productivity. Ecol Lett 14:1001–1009
Weston LA, Ryan PR, Watt M (2012) Mechanisms for cellular transport and release of
allelochemicals from plant roots into the rhizosphere. J Exp Bot 63:3445–3454
Wieland G, Neumann R, Backhaus H (2001) Variation of microbial communities in soil, rhizo-
sphere, and rhizoplane in response to crop species, soil type, and crop development. Appl
Environ Microbiol 67:5849–5854
Wiktorowska E, Długosz M, Janiszowska W (2010) Significant enhancement of oleanolic acid
accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L. Enzym
Microb Technol 46:14–20
Wilson D (1995) Endophyte: the evolution of a term, and clarification of its use and definition.
Oikos:274–276
Yajima A (2014) Recent progress in the chemistry and chemical biology of microbial signaling
molecules: quorum-sensing pheromones and microbial hormones. Tetrahedron Lett
55:2773–2780
Yao LH, Jiang YM, Shi J, Tomás-Barberán FA, Datta N, Singanusong R et al (2004) Flavonoids in
food and their health benefits. Plant Foods Hum Nutr 59:113–122
Yu K, Pieterse CM et al. (2019) Beneficial microbes going underground of root immunity. Plant
Cell Environ 42:2860–2870
Zaat SA, Schripsema J, Wijffelman CA, Van Brussel AA, Lugtenberg BJ (1989) Analysis of the
major inducers of the Rhizobium nod A promoter from Vicia sativa root exudate and their
activity with different nodD genes. Plant Mol Biol 13:175–188
Zahir ZA, Abbas SA, Khalid M, Arshad M (2000) Substrate dependent microbially derived plant
hormones for improving growth of maize seedlings. Pak J Biol Sci 3:289–291
Zahran HH (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and
in an arid climate. Microbiol Mol Biol Rev 63:968–989
Zhang J, Subramanian S, Stacey G, Yu O (2009) Flavones and flavonols play distinct critical roles
during nodulation of Medicago truncatula by Sinorhizobium meliloti. Plant J 57:171–183
Microbial Consortia for Plant Disease
Management and Sustainable Productivity 17
Shamarao Jahagirdar, Gurudatta Hegde, P. U. Krishnaraj,
and D. N. Kambrekar
Abstract
Crop diseases take heavy toll on agriculture. The estimated annual loss due to
various diseases ranged from 15% to 20% of the total production. Apart from the
yield losses in commercial productions, the losses in storage and perishables are
equally significant. Out of several management options of disease control in
commercial production (row crops, vegetables, and horticultural), potential use
of microbial consortia as a holistic approach for integrated management option
has been explored. The various aspects of microbial consortia such as develop-
ment of formulation, strain compatibility, mechanism of action, and delivery
methods are discussed in this chapter.
Keywords
Plant growth–promoting fungi · Disease suppression · Plant growth promotion ·

Biological control
Agricultural practices are getting innovative with advancement in science and

technology. Even though revolutionary development has occurred in pesticide
industry with the advent of many new molecules, the risk of pesticide residues,
resistance development, and environmental safety are still major concerns and have
opened the doors for new eco-friendly, sustainable approaches like biological
control and integrated disease management. The use of plant growth–promoting
fungi (PGPF) occupies a small but growing niche in the development of organic
agriculture. Soil is enriched with microscopic life forms including bacteria, fungi,
actinomycetes, protozoa, and algae. The interaction between soil, plants, and
microbes may be beneficial, harmful, or neutral. Plant growth–promoting fungi
S. Jahagirdar (*) · G. Hegde · P. U. Krishnaraj · D. N. Kambrekar

University of Agricultural Sciences, Dharwad, India

https://doi.org/10.1007/978-981-15-6275-4_17
368 S. Jahagirdar et al.
(PGPF) are a class of soil-borne filamentous fungi that have beneficial effects on
plants without causing any disease. Trichoderma, Aspergillus, Penicillium, and
some endophytes have been harnessed as PGPFs in agriculture.
PGPFs produce substances such as plant hormones (e.g., indole-3-acetic acid –
IAA), which help plants to utilize organic matter through mineral solubilization
(N, P, and Fe) and to suppress plant pathogens in the rhizosphere by various
mechanisms, such as the production of hydrolytic enzymes, aggressive
mycoparasitism, competition for saprophytic colonization, and the induction of
plant systemic resistance. Colonization of the root of plants is one of the most
important characteristics of PGPF that helps them to interact with plants to enhance
growth and development apart from protection against phytopathogens
(Hyakumachi 1994).
The presence of native population of microorganisms in rhizosphere soil offers
strong competition to seed-inoculated rhizobia that does not allow the inoculum to
form nodules that results in low or failure of inoculation response (Kumar and
Chandra 2008). The major approaches for biological control of plant diseases have
focused on (i) altering genetic make of rhizosphere components with an aim of
disease suppression operable against more than one pathogens; (ii) favorable
rhizospheric environment for biological control agent and inhibit other competitive
microflora; and (iii) to develop microbial mixtures or consortia with superior bio-
control activity (Janisiewicz 1988). Mixtures of two or more antagonists will
increase the efficacy or decrease the variability associated with biocontrol treatments
(Haggag and Nofal 2006).
A microbial consortium is a group of different species of microbes that act
together as a community. For developing a consortium, one can choose
microorganisms that are resistant to environmental shock, fast acting, synergistically
active, producing natural enzymatic activity, easy to handle, having long shelf life,
good sustainability, nonpathogenic, noncorrosive of consistent quality, and econom-
ical. Combinations of biocontrol strains are expected to result in a higher level of
potential to suppress multiple plant diseases. Commonly, control is based on the use
of single biocontrol agents. This strategy must be changed because from the ecolog-
ical point of view the disease is part of a complex agroecosystem. As opined by
Fravel (2007), a holistic view of this system can help take correct decisions about
management. Therefore, a special approach for improving the PGPR efficiency is the
use of mixtures containing different genera or species that presents additive or
synergistic effects such as nitrogen-fixing bacteria and mycorrhiza helper bacteria
(MHB). Another strategy is to use PGPR, mixed or alternated with fungicides,
integrating biological and chemical methods. The mutual actions between AMF
and Bacillus spp. led to benefits in terms of plant development in papaya infected
with Meloidogyne incognita. In the absence of the pathogen, PGPRs enhance the
mycorrhizal effect in those plants treated with G. mosseae (Vega et al. 2005). There
are several reports indicating the combined influence of fluorescent pseudomonads
and Bacillus spp. along with other BCAs in effective management of crop diseases
(Srinivasan and Mathivanan 2011).
17 Microbial Consortia for Plant Disease Management and Sustainable Productivity 369
Individual application of Rhizobium sp. and PGPRs alone significantly increased

only N while PSB increased N and P uptake in grain and straw significantly over the
uninoculated control. However, their mixed application (i.e., Rhizobium sp. + PSB,
Rhizobium sp. + LK-786 and Rhizobium sp. + LK-884) was comparable to their
inoculation alone in N uptake by grain and N and P uptake by straw. P uptake by
grain was significantly better with Rhizobium sp. + PSB than the Rhizobium sp. and
PSB alone inoculation (Kumar and Chandra 2008).
In recent years, more emphasis has been laid on the combined use of biocontrol
agents with different mechanisms of disease control to achieve more consistent
results introduced biocontrol agents. Attempts were also made to use a consortium
of biocontrol agents that enables persistent suppression of plant pathogens (Chaube
and Sharma 2002). Antagonistic bacteria and fungi isolated from the rhizosphere soil
were evaluated for control of M. phaseolina and S. sclerotiorum infections in
Glycine max. In vitro compatibility of the identified biocontrol agents were exploited
for effective management of soil borne diseases with an objective of evaluating
potential of consortium biocontrol agents in suppression of soil-borne pathogens of
Glycine max as well as in greenhouse studies. Jetiyanon and Kloepper (2002)
discovered that the use of mixtures of PGPR strains with high potential for inducing
systemic resistance against diseases of several different plant hosts in the green-
house. Jetiyanon et al. (2003) confirmed these findings in the field by application of
mixtures of two PGPR strains that more consistently protected several different crop
species against multiple diseases in field tests in Thailand when compared to single
strain. These experiments were conducted under the multi- or intercropping agricul-
tural conditions prevalent in Thai agriculture. During both rainy and dry seasons,
mixtures of B. amyloliquefaciens IN937a and B. subtilis IN937b significantly
protected against all the tested diseases including southern blight of tomato (caused
by Sclerotium rolfsii Sacc.), mosaic viral disease of cucumber (caused by CMV), and
anthracnose of long cayenne pepper (caused by Colletotrichum gloeosporioides
Penz.). Shelf life of the formulations helps in the development of commercial
formulations of biocontrol agents. Formulations should support the viable nature
of the product for the increased period of storage. Biocontrol product should have the
minimum shelf life of 8–12 months for industrialization. Carrier material should not
affect the viable nature of the biocontrol agent. Hence, there is a need for more
concentrated research to enhance the shelf life of the formulation by developing
superior strains that support the increased shelf life, or the organic formulations that
support the maximum shelf life with low level of contaminants in order to make
biocontrol as a commercial venture.
Cook et al. (1996) reported eight species of microorganisms registered by
U.S. Environmental Protection Agency for commercial use against soil-borne
plant pathogens in the United States. These include two fungi (Gliocladium virens
G-21 and Trichoderma harzianum KRL-AG2), three Gram-negative bacteria
(Agrobacterium radiobacter K84, Pseudomonas fluorescens EG1053, and
Burkholderia cepacia type Wisconsin), and three Gram-positive bacteria (Bacillus
subtilis GB03, B. subtilis MBI 600, and Streptomyces griseovir-idis K61). Other
than A. radiobacter K84, all others are used to manage damping-off diseases and
improve stand establishment and seedling vigor. Thirty isolates of bacteria and six
isolates of Trichoderma were isolated from fertile agricultural soil and evaluated for
their antagonistic activity against phytopathogens like Macrophomina phaseolina
and Sclerotinia sclerotiorum, under in vitro conditions. Different isolates showed
varying degrees of antagonism. The three most antagonistic bacteria Pseudomonas
aeruginosa (MBAA1), Bacillus cereus (MBAA2), and Bacillus amyloliquefaciens
(MBAA3) and one fungi Trichoderma citrinoviride (MBAAT) were selected as the
most effective isolates as biocontrol agents. The present study was undertaken to
develop a plant growth–promoting microbial consortium to reduce the disease
incidence in Glycine max both under in vitro and in vivo conditions. Biocontrol
attributes such as ammonia, siderophore, enzymes like β-1,3 glucanase, chitinase,
and cellulase were more in consortia when compared to single isolates. Plants treated
with consortia + pathogen showed lower disease incidence in comparison to single
antagonist + pathogen and pathogen-infested control (p 0.05). Maximum disease
suppression was noticed in potted plants treated with
S. sclerotiorum + MBAA1 + MBAAT showing only 15.8% disease incidence
when compared to Sclerotinia-infested control (97%) incidence. Seed bacterized
with MBAA1 + MBAAT exhibited enhanced seed germination of G. max up to 68%
along with subsequent increase in other plant growth parameters. Considerable
increase in seedling vigor index (1863.2) and chlorophyll content (13.518 mg/g)
was observed in seeds treated with MBAA1 + MBAAT in plants infected with
M. phaseolina (Thakkar and Sharaf 2015).
Diverse mechanisms are involved in the suppression of plant pathogens more
often indirectly connected with plant growth. Plant growth–promoting
microorganisms (PGPM) and biological control agents (BCA) possess secondary
beneficial effects that would increase their usefulness as bioinoculants, regardless of
the need for their primary function. Indeed, PGPMs such as Rhizobium spp. can
promote plant growth and productivity (primary effect) but have now been shown to
also play a role in reducing disease (secondary effect). Conversely, BCAs such as
Trichoderma spp. and Pseudomonas spp. not only suppress the disease (primary
effect) but have recently demonstrated stimulation of plant growth (secondary effect)
either in the presence or absence of a pathogen. Based on these beneficial plant–
microbe interactions, it is possible to develop microbial inoculants for use in
agriculture. Depending on their mode of action and effects, these products can be
used as biofertilizers, plant strengtheners, phytostimulators, and biopesticides. The
use of microorganisms and the exploitation of beneficial plant–microbe interactions
offer promising and environmentally friendly strategies in conventional and organic
agriculture worldwide. However, PGPF-inoculated crops make up only a small
fraction of current agricultural practices due to lack of commercialized and effective
products. For the extensive commercialization of PGPFs in future days, a number of
issues need to be taken care of that include (i) development of efficient strains of
PGPFs with effective biological activities; (ii) the release of genetically engineered
strains to the environment with the assurance of environmental safety; (iii) a better
understanding of the advantages and disadvantages of use of PGPFs; (iv) selection of
PGPF strains that function optimally under all environmental conditions;
(v) development of more efficient means of applying PGPFs to plants in various

conditions; and (vi) a better understanding of the potential interactions between
PGPFs and other soil fungi. Efforts has to be channelized more toward understand-
ing the diversity of PGPFs and use of more suitable strains pertaining to suitable
ecology and agriculture ecosystem rather than a blind recommendation with less
effective impact on plant growth and development.
Modern agriculture is facing new challenges in which ecological and molecular
approaches are being integrated to achieve higher crop yields while minimizing
negative impacts on the environment. In this context, enhancing plant growth and
plant resistance by using beneficial microorganisms is currently considered as an
important key strategy (Pineda et al. 2010). Approximately 300,000 plant species
growing in unexplored area on the earth are host to one or more endophytes (Strobel
and Daisy 2003; Strobel et al. 2004) and the presence of biodiverse endophytes in
huge number plays an important role on ecosystems with greatest biodiversity.
Endophytes provide a broad variety of bioactive secondary metabolites with unique
structure, including alkaloids, benzopyranones, flavonoids, phenolic acids,
quinones, steroids, terpenoids, tetralones, xanthones, and others (Tan and Zou
2001).
Plants are associated with a diverse community of microorganisms. The
microorganisms residing within the plants or endophytes are unique in their
adaptations to specific chemical environment of host plant. Endophytes are
microorganisms (bacteria, fungi, and unicellular eukaryotes) which can live at
least part of their life cycle inter- or intra-cellularly inside of plants usually without
inducing pathogenic symptoms. This can include competent, facultative, obligate,
and opportunistic endophytes. Endophytes can have several functions, and these
may change function during their lifecycle (Murphy et al. 2015).
Biological control is gaining momentum in the management of sunflower necro-
sis virus disease (SNVD) because at present no effective method is available. In
glasshouse experiment-I, six different plant growth–promoting microbes (PGPMs) –
Streptomyces sp. PM5, Trichothecium roseum MML005, Bacillus licheniformis
MML2501, Streptomyces fradiae MML1042, Pseudomonas aeruginosa
MML2212, and Bacillus sp. MML2551 – and 2% Morinda pubescens fruit extract
applied individually (seed + foliar applications) along with sunflower necrosis virus
(SNV) were evaluated in sunflower. Among the treatments, B. licheniformis (Bl),
Bacillus sp. (Bsp), P. aeruginosa (Pa), and S. fradiae (Sf) effectively increased the
plant growth and significantly increased the reduction of virus titre, ranging from
32.5% to 52.5%. In experiment-II, the above four effective PGPMs (Bl, Bsp, Pa, and
Sf) were developed as consortia in all possible combinations in this study and were
applied along with SNV against SNVD. All the consortial treatments significantly
reduced SNVD in virus titre with disease reduction and concomitant increase in
growth promotion when compared to control. In experiment-III, the best PGPM
consortia (PGPMC) were applied as seed + soil inoculations along with SNV to
study the induction of systemic resistance enzymes. The four culture consortium
significantly reduced the SNVD symptoms and virus titer with a concomitant
increase in plant growth promotion and ISR enzymes compared to control. In
experiment-IV, based on biocontrol efficacy and ISR against SNVD from the
experiments I to III, the two more dominant PGPMC treatments were selected and
evaluated against SNVD under field conditions. From these results, Bl + Bsp + Pa + Sf
effectively reduced the SNVD and improved the plant growth and yield parameters
with additional seed yield with income and benefit–cost ratio when compared to
farmer’s practice. In conclusion, PGPM (Bl, Bsp, Pa and Sf) was found to be very
effective against SNVD under glasshouse and field conditions.
17.1 Research Efforts in Management of Crop Diseases by

Exploitation of Microbial Consortia and ITK Measures
Regarding validation of Indigenous Technology Knowledge, UAS, Bengaluru, is the

pioneer research institute in India due to its contributions in plant protection.
Management of soil-borne plant pathogens is very difficult, especially in Fusarium
group which is soil inhabitant that survives in soil for more than 50 years. The
management through chemical method is often uneconomical.
The cow milk, curd, ghee, cow dung, and cow urine have been used individually
used for curing many ailments as described in ancient text. It is know that cow ghee
and curd contain certain living entities and antimicrobial substances. The
Panchagavya is the product of five cow products such as milk, curd, ghee, dung,
and urine. In traditional Hindu families, it is also taken as Panchamratha in little
quantity for purification both external and internal environment of systems. The
innovative research on the use of modified Panchagavya mixture (MPG-3) was
carried out on three soil-borne diseases like Fusarium wilt of tomato and banana,
and also foot rot of black pepper. The traditional Panchagavya was modified by
adding yeast and common salt, and all the three formulations were tested. The
components of three MPG-3 were most effective in managing all these plant
diseases, which included 2 ml of ghee, 5 ml of curds, 5 ml of milk, 40 g dung,
and 48 ml of urine mixed with 2 g yeast and 2 g salt for 100 ml preparation. These
components were mixed by adding one after the other in plastic container and kept
for fermentation for 7 to 10 days with closing of plastic container. The addition of
salt is to reflect Jim Martin’s living water promoting microbial activity which is
further augmented with addition of yeast. The fermented preparation was diluted ten
times with water and filtered through two layers of muslin cloth to obtain clear
filtrate. The filtrate was used in different delivery methods of seedling dip for 30 min
and soil drenching for the pre-infested soil with the pathogen in the investigations.
(i) Management of Panama disease of banana: In case of Panama disease of

banana, MPG-3 was used at 101 dilution along with different bioagents like
Trichoderma viride (0.25%), Pseudomonas fluorescens (1 h dip, 108 cells/ml),
and Bacillus subtilis (1 h dip, 106 cells/ml). The MPG-3 gave better influence
on plant height, number of leaves, maximum root length, and pseudo stem girth
etc. There was reduction in Fusarium population in MPG-3, which provided
encouraging results compared with seedling dip. The population of Fusarium
oxysporum f. sp. cubense declined significantly to 11.8 104 cfu/g after

150 days of planting. These results indicate the promise shown by MPG-3 in
eco-friendly and cost-effective management of Fusarium wilt (Jahagirdar 1995;
Jahagirdar et al. 2000).
(ii) Management of foot rot of black pepper: Developed and standardized
effective IDM package: Soil application of T. viride (75 g/pt) + spraying with
metalaxyl (1.25 g per L) + Akomin (4 ml per L) or MPG 3 (101) for the
management of foot rot of black pepper (Jahagirdar 1995; Jahagirdar et al.
2000).
(iii) Management of damping off of tomato in nursery and main field: The
research work carried out UAS, Bangalore, clearly demonstrated the role of
MPG-3 as PGPR component and ISR activity against Fusarium wilt of tomato
(Padmodaya 1994).
(iv) Management of tobacco mosaic virus through organics: Tobacco Mosaic
Virus (TMV) is the major stumbling block for successful cultivation of bidi
tobacco in Nipani area. Identification of resistant source against such systemic
biotic infection is a challenging task for plant pathologists and plant breeders.
In order to give a boost to ruling cultivators which are susceptible for TMV,
application of Virosin @ 2% (27.7% disease incidence) followed by bougain-
villea leaf extract @ 5% (30.2%) incidence and neem 1500 ppm(31.8%)
incidence. Among ITK measures, Panchaghavya @ 5% (37.7%) followed by
cow urine @ 10% (37.8%) incidence. The untreated check recorded maximum
incidence of 56.5% incidence. There was no significant difference among the
treatments with respect to growth parameters. However, higher plant height,
leaf length, and leaf breadth were recorded in Virosin, neem 1500 ppm, and
cow urine application indicating role-induced systemic resistance. Maximum
cured leaf yield (1206 kg/ha) was recorded in cow urine @ 10% followed by
Virosin @ 2% (1157 kg/ha). Among quality parameters nicotine percentage
ranged from 2.66 to 4.16 with maximum (4.16) in neem leaf extract followed
by 3.77% in buttermilk @ 5%.The reducing sugar ranged from 5.63% to
10.14% with maximum (10.14%) in neem @1500 ppm followed by 9.78% in
cow urine @ 10%. The chloride percentage was within the limit of <1 except
buttermilk (1.07%). Thus, the investigations opened a new window of oppor-
tunity in managing TMV infections through ITK measures enhancing both leaf
yield and quality parameters in bidi tobacco (Jahagirdar et al. 2008).
(v) Management of Asian soybean rust in India: The Asian Soybean rust,
Phakopsora pachyrhizi Syd, is an economically important disease which
causes significant yield loss in India. Lack of resistant cultivars, growing
concern over use of chemical pesticides, and increasing area under organic
soybean cultivation has led to exploitation of Indigenous Technology Knowl-
edge (ITK) in the management of Asian Soybean Rust. The pooled analysis
over 2 years revealed that among the ITK measures application of cow urine @
10% + Pongamia pinnata oil @ 0.5% recorded minimum Percent Disease
Index (PDI) of 37.9 followed by cow urine @ 10% alone (40.3). The chemical
elicitors like MnS04, Muti-k, or plant-based extracts like A. vesica. Pongamia
pinnata oil and bioagent like Trichoderma harzianum along with cow urine are
being used in developing Integrated Disease Management strategies against
Asian soybean rust in India which will help in reducing the chemical pesticides
in long-term sustainable management. The present findings draw on the first
line of research on large-scale application of Indigenous Technology Knowl-
edge in suppressing rust and enhancing both yield and quality parameters
(Jahagirdar et al. 2009).
(vi) Development of bio-intensive integrated disease management strategies
against soybean rust: In Karnataka, area under soybean is increasing and
the crop has attained its multifold dynamism due to its cultivation in both
Kharif and summer seasons. This has mainly affected the epidemiology of rust
in the region. The disease has been observed in most severe form in major
soybean-growing areas in northern Karnataka districts. Till today there are no
promising resistant cultivars against this disease. Under this background devel-
opment of eco-friendly integrated management becomes the key factor for
successful management of the disease in the region.
The results of 2 years’ study (2009 and 2010) on Development of Biointensive

Integrated Disease Management strategies against soybean rust revealed the signifi-
cant superiority of seed treatment with Trichoderma harzianum @ 6 g/kg + spray
with cow urine @10% + T.harzianum @ 0.5% recorded minimum (35.1) Percent
Disease Index (PDI) followed by 37.4, 38.9 PDI in case of spray with cow urine @
10% + potassium phosphonate @ 0.3% and neem oil @ 1%, respectively. However,
minimum PDI was recorded in Hexaconazole @ 1 ml/l (30.5) which is statistically
on par with each other. The maximum incidence of 87.8 PDI was recorded in
untreated check. The maximum seed yield of 18.06q/ha was recorded in
Hexaconazole@0.1% followed by ST with Trichoderma harzianum @6 g/kg + Spray
with Cow urine @ 10% + T. harzianum @ 0.5% (17.15q/ha) which are statistically
on par with each other. The minimum seed yield was recorded in untreated check
(9.06q/ha). The role of biochemical parameters in triggering defense genes and
increasing in seed yield apart from bringing down the disease pressure has been
demonstrated successfully in the outcome of this project. Among the biointensive
strategies, reducing sugars was maximum(0.737%) in seed treatment with
Trichoderma harzianum @ 6 g/kg + spray with cow urine @ 10% + T. harzianum
@ 0.5% followed by 0.707% in case of seed treatment with Pseudomonas
fluorescens @ 10 g/kg + spray with cow urine @ 10%+ Pusedomonas fluorescens
@ 0.5%.The minimum reducing sugars was recorded in untreated control (0.071%).
With respect to nonreducing sugars, seed treatment with Pseudomonas fluorescens
@ 10 g/kg + spray with cow urine @ 10% + Pseudomonas fluorescens @ 0.5%
recorded maximum (10.53%) followed by 9.59% in case of Trichoderma harzianum
@ 6 g/kg + spray with cow urine @10% + T. harzianum @ 0.5%. The minimum
nonreducing sugars percentage was recorded in untreated control (1.22%). We also
studied role of different enzymes in triggering the host defense by use of these ITK
measures by employing Lowry’s Method and Poly Acrylamide Gel Electrophoresis
(PAGE) for assessing the total protein and estimation peroxidase, polyphenol
oxidase, and catalase activity. The peroxidase activity ranged between 25 and
70 Kda. The peroxidase activity was better in all the biointensive treatments,
indicating triggering of host defense genes. The maximum peroxidase activity was
recorded in cow urine @ 10% and neem oil @ 1%. We also studied the expression of
polyphenol oxidase which is the key factor for upregulation of defense genes in
Induced Systemic Resistance (ISR) against rust pathogen. The maximum polyphe-
nol activity was recorded in cow urine @ 10% + neem oil @ 5%, cow urine @ 10%
+ Trichoderma viride @ 0.5%, and neem oil @ 1%. The polyphenol activity ranged
between 55 and 110 Kda. There was no expression of catalase activity in any names
of treatments. This signifies the absence of catalase pathway in ISR against soybean
rust. The studies brought for the first time a new information on salicylic acid–based
pathway in inducing defense in the soybean using ITK measures. This information
will be a key factor in developing ISR elicitors against soybean rust. This helps to
reduce pesticide application by the farmers for managing soybean rust and
minimizes the cost of production and also helps plant growth promotion and ISR
activity against soybean rust. The expression of these defense genes ultimately
helped in realizing maximum seed yield on par with chemical control (Shamarao
Jahagirdar et al. 2013).
Regular collection of samples from rust-infected areas only yielded uredospores
and failed to get telial stage. The identification of telial stage still forms the basis for
understanding epidemiology of the disease in the region.
17.1.1 Technology Adoption and Spread
Thus, seed treatment with Trichoderma harzianum @ 6 g/kg + spray with cow urine
@ 10% + T. harzianum @ 0.5% or cow urine @ 10% + potassium phosphonate @
0.3% or neem oil @ 1% be recommended for management soybean rust in
Karnataka that helps to minimize the use of hexaconazole.
(vii) Endophytes in management of soil-borne diseases of soybean: The antago-

nistic effect of 30 bacterial endophytes of soybean collected from northern
Karnataka and parts of Maharashtra against Sclerotium rolfsii, Rhizoctonia
bataticola and Fusarium oxysporum were assayed in vitro through dual culture
plate technique. The bacterial endophytes RB-KK-6 (40.78%), SB-BS-6
(50.08%), and LB-BU-1 (47.02%) were found effective against S. rolfsii and
the isolates SB-DG-11 (47.41%) and LB-BiN-8 (41.22%) were effective
against R. bataticola. The effective bacterial endophytes against
F. oxysporum were RB-HS-1 (41.99%), SB-BiJ-9 (40.07%), LB-BU-1
(54.20%), and LB-BV-2 (51.64%). Based on molecular characterization, the
effective bacterial endophytes were identified as Acinetobacter sp. (RB-HS-1),
Alcaligenes faecalis (RB-KK-6), Stenotrophomonas sp. (SB-BiJ-9), Bacillus
pumilus (SB-DG-11 & LB-BiN-8), Paenalcaligenes sp. (LB-BU-1), Bacillus
cereus (SB-BS-6), and Brevibacillus sp. (LB-BV-2) (Brunda et al. 2018)
(Fig. 17.1).
376
Fig. 17.1 Diversity analysis studies of endophytes of soybean

S. Jahagirdar et al.
(viii) Eco-friendly approaches in the integrated management of root knot

nematode in bidi tobacco – 2003 to 2007: Preamble: Different types of
tobacco are being cultivated in India under different agro-climatic conditions.
In India, various types of tobacco are being cultivated, and among these the
Nipani area of Belgaum district of Karnataka is known for production of
quality bidi tobacco. In this region, continuous cultivation of tobacco has led
to building up populations of Meloidogyne incognita (Kofoid and White)
Chitwood. The disease has become a constraint for tobacco cultivation in
the area. It infects at any stage of the crop and causes considerable loss in
quality and yield of tobacco. Bidi tobacco is bread and butter of Nipani
farmers of northern Karnataka. Our 4-year research efforts finally gave solid
recommendation as poultry manure (1 t/ha) mixed with Carbofuran 3G (5 kg/
ha) was the most effective, suitable, eco-friendly, and economically viable
strategy for the management practice of root-knot disease of bidi tobacco
which has reduced excess use of carbofuran in the area (Jahagirdar and
Hundekar 2007a, b, 2008).
17.2 Classical Examples of Local Practices Adopted for Plant

Disease Management Are as Follows
• Regulation of shade in the orchard for the management of coffee leaf rust and
blister light of tea.
• Growing of windbreakers like silver oak, casuarina, jackfruit, etc. to avoid sun
scorching of young shoots of plantation crops.
• Tying of areca nut seedlings with coconut and areca nut fronds to protect them
from western sun scorching.
• Lime pasting on areca trees to avoid ill effects due to sun scorching.
• Watering nursery beds in early morning for higher seedling vigor and stand,
particularly followed in chili and brinjal.
• Burning of leaf litter and farm waste to overcome certain soil-borne pathogens in
the seed bed nursery.
• Raised beds, fields, and ridges used to manage some soil-borne pathogens. In
Mexico, raised beds are called as Chinampas or floating garden and were used to
control Pythium, Phytophthora, and other soil-borne pathogens.
• Collection and burning of plant residues and stubbles in the field to tackle the
problem of soil-borne pathogens.
• Flooding with water to overcome the problems of soil-borne pathogens by
creating anaerobic conditions. In our studies, flooding for 85 to 100 days brought
down significantly the Fusarium oxysporum f. sp. cubense population causing
panama disease of banana.
• Earthing up to overcome the problem of pythium damping off in nursery in brinjal
and tomato.
• Kotte tying for areca bunches to overcome problem of koleroga of areca nut.
• Planting across the wind direction helps to manage some airborne diseases.
• Mixed cropping of jowar with tur to prevent the movement of mites which
transmit sterility mosaic of pigeon pea and to minimize tur wilt.
• Manipulation of planting time/sowing to overcome problems of foliar diseases,
for example, Tikka disease of groundnut and anthracnose of chili.
• Ploughing in summer to reduce the problem of nematode infestation and soil-
borne pathogens.
• Crop rotation with legumes, cereals, and millets to overcome the problem of soil-
borne plant pathogens.
• Mulching with green manure to overcome problem of soil-borne pathogens in
paddy.
• Saltwater treatment to overcome the problem of seed-borne diseases, for example,
Bunt and seed gall in wheat.
• Cultivation of covered beans and combination of mulch and beans effectively
prevented bean blight in north Costa Rica by traditional farmers through a system
called tapaga.
• Maize earheads benching in Mexico to manage seed-borne fungal pathogens.
• To tackle problem of stem bleeding in areca nut, tying of paddy thread prepared
out of hay near the crown region of coconut and placement of 1 kg of salt
(Jahagirdar et al. 2003a, b, c).
17.3 Role of Biological Control in Organic Agriculture
Application of bioagents in management of crop diseases is generally termed as

biological control. Many workers have tried to define biological control. The most
commonly accepted definition by Baker and Cook (1974) specified: “reduction of
inoculum density or disease producing activities of a pathogen or parasite in its
active or dormant stage by one or more organisms accomplished naturally or through
manipulation of environment, host or antagonist, or mass introduction of one or
more antagonists.” Further, Cook and Baker simplified biological control as the
reduction of the amount of inoculum or disease-producing activity of a pathogen
accomplished by or through one or more organisms other than man.
Mukhopadhyay (1987) underlined that “Biological control of soil borne plant
pathogens by Trichoderma spp. and other bioagents as a vital area of plant patho-
logical research all over the world these days.” Biological plant protection is an
integral component of ecofriendly management of plant diseases all over the globe.
It is now widely accepted as a distinct possibility for the future and can successfully
be exploited within the frame work of Integrated Pest/Disease Management System.
17.3.1 Why We Are Giving Importance to Biological Control of Plant

Diseases?
Following are some of the points presently supporting management of plant diseases
through bioagents.
1. They avoid environmental pollution of soil, air, and water unlike in chemical
control.
2. They avoid the residual toxicity of crop products unlike in chemical control.
3. They avoid adverse effects on beneficial microorganisms including antagonist in
the soil whereas chemical controls are lethal.
4. They are comparatively less expensive compared to chemical control.
5. There is no development of resistance by the pathogens unlike in chemical
control.
6. Bioagents application is usually once and do not need repeated applications
while chemicals have to be applied at regular intervals.
7. Bioagents are more effective especially for soil-borne diseases whereas
fungicides are generally uneconomical and fail to reach target site.
8. Biological control is the only option to tackle problem of virus diseases in the
absence of host plant resistance.
9. Biological control is risk-free management of plant diseases when compared to
chemical control, that is, phytotoxicity and residue problems.
10. They have become an integral part of modern large-scale agriculture for sus-
tainable productivity.
17.3.2 Advanced Approaches to Biological Control
The four fundamental approaches of biological control are as follows:
(a) Destruction of propagules or biomass of pathogen by hyper-parasitism or

predation.
(b) Prevention of inoculum formation entering into disease-free areas.
(c) Reduction in pathogen virulence by competitive saprophytic ability.
(d) Reduction of vigor of virulence or pathogen by agents such as mycoviruses or
hypo-virulence determinate.
Biological protection agents’ infection is achieved by
(i) Protection of planting material

(ii) Protection of roots with biological seed treatment
(iii) Biological protection of foliage and flowers
(iv) Inoculation of pruning wounds with antagonists
17.3.3 Mechanisms of Biological Control
Antagonism includes antibiosis, competition, and mycoparasitism, and the mecha-

nism of biological control of plant diseases operates through one or both or all of
these together or singly. In addition, the mycorrhizae, plant growth–promoting
rhizobacteria (PGPR), cross-protection, and induced resistance are also operating
during the biological control process. In addition, non-pathogenic strains/avirulent
strains, mycoviruses, and hypovirulences are being used in suppressing the plant
pathogens either through ISR/SAR approaches, HR reaction, or RNAi-mediated
resistance.
Antibiosis Antibiosis is the inhibition of pathogen by the metabolic (antibiotic)

product or products of the antagonist. The antagonist releases antibiotics or other
metabolic products (enzymes), which are harmful to the pathogen and inhibit its
growth.
Competition It is the endeavor of two or more microorganisms to gain the measure

each wants from supply of substrate in the specific form and under specific
conditions in which that substrate supply is not sufficient for both. In essence the
competition is for nutrients (high energy carbohydrates and nitrogen), and also for
space and oxygen, but not for temperature, PH, and water potential. The antagonists
grow very fast and utilize all the food and occupy the space, and thus make the
pathogen weak. Heterotrophic rhizobacteria like fluorescent pseudomonas compete
for iron with plant pathogens, very efficiently use the iron, and produce siderophores
(microbial iron transport agents), which complex the iron and thus affect them
adversely.
Mycoparasitism and Predation Mycoparasitism (¼ Hyperparasitism) is defined

as parasitism of one fungus by another. Several necrotrophic mycoparasites have
potential biocontrol agents. The mechanism of mycoparasitism includes different
kinds of interactions like coiling of hyphae around the pathogen, penetration,
production of haustoria, and lyses of hyphae. Recently, it is also postulated that
necrotrophic parasites kill susceptible fungi by the action of toxins, antibiotics and or
enzymes.
Induced Systemic Resistance In the later part of the 1990s, the research on plant
growth–promoting rhizobacteria (PGPR) and induced systemic resistance (ISR)
clearly gave a hint on the role of useful microbes in imparting resistance to plants
for specific group of pathogen. Free-living root-colonizing bacteria (rhizobacteria)
have been studied for the past century as possible inoculants for increasing plant
productivity and controlling microbial pathogens. Soil or seed applications with
PGPR have been used to enhance the growth of several crops (Glick 1995), as well
as to suppress the growth of plant pathogens. PGPR that colonize root systems
through seed applications and protect plants from foliar diseases include Pseudomo-
nas spp., Bacillus spp., Paenibacillus spp., and Serratia sp. The mechanisms for
plant growth promotion and induced systemic resistance (ISR) by PGPR have been
extensively studied in the past decade. There are several determinants for
mechanisms of growth promotion that include bacterial synthesis of the plant
hormones (indole-3-acetic acid (IAA), cytokinin, and gibberellins), breakdown of
plant-produced ethylene by bacterial production of 1-aminocyclopropane-1-carbox-
ylate (ACC) deaminase, and increased mineral and N availability in the soil.
Recently, the phenomenon that PGPR elicit plant defense has also been found to
lead to a state of ISR in the treated plant. ISR occurs when the plant’s defense
mechanisms are stimulated and primed to resist infection by pathogens ISR is
different from systemic acquired resistance (SAR) that triggers systemically plant
defense response following hypersensitive response after inoculation of plant
pathogens. Previous works demonstrated that several bacterial determinants such
as siderophores, salicylic acid (SA), and lipopolysaccharides (LPS) contributed to
ISR (Choong-Min Ryu et al. 2005).
17.3.4 Role of Biological Control in the Integrated Disease

Management (IDM)
Biocontrol agents form an effective component in the integrated management of

diseases. Under high disease pressure or pathogen population pressure, the biocon-
trol may be less effective and needs other practices also to completely manage the
disease. Jahagirdar et al. (2001) reported the effectiveness of seed treatment with
biocontrol agent if integrated with other management practices, that is, the use of
moderately resistant cultivar + biocontrol + FYM helped in managing the disease.
The effectiveness of biocontrol agents in other crops against soil-borne pathogens
are presented in table for reference.
Success of Biological Agent Depends on the Following

1. Selection of virulent strain of antagonist.
2. Fast growing and highly sporulating on the mass culture media in case of
facultative before applying.
3. Preventive application to provide enough time for interaction between antagonist
and target pathogen.
4. Optimum soil temperature, moisture for establishment, and growth of antagonist.
5. For survival and multiplication of the antagonist sufficient organic matter (food
base) should be present in the soil.
6. Regular assessment of population of the target pathogen and antagonist from time
to time on selective media.
7. Ease in handling, production, storage of product and should be cheap and
available.
8. Integrated strategy of biocontrol with tolerant/resistant varieties rather than
chemical control alone.
Future Line of Research in Respect of Biological Control

1. Isolation and evaluation of native antagonists, their multiplication, and prepara-
tion of seed treatment formulations.
2. Mapping up of population dynamics of pathogen and biocontrol agents in various
geographical areas of production systems.
3. Application biotechnological techniques, genetic engineering, protoplast fusion
technique, etc., may be employed in developing efficient strains of antagonists
which are site specific, area specific, and crop specific would further pave way for
faster developments in biological control.
Inference
Scientists dealing with management of crop diseases are beginning to evince keen
interest in indigenous technology knowledge. Recently this knowledge is being
made available, and research endeavors are reoriented toward validation of indige-
nous methods encouraging Integrated Disease Management (IDM) practices. Identi-
fication of ideal bioagent for its per se performance under different regions in the
backdrop of climate would surely lay the foundation for effective management of
diseases without affecting natural ecosystem adversely.
References
Baker K F and Cook R J (1974) (original ed.) Biological Control of Plant Pathogens. W H Freeman.
San Francisco. Reprinted ed. 1982. Am. Phytopathological Society. St Paul MN 433pp
Brunda KS, Jahagirdar S, Kambrekar DN (2018) Antagonistic activity of bacterial endophytes
against major soil borne pathogens of soybean. J Expt Zool 6:43–46
Chaube HS, Sharma J (2002) Integration and interaction of solarization and fungal and bacterial
bioagents on disease incidence and plant growth response of some horticultural crops. Plant Dis
Res 17:201
Choong-Min Ryu MA, Paul WP, Kloepper JW (2005) Invisible signals from the underground:
bacterial volatiles elicit plant growth promotion and induce systemic resistance. Plant Pathol J
21:7–12
Cook RJ, Brucart WL, Coulson JR, Goettel MS, Humber RA, Lumsden RD, Maddox JV,
McManus ML, Moore L, Meyer SF, Quimby PC, Stack JP, Vaughn JL (1996) Safety of
microorganisms intended for pest and plant disease control: framework for scientific evaluation.
Biol Control 7:333–351
Fravel D (2007) Commercialization of biocontrol agents for use against plant pathogens. In: IX
Reuniao Brasileira sobre Controle Biológico de Doenças de Plantas, Campinas, S. Paulo, Brasil,
CD-ROM, pp 1–2
Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol
41:109–117
Haggag WM, Nofal MA (2006) Improving biological control of Botryodiplodia disease in some
Annona cultivars by combining biological agents in Egypt. Biol Control 38:341–349
Hyakumachi M (1994) Plant-growth-promoting fungi from turf grass rhizosphere with potential for
disease suppression. Soil Microorganisms 44:53–68
Jahagirdar G (1995) Studies on panama disease of banana caused by fusariutn oxysporum f. sp.
cubense with special reference to biological control. M.Sc. (Agri) thesis, University of Agricul-
tural Science, Bangalore, p. 82
Jahagirdar S, Hundekar AR. (2007a). Ecofriendly strategies in the integrated management of root
knot nematode of bidi tobacco P.181 182. In: Proceedings of Third Asian Conference on Plant
Pathology held at Gadjah Mada University, Indonesia from 20–24, August, 2007, 344pp
Jahagirdar S, Hundekar AR (2007b) Eco-friendly strategies in the integrated management of root
knot nematode in bidi tobacco-2003 to 2004, (Part I). Indian J Plant Proctn 35:329–330
Jahagirdar S, Hundekar AR (2008) Eco-friendly strategies in the integrated management of root-
knot nematode of bidi tobacco, 2003–2006. Plant Disease Management Reports Vol 2 (APS,
Online Journal)
Jahagirdar S, Siddaramaih AL, Chandrappa HM (2000) Eco friendly integrated management of foot
rot of black pepper (Piper nigrum L.). Mysore J Agric Sci 34:47–54
Jahagirdar S, Patil MS, Indira S (2001) Biological control of charcoal rot of sorghum caused by
Macrophomina phaseolina. Agril Sci Dig 21:153–156
Jahagirdar S, Biradar BD, Rao TG (2003a) Application of Pseudomonas fluorescens as low cost
eco-friendly technology for management of charcoal rot of rabi sorghum caused by
Macrophomina phaseolina in Northern Karnataka. Paper presented in 6th international PGPR
workshop held at IISR, Calicut from 5–10, Oct. 2003, 634–637pp
Jahagirdar S, Kulkarni S, Nageshwar TG (2003b) Present status and future research needs on the
management of charcoal rot of sorghum. P.15 In National symposium on Recent Advances in
the Diagnosis and management of Plant Diseases for meeting Global challenges held at Dept. of
Plant Pathology, UAS, Dharwad from 18–20, December, 2003, 118pp
Jahagirdar S, Ravikumar MR, Siddaramaiah AL (2003c) Traditional methods in management of
plant diseases. Agric Rev 24:142–146
Jahagirdar S, Kajjidoni ST, Matiwade PS, Devappa V (2008) Management of tobacco mosaic virus
in bidi tobacco in Karnataka through organics. Proceedings of National symposium on Plant
Protection held at UAS, Bangalore from 4–6, December, 2008
Jahagirdar S, Patil PV, Basavaraja GT (2009) Role of Indigenous technology Knowledge in the
management of Asian Soybean rust caused by Phakopsora pachyrhizi in India. In the
proceedings of World Soybean Research Conference VIII held at Beijing, China from 10–15,
August, 2009
Jahagirdar S, Khedekar S, Bhat R, Basavaraja GT, Bhat Y (2013) Biointensive management of
Asian soybean rust caused by Phakopsora pachyrhizi Syd. Res Crops 14:182–188
Janisiewicz WJ (1988) Biocontrol of postharvest diseases of apples with antagonist mixtures. Am
Phytopathol Soc. https://doi.org/10.1094/Phyto-78-194
Jetiyanon K, Kloepper JW (2002) Mixtures of plant growth-promoting rhizobacteria for induction
of systemic resistance against multiple plant diseases. Biol Control 24:285–291
Jetiyanon K, Fowler WD, Kloepper JW (2003) Broad-spectrum protection against several
pathogens by PGPR mixtures under field conditions in Thailand. Plant Dis 87:1390–1394
Kumar R, Chandra R (2008) Influence of PGPR and PSB on Rhizobium leguminosarum pv. viciae
strain competition and symbiotic performance in lentil. World J Agric Sci 4:297–301
Mukhopadhyay AN (1987) Biological control of soil borne plant pathogens by Trihoderma spp.
Indian J Mycol Plant Pathol 17:1–10
Murphy BR, Doohan FM, Hodkinson TR (2015) Fungal root endophytes of a wild barley species
increase yield in a nutrient-stressed barley cultivar. Symbiosis 65:1–7
Padmodaya B (1994) Integrated management of Fusarium wilt of tomato, Ph. D thesis submitted to
Dept. of Plant Pathology. UAS, Bangalore
Pineda A, Zheng S-J, van Loon JJA, Pieterse CMJ, Dicke M (2010) Helping plants to deal with
insects: the role of beneficial soil-borne microbes. Trends Plant Sci 15:507–514
Srinivasan K, Mathivanan N (2011) Plant growth promoting microbial consortia mediated classical
biocontrol of sunflower necrosis virus disease. J Biopest 4:65–72
Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products.
Microbiol Mol Biol Rev 67:491–502
Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorganisms.
J Nat Prod 67:257–268
Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep
18:448–459
Thakkar A, Saraf M (2015) Development of microbial consortia as a biocontrol agent for effective
management of fungal diseases in Glycine max L. Arch Phytopathol Plant Protect 48:459–474
Vega FE, Pava-Ripoll M, Posada F, Buyer JS (2005) Endophytic bacteria in coffea arabica L. J
Basic Microbiol 45:371–380
Microbial Biofilm: Formation, Quorum
Sensing, and Its Applications in Plant 18
Disease Management
Pravallikasree Rayanoothala, M. Divya, Sunita Mahapatra,

and Srikanta Das
Abstract
In search of an eco-friendly plant disease management, a ray of hope for

sustainability was created after the recognition of microbial strategy on plant
surfaces was adapted under adverse environmental conditions in the early 1970s,
which are the microbial aggregations termed as biofilms. The assemblage of
microbes on plant surfaces are formed due to microbial adhesion, growth, and
expansion process, which in turn depends on surface tension, texture, and wetta-
bility. The microbial cells in biofilm communicates by various signaling
molecules in order to modulate their functional mechanism by controlled release
of antibiotic and toxins and in regulation of gene expression through quorum
sensing. The microbes that are capable of forming biofilms include various
bacteria, yeast, fungi, and symbionts which are not only antagonistic to
phytopathogens but also help in enhancement of plant growth and development
by acting as a sink for nutrients as a function of site of colonization of plant parts.
Keywords
Biofilm · PGPR · Microbes · Quorum sensing · Biocontrol · Plant diseases
18.1 Introduction
Plants come across a number of pests and diseases, a scenario which can be
compared to a battleground in which plants struggle against pathogens for their
survival. In several strategies adopted to combat phytopathogens, using of synthetic
chemicals became quite common. As most of them cause environmental threat due
to their broad-spectrum activity, it is one of the major ecological challenges for plant
P. Rayanoothala · M. Divya · S. Mahapatra (*) · S. Das

Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, Nadia, West Bengal, India

https://doi.org/10.1007/978-981-15-6275-4_18
386 P. Rayanoothala et al.
pathologists as well as microbiologists in near future. Therefore, there is an urge to

replace such environmental harmful chemicals with environment-friendly
substitutes like biocontrol agents. These beneficial microorganisms are involved in
plant disease control, which is considered as one of the most promising methods for
rational, ecological, and eco-friendly crop management practices and also ensures
reproducible performance in natural environments. When the combination of differ-
ent microorganisms with different modes of action are imposed in both in vitro and
in vivo, the efficacy of different biocontrol agents can be enhanced such that every
component can be individually colonized without posing negative effect on devel-
opment of the others. Therefore, mono strain of a biocontrol agent having several
mechanisms to reduce disease incidence is a prerequisite in adopting any biocontrol
agent against agricultural pathogens. Despite all the research work on the biological
control of oomycetic soil-borne plant diseases, there are still no commercially
thriving examples against such oomycetic diseases. There are several reasons for
the lack of adoption of biocontrol management by the growers, which may be due to
insufficient knowledge on the mechanism of pathogenesis and mode of action of
biocontrol agents and plant pathogens. Biocontrol microorganisms do not generally
perform well in uncontrolled conditions in soil to compete with chemical fungicides
so, there is a need to understand the mechanism of biocontrol agents. Considering
safety issues associated with the use of biocontrol agents, four possible adverse
effects are generally identified such as dislocation of nontarget organisms, allergenic
to animals and humans, toxicity and pathogenicity, and genetic stability (Cook et al.
1995). Except for allergenic activity, all the factors that enhance inoculant survival
and efficacy also increase their reputed adverse effect, therefore a minimal potential
biohazard is inherent to any employment of biocontrol agent (Migheli 2001).
Furtherance, this chapter emphasizes the need of thorough risk evaluation for the
safe use of any novel biocontrol agent.
Recently, the capacity to form biofilms on plant parts was recognized as a
possible mechanism of biocontrol management (Scherm et al. 2001; Ortu et al.
2005). Generally, microorganisms exist in multicellular aggregates in natural envi-
ronment which is usually described as biofilms in which the individual cells have
intimate contact with other cells. Its formation is a major bacterial adaptive strategy
to environment in aquatic and also on other solid surfaces. Cells adhere to each
other’s surface through a complex matrix medium comprising a variety of extracel-
lular polymeric substances (EPS) including exopolysaccharides, proteins, and DNA.
The recognition of aggregated microbes surrounded by EPS adhering to the
surfaces or located in tissues is not new to human beings, but a well-known
phenomenon since Leeuwenhoek and Pasteur described it in environmental and
technical microbiology. The concept of biofilms, however, was initiated in the
early 1970s due to the observation of heaps of Pseudomonas aeruginosa in lung
tissue and sputum in cystic fibrosis–infected patients. The term “biofilm” was
introduced into medical industry by J. W. Costerton in Costerton 1995. This chapter
emphasizes the importance of biofilm formation in plant disease control. Microbes
have many benefits of biofilm, for example, it acts as a sink for the nutrients in the
18 Microbial Biofilm: Formation, Quorum Sensing, and Its Applications in Plant. . . 387
rhizosphere, aids bacteria to survive under unfavorable conditions, etc. Besides that,
it helps in exchanging the genetic material.
18.2 Biofilm: Definition and Concept
Biofilms have great realistic importance in agricultural, medical, and industrial

sectors and its formation plays a predominant role in microbial lifestyle. Biofilm is
defined as “Highly structured and surface attached closed communities of the cells
enclosed within a self-produced extracellular polymeric matrix substance” (Branda
et al. 2005). Bacterial cells produce a mixture of extracellular polymeric substances
(EPS) as well as different exopolysaccharides, DNA, and proteins while attached on
the surface, and they have distinct physiological construction cells within it that vary
from each other, and up- and downregulation of genes also may vary from cell to cell
(Ramey et al. 2004). A biocontrol bacterium can affect plant growth by various
mechanisms (Glick et al. 1999; Timmusk and Wagner 1999; Timmusk et al. 2005;
Perneel et al. 2007; Rezzonico et al. 2007; Tran et al. 2007). Plant root exudates and
root electrical signals selectively influence bacterial colonization and biofilm forma-
tion (West et al. 2002; Bergsma-Vlami et al. 2005; Kiely 2006; Van Loon 2007). The
colonization rarely occurs as individual cells. Complex multicellular communities
such as biofilms and fruiting bodies are commonly coexisting forms in nature
(Davey and O’Toole 2000; Palkov’a and V’achov’a 2006; Ngo Thi and Naumann
2007). Biofilms are formed due to cellular recognition of specific or nonspecific
attachment sites on the surface, nutritional signal, or by exposure of planktonic cells
to sub-inhibitory concentrations of antibiotics (Watrick and Kolter 2000).
The microbial biofilms were protected by EPS surrounding them as they consist
of carbohydrates, nucleic acid, proteins, and various other substances and also have
structural and functional role for biofilm communities under diverse conditions. As
extracellular polymeric matrix substance acts as an anion exchanger, there is a
massive restriction of entry for foreign microbes and other compounds into the
biofilm. It also protects the bacteria from various environmental stresses like desic-
cation, osmotic shock, pH change, and UV radiation and acts as a sequester for metal
ions and different toxins. Microbes in biofilm formation work in a syntrophic
manner, and because of multispecies approach of biofilm lead to the effective
nutrient ability by syntrophism and anaerobic degradation of compounds (Rafique
et al. 2015).
In order to cope up with altering environmental circumstances, microbes in the
biofilm carry out exchange of genetic material. This results in genetic diversity in
which microbial communities obtain new genetic material, followed by transcribing
it to genes. Biofilm has the potential to develop a barrier of EPS against antimicrobial
diffusion molecules (Rafique et al. 2015).
The mechanism initially reported by Thomashow’s group (Weller and
Thomashow 1994) has gained less attention due to difficulties in studying natural
systems. However, biofilms could have the potential find for combating against
under natural conditions.
18.2.1 Identification of Biofilm
Biofilm configurations may range in complexity from flat, relatively featureless films
to tightly clustered aggregates to complex heterogeneous cellular arrangements such
as towers and streamers. The biofilm forming cells responds to waste and nutrient
product diffusion gradients and modulate their metabolism which depends on their
site of colonization within the biofilm and thus engage in cell–cell communication
(Ramey-Hartung et al. 2005).
The biopolymers in biofilm were purified with several precipitation steps using
ethanol and cetyltrimethylammonium bromide and the carbohydrates were analyzed
using various color reactions, infrared spectroscopy, and high performance liquid
chromatography which revealed that the biopolymer is a homo polysaccharide and it
consists of various sugars such as glucose, galactose, mannose, and xylose, and such
secretion is important in P. polymyxa biofilm development (Haggag 2007).
Infrared microspectroscopy assays were used for the characterization and detec-
tion of antibiotics in addition to the development of major biofilm-forming
metabolites. Furthermore, atomic force microscopy assays are being used for under-
standing of physical properties and persistence of biofilm on solid surfaces which are
important for agricultural applications (Fig. 18.1) (Haggag 2007).
18.2.1.1 Sites of Biofilm Formation on Plants

The activity and formation of biofilm mainly depends on the microenvironment of
the plant parts which differs in saturation levels, nutrient availability, and surface
chemistries (Table 18.1).
The gram-positive bacteria – Bacillus spp., Listeria monocytogenes, Staphylo-
coccus spp., lactic acid bacteria, and gram-negative bacteria – E. coli and
P. aeruginosa also forms biofilms. Some nitrogen-fixing symbionts form biofilms
on legume roots and other inert surfaces, for example R. leguminosarum and
Sinorhizobium meliloti.
18.2.1.2 Agents and Factors Involved in the Formation of Biofilms

Microbial biofilm formation and its growth are impacted by several aspects such as
nature of fabricated material, texture, surface tension, and wettability (Rafique et al.
2015).
Rough surfaces offer more chances for the attachment of microbe to form biofilm
and also influenced by the nutrients release and exudation at different sites and
hydration levels on various sites of colonization. Also, it was observed that in moist
and nutrient-rich sites the bacteria can grow into aggregates and biofilms (Rafique
et al. 2015).
One of the agents involved in biofilm formation are certain proteins present on
outer membrane on the surface. In recent reports, a cell surface protein called Lap A
Table 18.1 Site of colonization of biofilms

Site of
S. No colonization Bacteria
1) Aerial tissue Erwinia amylovora (Monier and Lindow 2003), E. chrysanthemi,
P. fluorescens, P. syringae pv. syringae (Rojas et al. 2002)
2) Vascular Clavibacter michiganensis subsp. sepedonicus (Marques et al. 2003),
tissues Pantoea stewartii subsp. stewartii (Leigh and Coplin 1992), Ralstonia
solanacearum, Spiroplasma spp. (Kang et al. 2002), Xanthomonas
campestris pv. campestris, Xylella fastidiosa (Purcell and Hopkins
1996; Newman et al. 2003, 2004)
3) Roots Agrobacterium tumefaciens (Gage 2004), Azospirillum brasilense
(Burdman et al. 2000), Bacillus cereus, P. aeruginosa, P. fluorescens,
P. putida, Rhizobium spp., R. leguminosarum, Biovar trifolii
(Espinosa-Urgel et al. 2002; Williams et al. 2008)
4) Seeds and Escherichia coli (Fett and Cooke 2003), P. fluorescens, P. putida,
sprouts Salmonella (Espinosa-Urgel et al. 2002)
(Large adhesion protein A) of 900-kDa is identified to affect colonization. Similarly,

in P. putida, homologues to Lap A, KT2440 are reported to involve in the seed
adhesion and competitive root colonization (Rafique et al. 2015).
In A. tumefaciens transcription factor SinR was reported to be a regulator in
biofilm formation which is an oxygen-responsive regulator of fumarate and nitrate
reductase (FNR) family of proteins (Rafique et al. 2015).
18.2.2 Stages of Biofilm Formation
Microbial biofilm formation includes five different stages (Monroe 2007) during
12-day incubation period. Spectroscopic and microscopic equipment can be used for
the identification of different developing stages of bacterial biofilms (Fig. 18.2). The
biofilm-forming microbes follow the principle of Brownian motion, which attaches
to the surface and readily removes from the surface which follows mild rinsing
(Rafique et al. 2015).
In biofilm formation, initiation stage is slow and persists for short period of time,
which is induced by environmental signals. This stage is reversible, and there is a
chance of detachment of cells. Their individuals exhibit logarithmic growth rate
followed by irreversible growth stage. The biofilm formation cells induce chemical
signals to communicate with adjacent cells. Motility is decreased, cell aggregates are
formed, and cell aggregates become progressively layered (Monroe 2007). The
genetic mechanism for EPS production is activated that can able to trap nutrients
and planktonic bacteria. In maturation stage, cells are progressively layered and
attain a thickness of >10 μm. Followed by dispersion happens either by shedding of
daughter cells from actively growing cells and depletion of nutrient levels or quorum
sensing or by detachment of biofilm aggregates by physical forces (Monroe 2007).
Fig. 18.1 Biofilm analysis
18.2.2.1 Composition of a Biofilm

Biofilm is composed of many substances of which the function of
exopolysaccharides, proteins, extracellular DNA (e-DNA), lipid components of
biofilm matrix were widely studied.
Fig. 18.2 Flow chart

representing five stages of Initial attachment
biofilm formation
Irreversible attachment
Maturation I
Maturation II
Dispersion
Though the function and chemical composition of each exopolysaccharides

varies with different species, most of them are polyanionic molecules due to
presence of uronic acids and sugar having substituents such as pyruvate, sulfate,
or phosphate. EPS regulates the carbon source (Amellal et al. 1998) and stabilizes
the biofilm structure by enhancing the water retention in microbial environment
(Bogino et al. 2013).
Proteins are major constituents in biofilm matrix as they perform as extracellular
enzymes and they have a role in degradation and recycling of biopolymers as well;
thus they enable the nutrients to retain in the matrix. Proteins help in shaping and
dispersion of cells from the biofilm structure by modifying other exopolymers. Some
of the proteins in the biofilm matrix have structural functions, for example, lectins
that bind bacterial cells to polymeric matrix (Bogino et al. 2013). Glucan-binding
proteins in Streptococcus mutans, LecA and LecB in P. aeruginosa, Lectins in
A. brasilense in P. aeruginosa, a large quantity of matrix proteins was found in
outer membrane vesicles, a typical biofilm component in this species. Amyloids are
another type of matrix protein which is common with extracellular adhesin function
(Bogino et al. 2013).
Extracellular DNA (e-DNA) plays an important role for exchange of genetic
material as a part of evolution process. In some gram-positive bacteria, e-DNA is
found to be involved in adhesion to hydrophobic surfaces (Bogino et al. 2013).
In biofilms, generally lipids act as biosurfactants with some functions such as
surface activity, dispersal, and bioavailability of hydrophobic substances,
antibacterial or antifungal properties, and bacterial attachment and detachment
(Bogino et al. 2013). Lipopolysaccharides from bacterial outer membranes have
been reported to be involve in the induction of induce systemic reaction (Peer and
Schippers 1992).
18.2.3 Quorum Sensing
Biofilm cells communicate with each other to modulate/transform their metabolic

functions through a mechanism known as quorum sensing. Greenberg introduced the
term “quorum sensing.” In biofilm formation, the contribution of this mechanism is
not clearly understood as it is distinct among various bacterial species. Quorum
sensing (QS) is a density-dependent cell signaling mechanism and is said to be
involved in the biofilm formation, bacterial pathogenicity, and virulence. It can be
defined as cell–cell interactions which are mediated by diffusible chemical signal
molecules called autoinducers (Als) (Hooshangi and Bentley 2008). A high popula-
tion density provides us with a chance to perform certain processes that single cells
cannot carry out efficiently (Danhorn and Fuqua 2007; An et al. 2006). They can
intercommunicate with each other by quorum sensing and can able to confuse the
pathogenic bacteria.
The quorum sensing response can be observed after the phosphorylation of a
response regulator protein. In yeast strains, bicarbonate, acetaldehyde, and ammonia
are known as cell-to-cell signaling molecules (Rutherford et al. 2011; Hooshangi and
Bentley 2008). Besides, farnesol, which is found in the biofilm matrix produced by
P. aeruginosa, is another signal molecule that provides the inhibition of Candida
albicans. The mechanism of quorum sensing was first discovered in bacterial cells.
The fungal quorum sensing mechanism has been studied in recent years (Rutherford
et al. 2011 and Hooshangi and Bentley 2008).
There are three classes of signaling molecules associated with QS in bacteria
(Hooshangi and Bentley 2008):
1. Oligopeptides class of signaling molecules used by gram-positive bacteria as a

means of communication (Danhorn and Fuqua 2007).
2. Acyl homoserine lactones (AHLs) class of signaling molecules used by species-
specific gram-negative bacteria as a means of communication (Danhorn and
Fuqua 2007).
3. Autoinducers-2 (AI-2).
18.2.3.1 QS-Signaling Pathways

The signal molecules are produced and released into the medium by microorganisms
and the extracellular concentration reaches a threshold level (Hooshangi and Bentley
2008). Then the signal molecules lead the changes in the behavior of the microbial
population due to activation of transcriptional regulator and the gene expression has
been carried out by the regulators (Hooshangi and Bentley 2008). The expression of
quorum sensing molecules varies in accordance with microbial diversity.
In gram-negative bacteria, the autoinducer system is synthesized by homoserine
lactones (HSL), which are fatty acid derivatives and synthesized by LuxI and LuxR
homologues and the complex of quorum sensing mechanism is called LuxI/LuxR
systems (Rutherford et al. 2011). In gram-positive bacteria, the autoinducers are
amino acids and secreted short peptides, unlike the system observed in gram-
negative bacteria for quorum sensing (Rutherford et al. 2011).
In general, the signal molecules found in the filamentous fungi such as Aspergil-
lus and Penicillium species are secondary metabolites. Quorum sensing can be also
observed in the different genus of microorganisms such as bacteria–fungi and yeast–
fungi interactions (Annous et al. 2009).
18.2.4 Advantages of Biofilms to Microbes
(1) Bacterial biofilms which are formed on plant roots not only protect the sites of
colonization, but also acts as sink for the nutrients in the rhizosphere, hence
reducing the root exudates nutritional elements availability for pathogen stimu-
lation or their colonization on the root (Weller and Thomashow 1994).
(2) Biofilm assists bacteria to survive under unfavorable environment and
nutritional conditions.
(3) It gives resistance to biofilm agents. It increases local nutrients concentration and
thus helps in the plant growth and development.
(4) It gives an opportunity for exchange of genetic material.
(5) It has the ability to intercommunicate between bacteria population of same and
or different species.
(6) It produces growth factors across species boundaries.
18.2.5 Role of Microbial Biofilm in Biocontrol of Plant Diseases
The peanut seeds that are pretreated with the exopolysaccharide-producing strains of
Paenibacillus polymyxa (B5 and B6) showed inhibitory effect against A. niger,
which can cause crown rot disease than untreated seeds due to combination of
antibiosis, induction of resistance, and exopolysaccharides production at rhizoplane
of peanut plants. The activity of plant defense enzymes 1, 3-glucanase and chitinase
were significantly stimulated in treated roots which are positively correlated with
resistance to pathogens (Timmusk and Wagner 1999). The resistance was triggered
against A. niger by the peanut plants sown from previously treated seeds with
P. polymyxa strains (Haggag 2007).
A biofilm-producing strain of a yeast Pichia fermentans was found to have dual
nature which controls brown rot disease on apple caused by a phytopathogenic
isolate of Monilinia fructicola, in its yeast-like shape. But when the same strain
applied to peach fruit, it showed unexpected pathogenic behavior due to its transition
from budding growth to pseudohyphal growth even in the absence of M. fructicola,
suggesting that the pseudohyphal growth plays a major role in the expression of
potential pathogenicity of P. fermentans. Also showed that the biocontrol exerted by
this strain should not depend upon the production of toxic metabolites (Giobbe et al.
2007).
The antagonistic properties of rhizobacterium, P. polymyxa strains (B2, B5, and
B6) toward Phytophthora palmivora and Pythium aphanidermatum on Arabidopsis
thaliana was studied in liquid assays and soil assays. In liquid assays, when
A. thaliana was pretreated with bacterial strains, all the strains of P. polymyxa
(B2, B5, and B6) reduced the zoospore colonization of P. palmivora and
P. aphanidermatum. B2 and B5 isolates produced significant protection toward
both the pathogens by producing highest amount of mycoidal substances and high
rate of survivability of A. thaliana plants. The latter assay showed the incompetence
of P. polymyxa strains to reduce the zoospore colonization of P. aphanidermatum
compared to the liquid assay. Among all the isolates of P. polymyxa, B6 was less
potent in reducing the colonization of oomycete plant pathogens in both the assays
(Timmusk et al. 2005).
In the enlightenment process of benefits of PGPR in management of plant disease,
Bacillus lipopeptides (surfactins, iturins, and fengycins) were studied for their
antagonistic activity for a wide range of phytopathogens, and further in-depth studies
have shed light on the fact that these lipopeptides can also influence ecological
fitness of the producing strain colonization by stimulating host defense mechanisms
(Bais et al. 2004). When B. subtilis strain 6051, a wild-type, was treated to
Arabidopsis root surfaces against P. syringae with confocal scanning laser micros-
copy, it revealed the biofilm formation process includes the surfactin, a lipopeptide
antimicrobial agent secretion. The mutant strain, M1 (deletion of surfactin synthase

gene) of B. subtilis was found ineffective as a biocontrol agent against P. syringae in
both infectivity and in biofilm formation on either roots or on inert surfaces (Bais
et al. 2004).
18.2.6 Summary of the Chapter
The colonization of biocontrol agents on the plant surfaces plays a crucial role in
plant disease control. Generally, bacteria persist in natural environment by forming
biofilms (Davey and O’Toole 2000). The beneficial rhizobacterium is B. subtilis,
which is ubiquitous in soil, promotes plant growth, protects against fungal pathogen,
and plays a vital role in the degradation of organic polymers in the soil (Emmert and
Handelsman 1999). On an average, it contributes 4–5% of its genome to antibiotic
synthesis and capable of producing more than 24 antimicrobial compounds which
are structurally diverse (Stein 2005). Bacillus subtilis forms adhering biofilms on
inert surfaces under the variety of transcriptional factors (Hamon and Lazazzera
2001). Biocontrol ability of a wildtype B. subtilis strain 6051 against P. syringae was
demonstrated by using an infection model (Kinsinger et al. 2003). Root-associated
Pseudomonas sp. act as biocontrol agents and also promote plant growth. Pseudo-
monas putida responds rapidly to root exudates in the soil converging at root sites
and establish stable biofilms (Espinosa-Urgel et al. 2002). Some microorganisms
inhabit leaf surfaces and forms biofilms. For instance, Burkholderia sp., FP62 is a
biocontrol agent of B. cinerea in geranium, forms biofilms in the phyllosphere
(Haggag 2007).
18.3 Conclusion
The need to fulfill the food, health, and high-yielding plants for growing population,
the use of microbes in plant disease management offers an attractive alternative to
the use of the synthetic chemicals and could be a hope for food security problem. The
microbial strain used in biocontrol management suppresses the pathogens without
leaving residues which could be an eco-friendly approach. Biofilm formation by
PGPR could serve as a novel model system which serves to study and understand the
microbial colonization on various sites of plants. However, as a coin has both sides,
both beneficial and harmful microbes exist on the plant surfaces, but it depends on
the extent to which a beneficial microbes compete with pathogens and aids in plant
growth promotion by reducing disease incidence.
There is a need to explore many other beneficial microorganisms and employ in
sustainable plant disease control for healthy farming and nation and research
findings are insufficient in understanding the intimacy of different genus of
microorganisms such as bacteria–fungi and yeast–fungi interaction. Need to empha-
size the mechanism of beneficial microbes completely before their release as bio-
control agents is also important. Recently, the isolates of P. fermentans were found
in the blood stream infections (Pfaller and Diekema 2004) and caused polyarthritis in
a patient suffering from alcoholism (Trowbridge et al. 1999).
The significance and impact of the chapter highlights the need to consider the
potential of biofilm formation for biocontrol assays in natural conditions and reveal
the need in advancement of research in this area for management of plant diseases in
a sustainable approach.
References
Amellal N, Burtin G, Bartoli F, Heulin T (1998) Colonization of wheat roots by an
exopolysaccharide-producing Pantoea agglomerans strain and its effect on rhizosphere soil
aggregation. Appl Environ Microbiol 64:3740–3747
An D, Danhorn T, Fuqua C, Parsek MR (2006) Quorum sensing and motility mediate interactions
between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures. Proc
Natl Acad Sci U S A 103:3828–3833
Annous B, Fratamico P, Smith JL (2009) Quorum sensing in biofilms: why bacteria behave the way
they do. J Food Sci 74:101–111
Bais HP, Fall R, Vivanco JM (2004) Biocontrol of Bacillus subtilis against infection of Arabidopsis
roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production.
Bergsma-Vlami M, Prins ME, Raaijmakers JM (2005) Influence of plant species on population
dynamics, genotypic diversity and antibiotic production in the rhizosphere by indigenous
Pseudomonas spp. FEMS Microbiol Ecol 52:59–69
Branda SS, Vik S, Friedman L, Kolter R (2005) Biofilms: the matrix revisited. Trends Microbiol
13:20–26
Bogino PC, Oliva MDLM, Sorroche FG, Giordano W (2013) The role of bacterial biofilms and
surface components in plant-bacterial associations. Int J Mol Sci 14(8):15838–15859
Burdman S, Okon Y, Jurkevitch E (2000) Surface characteristics of Azospirillum brasilense in
relation to cell aggregation and attachment to plant roots. Crit Rev Microbiol 26:91–110
Cook RJ, Thomashow LS, Weller DM, Fuiimoto D, Mazzola M, Bangera G, Kim D (1995)
Molecular mechanisms of defense by rhizobacteria against root disease. Proc Natl Acad Sci
USA 92:4197–4201
Costerton JW (1995) Overview of microbial biofilms. J Ind Microbiol 15:137–140
Danhorn T, Fuqua C (2007) Biofilm formation by plant-associated bacteria. Annu Rev Microbiol
61:401–422. https://doi.org/10.1146/annurev.micro.61.080706.093316
Davey ME, O’Toole GA (2000) Microbial biofilm: from ecology to molecular genetics. Microbiol
Mol Biol Rev 64:847–867
Emmert EAB, Handelsman J (1999) Biocontrol of plant disease: a gram positive perspective. FEMS
Microbiol Lett 171:1–9
Espinosa-Urgel M, Kolter R, Ramos JL (2002) Root colonization by Pseudomonas putida: love at
first sight. Microbiology 148:341–343
Fett WF, Cooke PH (2003) Reduction of Escherichia coli O157: H7 and Salmonella on laboratory-
inoculated alfalfa seed with commercial citrus-related products. J Food Prot 66:1158–1165
Gage DJ (2004) Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during
nodulation of temperate legumes. Microbiol Mol Biol Rev 68:280–300
Giobbe S, Marceddu S, Scherm B, Zara G, Mazzarello VL, Budroni M, Migheli Q (2007) The
strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown
rot on apple but is pathogenic on peach fruit. Fed Eur Microbiol Soc 7:1389–1398
Glick BR, Patten CL, Holguin G, Penrose DM (1999) Biochemical and genetic mechanisms used
by plant growth-promoting Bacteria. Imperial College Press, London
Haggag WM (2007) Colonization of exopolysaccharide-producing Paenibacillus polymyxa on

peanut roots for enhancing resistance against crown rot disease. Afr J Biotechnol 6:1568–1577
Hamon MA, Lazazzera BA (2001) The sporulation transcription factor Spo0A is required for
biofilm development in Bacillus subtilis. Mol Microbiol J 42:1199–1209
Hooshangi S, Bentley WE (2008) From unicellular properties to multicellular behavior: bacteria
quorum sensing circuitry and applications. Curr Opin Biotechnol 19:550–555
Kang Y, Liu H, Genin S, Schell MA, Denny TP (2002) Ralstonia solanacearum requires type 4 pili
to adhere to multiplesurfaces and for natural transformation and virulence. Mol Microbiol
46:427–437
Kiely PD (2006) Exploiting new systems-based strategies to elucidate plant-bacterial interactions in
the rhizosphere. Microb Ecol 51:257–266
Kinsinger RF, Shirk MC, Fall R (2003) Rapid surface motility and biofilm formation in Bacillus
subtilisis dependent on extracellular surfactin and potassium ion. J Bacteriol 185:5627–5631
Leigh JA, Coplin DL (1992) Exopolysaccharide in plant–bacterial interactions. Annu Rev
Loon LCV (2007) Plant responses to plant growth promoting rhizobacteria. Eur J Plant Pathol
199:243–254
Marques LLR, De Boer SH, Ceri H, Olsen ME (2003) Evaluation of biofilms formed by
Clavibacter michiganensis sub sp. sepedonicus. Phytopathology 93:57
Monier JM, Lindow SE (2003) Differential survival of solitary and aggregated bacterial cells
promotes aggregate formation on leaf surfaces. Proc Natl Acad Sci U S A 100:15977–15982
Monroe D (2007) Looking for chinks in the armor of bacterial biofilms. PLoS biology 5:e307.
https://doi.org/10.1371/journal.pbio.0050307
Migheli Q (2001) Genetically modified biocontrol agents: environmental impact and risk analysis.
Journal of Plant Pathology 83:47–56
Newman KL, Almeida RP, Purcell AH, Lindow SE (2003) Use of a green fluorescent strain for
analysis of Xylella fastidiosa colonization of Vitis vinifera. Appl Environ Microbiol
69:7319–7327
Newman KL, Almeida RP, Purcell AH, Lindow SE (2004) Cell–cell signaling controls Xylella
fastidiosa interactions with both insects and plants. Proc Natl Acad Sci U S A 101:1737–1742
Ngo Thi NA, Naumann D (2007) Investigating the heterogeneity of cell growth in microbial
colonies by FTIR microspectroscopy. Anal Bioanal Chem 387:1769–1777
Ortu G, Demontis MA, Budroni M, Goyard S, d’Enfert C, Migheli Q (2005) Study of biofilm
formation in Candida albicans may help understanding the biocontrol capability of a flor strain
of Saccharomyces cerevisiae against the phytopathogenic fungus Penicillium expansum. J Plant
Pathol 87(Special issue):300. (abstract)
Palkov’a Z, V’achov’a L (2006) Life within a community: benefit to yeast long-term survival.
FEMS Microbiol Rev 30:806–824
Peer VR, Schippers B (1992) Lipopolysaccharides of plant-growth promoting Pseudomonas
sp. strain WCS417r induce resistance in carnation to Fusarium wilt. Netherlands. J Plant Pathol
98(2):129
Perneel M, Heyrman J, Adiobo A, De Maeyer K, Raaijmakers JM, De Vos P, Hofte M (2007)
Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the
cocoyam rhizosphere with biocontrol activity. J Appl Microbiol 103:1007–1020
Pfaller MA, Diekema DJ (2004) Twelve years of fluconazole in clinical practice: global trends in
species distribution and fluconazole susceptibility of blood stream isolates of Candida. Clin
Microbiol Infect 10:11–23
Purcell AH, Hopkins DL (1996) Fastidious xylem-limited bacterial pathogens. Annu Rev
Ramey-Hartung B, Koutsoudis MD, von Bodman SB, Fuqua C (2005) Biofilm formation in plant-
microbe associations. Current Opinion in Microbiology 7:602–609. https://doi.org/10.1016/j.
mib.2004.10.014
Ramey BE, Matthysse AG, Fuqua C (2004) The FNR-type transcriptional regulator SinR controls
maturation of Agrobacterium tumefaciens biofilms. Mol Microbiol 52:1495–1511
Rezzonico F, Zala M, Keel C, Duffy B, Moenne-Loccoz Y, Defago G (2007) Is the ability of
biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4-diacetyl
phloroglucinol really synonymous with higher plant protection. New Phytol 173:861–872
Rojas CM, Ham JH, Deng WL, Doyle JJ, Collmer A (2002) HecA, a member of a class of adhesins
produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal
cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii
seedlings. Proc Natl Acad Sci U S A 99:13142–13147
Rutherford ST, Van Kessel JC, Shao Y, Bassler BL (2011) AphA and LuxR/HapR reciprocally
control quorum sensing in vibrios. Genes Dev 25:397–408
Rafique M, Hayat K, Mukhtar T, Anna Khan AA, Afridi MS, Hussain T, Sultn T, MFH M,
Imran M, Choudhury HJ (2015) Bacterial biofilm formation and its role against agricultural
pathogens. In: Mendez-Vilas A (ed) The battle against microbial pathogens: basic science,
technological advances and educational programs, pp 373–382
Scherm B, Ortu G, Muzzu A, Budroni M, Arras G, Migheli Q (2001) Genetically modified
biocontrol agents: environmental impact and risk analysis. J Plant Pathol 83:47–56
Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol
Timmusk S, Wagner EG (1999) The plant-growth promoting rhizobacterium Paenibacillus
polymyxa induces changes in Arabidopsis thaliana gene expression: a possible connection
between biotic and abiotic stress responses. Mol Plant-Microbe Interact 12:951–959
Timmusk S, Grantcharova N, Wagner EGH (2005) Paenibacillus polymyxa invades plant roots and
forms biofilms. Appl Environ Microbiol (11):7292–7300
Tran H, Ficke A, Asiimwe T, Hofte M, Raaijmakers JM (2007) Role of the cyclic lipopeptide
massetolide A in biological control of Phytophthora infestans and in colonization of tomato
plants by Pseudomonas fluorescens. New Phytol 175:731–742
Trowbridge J, Ludmer LM, Riddle VD, Levy CS, Barth WF (1999) Candida lambica polyarthritis
in a patient with chronic alcoholism. J Rheumatol 26:1846–1848
Watrick P, Kolter R (2000) Biofilm, city of microbes. J Bacteriol 182:2675–2673
Weller DM, Thomashow LS (1994) Current challenges in introducing beneficial microorganisms
into the rhizosphere. In: Molecular ecology of rhizosphere microorganisms: biotechnology and
release of GMOs. VCH, New York, pp 1–18
West PV, Morris BM, Reid B, Appiah AA, Osborne MC, Campbell TA, Shepherd SJ (2002)
Oomycete plant pathogens use electric fields to target roots. Mol Plant-Microbe Interact
15:790–798
Williams A, Wilkinson A, Krehenbrink M, Russo DM (2008) Glucomannan- mediated attachment
of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection. J
Bacteriol 190:4706–4715
Role of Endophytes in Plant Disease
Management 19
Sunanda Chakraborty, Debanjana Debnath, Sunita Mahapatra,
and Srikanta Das
Abstract
Sustainable agriculture and agri-food production can only be preserved for our
next generation by protecting different natural resources. So a thrust interest has
been developed to exploit internal colonisation of healthy plants that termed as
endophytes to execute in a systemic way against plant disease management. The
matter in this chapter is to have an impact on economic and environment by
limiting substantial side effects of abiotic and biotic factors by immediately
protecting them by living organisms, i.e. endophytes within the plant tissues.
The future implication of combinations of endophyte with commercial pesticide
both as seed and seedling treatment could have a synergistic effect against
multiple disease resistance under changing climate scenario.
Keywords
Endophytes · Sustainable agriculture · Colonisation · Disease resistance
19.1 Introduction
In the twentieth century, agricultural intensification has been achieved with the help
of advanced farm machineries, intensive tillage, high-yielding varieties and heavy
doses of fertilizers and pesticides (Foley et al. 2005). These practices, however, had a
detrimental effect on the soil as well as human health, with a reduction in soil
fertility, high water needs and increased resurgence and resistance to pest and
diseases. Therefore, alternative environmentally benign approaches need to be
utilised to maintain sustainable agricultural production while overcoming threats
that lead to various abiotic stresses, such as soil salinity, temperature extremes or
S. Chakraborty · D. Debnath · S. Mahapatra (*) · S. Das

Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, Nadia, West Bengal, India

https://doi.org/10.1007/978-981-15-6275-4_19
400 S. Chakraborty et al.
drought, as well as biotic stresses caused by pests and plant pathogens. So, the use of
microorganisms has come up as an essential alternative for improved plant perfor-
mance in integrated plant disease management (reviewed by Singh et al. 2011; Jha
et al. 2013). In this context, the plant endophytes are being extensively studied in the
recent years for their optimum utilisation in managing plant diseases while enhanc-
ing overall soil and plant health.
The word endophyte, first introduced by Anton de Bary (1866), is derived from
two Greek words, ‘endon’ meaning within and ‘phyton’ meaning plant, i.e. the word
endophyte literally means ‘in the plant’. Endophytes are microorganisms which
colonise healthy plant tissues intracellularly and/or intercellularly but do not cause
any apparent symptom of the disease. Endophytes were reported as early as 1904 but
did not receive much attention till the recent discovery of its beneficial role in
pharmaceutical and ecological aspects. It is now reported that endophytes are not
only instrumental in direct inhibition of plant pathogens by the production of
antibiotics and various enzymes but are also responsible for improving plant physi-
ology, production of beneficial secondary metabolites, induction of plant resistance,
improving soil fertility and phytoremediation.
As per Sturz and other associates (2000), endophytic bacteria can be isolated from
a large diversity of plants, not a single plant is devoid of endophytes. However, there
are only a few differences exists between endophytic microbes and other
microbiomes present in the rhizospheric soil (Hallmann et al. 1997; Rosenblueth
and Martinez-Romero 2004). But among them, those which are most beneficial still
have controversies around world. As both types of microbiomes are present together
in plant and soil rhizosphere, the more advantageous ones are very hard to detect.
But through many researches, it has been proved that endophytic population in
plants is conditioned by different biotic and abiotic factors, but endophytes perform
better compared to rhizospheric bacteria against biotic and abiotic stresses
(Hallmann et al. 1997).
In this review, we can address together in a same frame about types, ecology,
colonisation, mode of action, stress tolerance and recent formulations of different
endophytes that would be useful for agricultural sectors, for future research as well
as for commercial purposes.
19.2 Origin
The history of endophytes is very ancient. Studies on fossil record prove that
endophytes had close relationship with terrestrial land plants for >400 million
years ago (Krings et al. 2007). Now endophytes are known to be present in all
types of plant habitats,such as fern, lichen, mosses, shrub and grasses, along with
deciduous and coniferous trees (Sun and Guo 2012). Thus, endophytes have become
an integral part of many ecosystems. Historically, endophytes started gaining impor-
tance when toxicosis, caused by Neotyphodium coenophialum (family
Clavicipitaceae), was observed in cattle eating the grass, Festuca arundinacea.
The first report on the isolation of endophytic fungi was from the plants belonging
19 Role of Endophytes in Plant Disease Management 401
to the families Araceae, Bromeliaceae and Orchidaceae by Petrini and Dreyfuss

(1981). The abundance of endophytic fungus is variable from host to host, but at
least one species of endophytic fungus has been found in whichever plant was taken
for investigation (Faeth and Fagan 2002).
The hypothesis behind the bacterial endophytes is that they may originate from
the microflora belonging to the rhizosphere (root zone) and phyllosphere (above-
ground portion) (Sturz and Nowak 2000). In a nutshell, the bacterial endophytes are
special types of bacteria that were isolated from the internal parts of the plant such as
xylem tissues and colonise there but without having any harmful effect on the host
plant (Schulz and Boyle 2006).
19.3 Types of Endophytes
Endophytes are microorganisms that are associated with plants in various forms,
including bacteria (actinomycetes or mycoplasma) or fungi, which are colonised
inside the plant tissues intracellularly and/or intercellularly. Among the endophytes
the large population is shared by fungi alone, and others are bacteria and
actinomycetes. Endophytic fungi may be present in almost all parts of plants without
expressing any symptom. The interesting fact about endophytes is that a single
species of endophytes can occur in association with many plant species, while
many species of endophytes can exist in the same host plant species as latent or
with the interaction with other endophytes present in the same plant
(Zabalgogeazcoa 2008).
Among bacteria, more than 200 genera from 16 phyla have been reported to be
associated with endophytes, most of them belonging to the phyla Actinobacteria,
Proteobacteria and Firmicutes (Golinska et al. 2015). Bacterial genera, such as
Achromobacter, Acinetobacter, Agrobacterium, Bacillus, Brevibacterium,
Microbacterium, Pseudomonas, Xanthomonas, etc., have been reported as
endophytes (Sun et al. 2013). Bacterial endophytes have also been reported to
produce bioactive metabolites such as antimicrobial and antifungal compounds
including ecomycins and fusaricidins, respectively. Table 19.1 outlines the manage-
ment of biotic and/or abiotic stresses by prokaryotic endophytes on host plant
(Figs. 19.1 and 19.2).
Actinomycetes, belonging to the phylum Actinobacteria, are prokaryotic
microorganisms that possess mycelium and have the ability to form spores
(Chaudhary et al. 2013; Barka et al. 2016). Endophytic actinomycetes have been
reported to produce different various chemical compounds that have important
medicinal properties (Gayathri and Muralikrishnan 2013; Singh and Dubey 2015).
Among endophytic actinomycetes, Streptomyces is a dominant genus, which
produces approximately 76% of the antimicrobial and anticancer compounds that
have been reported so far (Berdy 2012). Antimicrobial compounds of biological
interest, such as coronamycin, naphthomycin (A and K), munumbicins (A and B),
clethramycin, cedarmycin (A and B), kakadumycins and saadamycin, have been
isolated from Streptomyces. Paclitaxel extracted from Kitasatospora sp. has been
Table 19.1 Role of bacterial endophytes in management of biotic and/or abiotic stresses in host
plants
Pathogen/
Name Host abiotic stress Changes in plants References
Burkholderia Grapevine Cold tolerance Altering Ait et al.
phytofirmans plants photosynthetic (2006),
activity and Fernandez
metabolism of et al. (2012)
carbohydrates
Pseudomonas Rice Salinity stress Accumulation of Jha et al.
pseudoalcaligenes tolerance higher (2011)
concentrations of
glycine betaine-like
compounds
Azospirillum spp. Maize plants Water stress Accumulation of the Tuteja
tolerance abscisic acid (ABA) (2007)
Achromobacter Catharanthus Salinity stress Production of ACC Karthikeyan
xylosoxidans roseus tolerance deaminase and et al. (2012)
AUM54 reduction of
ethylene levels
Pseudomonas Oryza sativa Salinity stress Increased glycerol Jogawat
indica concentration et al. (2016)
Paecilomyces Arabidopsis Cold tolerance Accumulation of Su et al.
formosus LHL10 thaliana pigments and (2015)
induced cold
response pathway
Pseudomonas Capsicum Osmotic stress Gene encodes the Sziderics
indica annum enzyme ACC et al. (2007)
oxidase
Pseudomonas Cucumber Colletotrichum Elicitation of ISR Wei et al.
fluorescens strain lagenarium (1991),
89B-61 Kloepper
and Ryu
(2006)
Pseudomonas Rice Pyricularia Elicitation of ISR Smith and
syringae oryzae Métraux
pv. syringae (1991)
Curtobacterium Citrus Xylella Elicitation of ISR Araujo et al.
flaccumfaciens fastidiosa (2002)
observed to inhibit food-borne microbes (Zhao et al. 2011; Gangwar et al. 2014;
Golinska et al. 2015).
Fungi are a group of heterotrophic organisms which are often associated in a
symbiotic relationship with a large number of autotrophic organisms (Saar et al.
2001). Table 19.2 outlines the management of biotic and/or abiotic stresses by fungal
endophytes on host plant.
Generally, fungal endophytes are categorised into two broad categories,
i.e. clavicipitaceous (C) and non-clavicipitaceous (NC), based on the evolutionary
relatedness, taxonomy, host plant range and ecological function. C endophytes are
Plant growth
promotion Plant Isolation of endophytic
bacteria and fungi
Increase Crop
yield
Increase Plant
fitness Inoculation
Enhanced
Biocontrol
nutrient uptake
agents
Plant
Phytohorm
Stress
on
regulator
Fig. 19.1 Schematic representation of role of endophytes on plant health
Tolerance to biotic
and abiotic factors
Plant growth development Plant protection
against pathogens
Physiology and Production of bioactive
metabolism compounds
Endophytes
Ecosystem productivity Nutrient absorption
and management
Agronomic application Plant defense activation
Fig. 19.2 Direct and indirect role of endophytes within the plant system
phylogenetically related to ascomycete fungi, and mainly they harbour the Poaceae
family. Till now seven genera of C endophytes have been identified from the grasses
of Poaceae family. Among them, the Neotyphodium genus (teleomorph Epichloë),
harbouring cool season C3 grasses, is the most important and extensively studied.
Neotyphodium species have also been found in cereals (Marshall et al. 1999). C
endophytes mainly occurred in the shoots and caused perennial systemic intercellu-
lar infection without expressing any symptom and remained endophytic for the
entire life cycle of the host (Rodriguez et al. 2009). However, they often vertically
transmitted to the next generation plant from inflorescence and seeds (Saikkonen
et al. 2010). Up to 20–30% of grass species worldwide are harboured by different C
endophytes, including Neotyphodium. On the other hand, NC endophytes have been
identified from almost all terrestrial plants especially ferns, conifers and angiosperms
(Rodriguez et al. 2009). The basic differences between the NC and C endophytes are
Table 19.2 Role of fungal endophytes in management of biotic and/or abiotic stresses in host
plants
Name Host Stress References
Fusarium culmorum Leymus mollis Salt tolerance Rodriguez et al. (2008)
Curvularia Dichanthelium Heat tolerance Redman et al. (2002)
protuberata lanuginosum
Piriformospora Brassica Drought tolerance Sun et al. (2010)
indica campestris ssp.
chinensis
Chaetomium and Wheat Puccinia and Dingle and McGee
Phoma Pyrenophora spp. (2003), Istifadah and
McGee (2006)
Acremonium Dactylis Helminthosporium Rivera Varas et al.
strictum glomerata L. solani Durieu and Mont (2007)
Piriformospora Barley Blumeria graminis Waller et al. (2005)
indica
Neotyphodium sp. Lolium pratense Barley yellow dwarf Lehtonen et al. (2006)
virus (BYDV)
Curvularia sp. Dichanthelium Tolerance to high soil Márquez et al. (2007)
lanuginosum temperatures
Trichoderma Oryza sativa Drought stress Pandey et al. (2016)
harzianum TH-56
Fusarium equiseti Barley Gaeumannomyces Macia-Vicente et al.
graminis var. tritici (2009)
Chaetomium sp. Wheat Puccinia recondita Dingle and McGee
(2003), Mahapatra
et al. (2020)
Piriformospora Wheat Pseudocercosporella Serfling et al. (2007)
indica herpotrichoides
Beauveria bassiana Cotton Rhizoctonia solani Griffin (2007)
strain 11-98
Beauveria bassiana Tomato Pythium myriotylum Griffin (2007)
L. lecanii (Zimm.) Coffee Hemileia vastatrix Vandermeer et al.
Zare and W. Gams Berk. and Broome (2009)
Chaetomium Wheat Puccinia tritici repentis Istifadah and McGee
globosum (2006), Mahapatra
et al. (2020)
Cordana sp. Wild banana Colletotrichum sp. Nuangmek et al.
(2008)
Fusarium Maize Ustilago maydis Lee et al. (2009)
verticillioides
that NC endophytes may not harbour host plant for the entire life cycle of the host
and that the endophyte itself may not remain endophyte for its own life cycle
(Rodriguez et al. 2009).
In the recent years, another new class of root-inhibiting fungi, i.e. dark septate
endophytes (DSE), has been identified, which belongs to heterogeneous ascomycete
fungi. It is characterised by dark-pigmented septate hyphae, which generally
colonises in the root tissue both intercellularly and intracellularly (Jumpponen and
Trappe 1998). Exophiala pisciphila isolated from maize and Harpophora oryzae
(Yuan et al. 2010) isolated from wild rice in China are some examples of DSE.
Endophytic fungi are reported to produce some of the popularly used antibiotics
and anticancer drugs. The Penicillium sp. produces penicillenols, which are cyto-
toxic to cell lines. Taxol is an effective and a very popular anticancer drug that is
extracted from endophytic fungi, Taxomyces andreanae. Many other antibacterial
and antifungal compounds, such as sordaricin (Fusarium sp.), jesterone
(Pestalotiopsis jesteri), clavatol (Torreya mairei), javanicin (Chloridium sp.), etc.,
are reported to be highly effective against a large number of food-borne infectious
microorganisms (Jalgaonwala et al. 2011). Pestacin, isolated from P. microspora,
has excellent antioxidant properties.
19.4 Colonisation
The nature of colonisation in the internal tissue has made the endophytes a valuable
tool for crop improvement performance in agriculture (Azevedo et al. 2000).
Endophytes, both prokaryotic and eukaryotic, often colonise the root tissues system-
ically in both inter- and intracellular manner. Bacterial endophytes enter into their
host via stomata (Roos and Hattingh 1983), nectarthodes (Rosen 1936), lenticels,
germinating radicles (Gagné et al. 1987), broken trichomes, wounds (Daft and Leben
1972), foliar damage from windblown soil particles, rain or hail and undifferentiated
meristematic root tissue. Colonisation by bacterial endophytes is primarily intercel-
lular (Hallmann et al. 1997), though some of them, such as Azoarcus spp., have been
found to colonise the root tissues intracellularly (Hurek et al. 1994). They are widely
distributed in the vascular bundles which aid in their distribution throughout the
plant (Kobayashi and Palumbo 2000), fungal endophytes colonise asymptomatically
in both inter- and intracellular manner (Barrow 2003).
Endophytic microorganisms may not be endophytic for their entire life cycle,
thereby encompassing not only symbiotic and communalistic species but also latent
pathogens and saprotrophs. Generally, nitrogen-fixing bacteria (rhizobia) produce
morphological changes in the roots by forming root nodules; otherwise, endophytes
remain silent without any morphological change in the system (Malfanova et al.
2013).
19.5 Mode of Action
19.5.1 Direct Inhibition of Plant Pathogens
Endophytes have been studied extensively in the recent years for their ability to
protect plants from various diseases as well as reduce the damage caused by plant
(Mejia et al. 2008). By producing antibiotics, competition and secreting lytic
Table 19.3 Bioactive compounds produced by endophytes

Fungal
endophyte Host Antibiotic Target pathogens References
Periconia sp. Taxus Fusicoccane Bacillus subtilis, Kim et al.
cuspidata diterpenes Staphylococcus (2004)
aureus, Klebsiella
pneumoniae,
Salmonella
typhimurium
Acremonium Maize Pyrrocidines A, Aspergillus flavus, Wicklow
zeae B Fusarium et al. (2005)
verticillioides
Muscodor Tropical tree Tetrahydofuran, Stachybotrys Atmosukarto
albus 2-methyl furan, chartarum et al. (2005)
2-butanone,
aciphyllene
Phomopsis Cassia Cadinane Cladosporium Silva et al.
cassiae spectabilis sesquiterpenes sphaerospermum, (2006)
Cladosporium
cladosporioides
Verticillium sp. Rehmannia Massariphenone, Pyricularia oryzae You et al.
glutinosa ergosterol P-2b (2009)
peroxide
Colletotrichum Pteromischum Collutellin Pythium ultimum, Ren et al.
dematium sp. Sclerotinia (2008)
sclerotiorum
Streptomyces Oryza sativa Efomycins M Plasmodium Supong et al.
sp. and G, falciparum (2016)
oxohygrolidin,
abierixin and
29-O-
methylabierixin
Aspergillus Carthamus Aspernolide F Candida Ibrahim et al.
terreus lanatus neoformans (2015)
enzymes, endophytes are able to suppress the plant pathogens directly. However,
these interactions are very complex and specific to species-species antagonism.
Antibiosis Many endophytes produce antifungal and antibacterial secondary

metabolites, which inhibit the growth and development of other microorganisms
including plant pathogens (Gunatilaka 2006). Antibiotics produced by a single
endophyte may be of various chemical compositions such as alkaloids, terpenoids,
polypeptides and aromatic compounds, which are harmful to the plant pathogens.
Chaetomium and Phoma, after inoculation in wheat, reduced the severity of foliar
disease caused by Puccinia and Pyrenophora spp. However, most interestingly,
same result has been observed when the filtrate of above-mentioned endophytes’
culture was applied in the wheat plants (Istifadah and McGee 2006). Phomopsis
cassiae, an endophytic fungus isolated from Cassia spectabilis, has been shown to
produce 3, 11, 12-trihydroxycadalene, a cadinane sesquiterpene, which has been
proved to be the most active antifungal compound against Cladosporium
sphaerospermum and Cladosporium cladosporioides (Silva et al. 2006).
Table 19.3 enlists some antibiotics that are produced by endophytic fungi.
Alkaloids too have a strong ability to suppress microorganisms. Endophytic

Alternaria spp. produces altersetin, an alkaloid, which has shown antibacterial
activity against large number of pathogenic gram-positive bacteria (Hellwig et al.
2002). Volatile organic compounds (VOCs) are also considered to be effective in
antibiosis. An endophytic fungus, Muscodor albus, has been reported to produce
many volatile organic compounds including 2-methyl furan, tetrahydofuran,
aciphyllene and 2-butanone, which possess antibiotic activities (Atmosukarto et al.
2005). In vitro, it was observed that endophytic fungi isolated from Artemisia annua
produced EtOAc and n-butanol, which acted as antifungal compounds, inhibiting
growth of a number of phytopathogens (Liu et al. 2001).
Lytic enzymes secreted from endophytes: Endophytes produce a wide array of
enzymes that aid in the hydrolysis of polymeric compounds including DNA, protein,
lignin, cellulose, chitin and hemi-cellulose (Tripathi et al. 2008). During
colonisation, endophytes secrete these enzymes to break down the plant cell wall.
At the same time, these enzymes possess the ability of hydrolysing the cell walls of
fungi and oomycetes. The enzymes include β-1,3glucanases, cellulases and
chitinases. It has been reported that β-1, 3-glucanase genes in Lysobacter
enzymogenes strain C3 helped in biological control activity towards Pythium
damping-off of sugar beet. Streptomyces has also exhibited antagonism to cacao
witches’ broom in vitro with the production of lytic enzymes (Macagnan et al. 2008).
The biocontrol fungus Talaromyces flavus Tf1 (Ascomycota: Eurotiales) produces
the enzyme glucose oxidase, whose reaction product, hydrogen peroxide, kills
microsclerotia of phytopathogenic Verticillium (Fravel 1988).
Mycoparasitism Some endophytes may be mycoparasites. Acremonium strictum

W. Gams is an endophyte which has been frequently isolated from Dactylis
glomerata L. and other grasses (Sánchez Márquez et al. 2007); recently it has been
shown that this fungus is a mycoparasite of Helminthosporium solani Durieu and
Mont., a potato pathogen (Rivera Varas et al. 2007). A significant increase in
resistance to dollar spot disease, caused by Sclerotinia homoeocarpa F.T. Benn.,
has been observed in Festuca rubra L. cultivars infected by Epichloë festucae. In the
case of viruses, the incidence of Barley yellow dwarf virus (BYDV) was lower in
Lolium pratense infected by Neotyphodium than in endophyte-free plants. Since
BYDV is transmitted by means of aphid vectors, toxic fungal alkaloids may be the
reason for this effect; in fact, aphid reproduction was lower in endophyte-infected
plants than in those free of endophyte. An endophyte of the plant Dichanthelium
lanuginosum (Elliott) Gould was found to confer tolerance to high soil temperatures
to the plant. Epichloë festucae virus 1 (EfV1) is another virus, which
asymptomatically infects the grass endophyte Epichloë festucae; in this case, it is not
known if the presence of the virus in the endophyte affects the plant host (Romo et al.
2007).
19.5.2 Indirect Effects to Enhance Plant Resistance
Plants experience a large number of stresses in the form of pathogens, pest, drought,
salinity, cold, etc. Morphological and biochemical changes such as lignifications,
cellular necrosis, hypersensitive response and phytoalexin production help in
responding to those stresses in an efficient manner. Since endophytes may evolve
from plant pathogens, they help in triggering plant defences against pathogens prior
to attack. The defence is mainly increased by resistance enhancement and production
of secondary metabolites.
19.5.2.1 Induction of Plant Resistance

Over the past two decades, researchers have extensively studied the responses
exhibited by plants when subjected to various biotic and abiotic stresses. In this
context, systemic acquired resistance (SAR) and induced systemic resistance (ISR)
have been widely studied. SAR is a pathogen-induced response, mediated by
salicylic acid and the accumulation of pathogenesis-related (PR) proteins, while
ISR is induced by some non-pathogenic rhizobacteria and is mediated by jasmonic
acid (Vallad and Goodman 2004; Tripathi et al. 2008). In some cases, ISR may also
be associated with expression of pathogenesis-related genes. This is seen in fungal
endophyte, Fusarium solani isolated from root tissues of tomato, which elicits
induced systemic resistance against the tomato foliar pathogen Septoria lycopersici
by triggering PR genes, PR5 and PR7, expression in roots (Kavroulakis et al. 2007).
Also, plants colonised by endophytes have the ability to initiate defence response
more rapidly as compared to non-colonised plants. This was evident when cucumber
(Cucumis sativus) and watermelon (Citrullus lanatus) previously exposed to a
non-pathogenic mutant of Colletotrichum magna exhibited increasingly higher
levels of peroxidase activity lignin deposition, and phenylalanine ammonia lyase
activity that resulted in the protection against disease caused by Colletotrichum
orbiculare and Fusarium oxysporum (Redman et al. 1999).
Endophyte Neotyphodium lolii was reported to enhance superoxide dismutase
(SOD) and peroxidase (POD) activities of host that resulted in reduced lesions on
infected leaves of the host (Tian et al. 2008). Thus, it can be deduced that the reactive
oxygen species (ROS) damage induced by pathogen infection is actively controlled
by antioxidative systems including SOD and POD, induced by the endophytes.
19.5.2.2 Stimulation of Plant Secondary Metabolites

Plant secondary metabolites do not play an important role in basic life functions but
are responsible for the adaptation of plants to their environment (Bourgaud et al.
2001). Among these compounds, plants produce phytoalexins (Smith 1996), which
may contain terpenoid, flavonoid, etc. Studies have shown that endophytic fungi
Fusarium spp. E4 and E5 help in promoting the growth of Euphorbia pekinensis as

well as increasing its terpenoid content (Yong et al. 2009). Further research has
revealed that Fusarium E5 elicitor could induce terpene production in E. pekinensis
cell suspension cultures and as well as increased production of defence-related
enzymes including phenylalanine ammonia lyase, peroxidase and catalase (data
have not been published).
The mechanism of endophyte elicitor induced plant secondary metabolite resis-
tance is similar to the stimulation of plant resistance. Fungal endophytes stimulate
the secretion of hydrolase of plant cell to limit the growth of fungi, which results in
the production of endophyte fragments that act as elicitors. These elicitors stimulate
plant defence and plant secondary metabolites, which help in effective suppression
of the attack by the pathogen.
19.5.2.3 Promotion of Plant Growth and Physiology

Endophytes help in the plant defence activities by enhancing the plant growth and
controlling the plant physiology (Giménez et al. 2007). An increase in plant growth
results in higher plant vigour that provides potential protection to pathogen challenge
(Kuldau and Bacon 2008). Studies conducted in the last two decades have revealed
that plants infected with endophytes show higher growth promotion tolerance to
unsuitable soil conditions and improved resistance towards drought stress
(Malinowski et al. 2004).
Lu et al. (2000) observed that Colletotrichum sp., an endophytic fungus, pro-
duced growth-regulating substances such as indole acetic acid (IAA) that helped in
controlling various plant processes (Lu et al. 2000). Similar results were observed in
the case of Fusarium sp., where E5 extract functioned as an auxin (Dai et al. 2008).
As defence responses are energy intensive in nature, the endophytes are also
believed to provide reducing equivalents and carbon skeletons with the help of
primary metabolic pathways (Bolton 2009). It is reasonable to believe that enhance-
ment of plant growth would lead to increased protection of the plant against
pathogens.
Volatile compounds such as 2, 3 butanediol and acetoin produced by bacteria
have been reported to be a newly discovered mechanism that helps in promoting
plant growth (Ryu et al. 2003). It was observed that endophytic bacteria produce
adenine ribosides that help in stimulating growth as well as mitigating browning of
pine tissues (Pirttilä et al. 2004). Hoffman et al. (2013) conducted in vitro experiment
on endophyte Pestalotiopsis and reported that endophytic bacteria identified as
Luteibacter enhanced indole-3-acetic acid (IAA) production, whereas the bacteria
and fungi were unable to produce IAA on the media when cultured alone.
19.5.3 Ecological Effects
The plant-pathogen-endophyte interaction is largely influenced by the endophytic

niche. Competition for nutrients and limited space leads to hyperparasitism and
predation between diverse microorganisms that live in endophytic niche, especially
between endophytes and pathogens. Endophytic recognition and colonisation rap-

idly occupy ecological niche and leave no space for pathogens, which would be the
common and main reason that fungal endophytes inhibit pathogen infection in plant.
Fungal endophytes have the ability to colonise inter- or intracellularly and often are
localised in a single cell. The colonisation of plant tissues by endophytes involves
several steps including host recognition, spore germination, penetration of the
epidermis and tissue colonisation (Petrini 1991). After endophytes are successfully
colonised in the host tissue, the endophytic niche becomes established. In the
endophytic niche, endophytes will obtain a reliable source of nutrition provided
from plant fragment, exudates and leachates and protect host against other
microorganisms. Fungal endophytes are generally thought to protect plant by rapid
colonisation and thereby exhausting the limited available substrates so that none
would be available for pathogens to grow (Pal and Gardener 2006). Furthermore, the
plants produce lignin and other cell-wall deposits to limit the growth of endophytes
and cause it to be a virulent (Harman et al. 2004). As a result, the cell wall becomes
re-reinforced after endophytic colonisation; thus, it becomes difficult for pathogens
to infest.
Hyperparasitism is another ecological strategy that endophytes provide to protect
host plant. In hyperparasitism, the pathogen is directly attacked by a specific
endophyte that kills it or its propagules (Tripathi et al. 2008). Fungal endophytes
parasitise around hyphae of pathogens by various means such as twisting,
penetrating the hyphae of pathogens and secreting lyase to decompose cell wall of
pathogens. For example, Trichoderma are able to parasitise hyphae of plant patho-
gen Rhizoctonia solani, and many of these observations are linked with biocontrol.
In contrast to hyperparasitism, microbial predation is a more general way to suppress
plant pathogens. Some endophytes show predatory behaviour under nutrient-limited
conditions. For example, Trichoderma produce a range of enzymes that are directly
used against cell walls of fungi to utilise the fragment of pathogens (Benhamou and
Chet 1997). It was also suggested that endophytes may protect their hosts by
enhancing stress tolerance to oxidative stress. Several studies demonstrated
increased production of antioxidant compounds (flavonoids and other phenolic
antioxidants) in endophyte-infected plants (Herrera-Carillo et al. 2009), which was
assumed to be triggered by production of reactive oxygen species (ROS) by
endophytes. ROS is produced to oxidise and denature host cell membranes, thereby
facilitating nutrient leakage from plant cells, which are subsequently absorbed by
fungal hyphae (White and Torres 2010). Consequently, host plant resistance to ROS
involving stresses, e.g. drought, diseases and metal toxicities, may likewise be
improved (White and Torres 2010). Furthermore, endophytes are known to be
producers of a vast variety of antioxidant compounds as well, such as phenolic
acids and their derivatives, isobenzofuranones, isobenzofurans as well as mannitol
and other carbohydrates. This antioxidant capacity may also contribute to the
enhancement of stress tolerance in their hosts (White and Torres 2010). Stress
tolerance conferred by some endophytes was shown to represent habitat-adapted
phenomena. Curvularia protuberata was found to dominantly colonise
Dichanthelium lanuginosum, thereby conferring heat tolerance to the plant growing

on geothermal soils of Yellowstone National Park, USA.
19.6 Crop Adaptation to Abiotic Stress Environment
Endophytes have been extensively studied in the recent decades for their overall
impact on the plant growth and physiology. It has been observed that endophytes not
only confer resistance to plants against invading plant pathogens but also aid in
adapting the plant to various stress conditions such as drought, extreme heat or cold,
salinity, water stress, etc. Endophytes are responsible for overall improvement in
plant vigour, which confers drought tolerance and reduced transplanting shock in
plants (Lazarovits and Nowak 1997). It was observed that grapevine plants exhibited
increased cold stress tolerance due to bacterial endophyte Burkholderia
phytofirmans PsJN, which was achieved by altering photosynthetic activity and
increased carbohydrate metabolism (Ait et al. 2006). In the presence of the bacte-
rium, the plant resulted in lower cell damage and accumulation of starch, proline,
phenolic compounds and other cold-stress-related metabolites (Naveed et al. 2014).
Endophytic bacterium Pseudomonas pseudoalcaligenes was reported for induce
tolerance to salinity stress in rice due to the accumulation of higher concentrations
of glycine betaine-like (Jha et al. 2011).
Water stress tolerance was demonstrated by Cohen et al. (2009) in maize plants
due to the production of the abscisic acid (ABA) by endophytic bacterium,
Azospirillum spp. ABA is known to be crucial for plant growth during stress
conditions, as it regulates plant water balance and osmotic stress (Tuteja 2007).
When ethylene is accumulated in plants as a result of stress, it has deleterious
effect on plant growth and health (Czarny et al. 2006). Endophytes may produce the
enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase that has no par-
ticular function in endophytic bacteria but significantly contributes to the promotion
of plant growth and improvement in stress tolerance due to cleaving of ACC, which
is an ethylene precursor (Campbell and Thompson 1996; Glick 2014). An endo-
phytic bacterium, Achromobacter xylosoxidans AUM54, was shown to impart
tolerance to its host plant Catharanthus roseus by producing ACC deaminase that
resulted in reduced ethylene levels (Karthikeyan et al. 2012). In addition,
Achromobacter xylosoxidans strain Ax 10 and Pantoea agglomerans Jp3-3 produc-
ing ACC deaminase were reported to alleviate stress of Brassica sp. plants that were
grown in copper-contaminated soils, along with improving copper uptake by the
plants (Ma et al. 2009).
19.7 Production of Bioactive Secondary Metabolites
Studies have demonstrated that endophytic fungi are able to produce a large number
of bioactive compounds, hence indicating that endophytes can be used as an
alternate source of these metabolites (Priti et al. 2009). The bioactive compounds
belong to a number of classes including terpenoids, alkaloids, cytochalasins,

flavonoids, polyketides and steroids. The best known example till date, for the
occurrence of ‘bioactive photochemical’ in endophytes is paclitaxel (Taxol®), an
anticancer drug, that has been extracted from endophytic fungus Taxomyces
andreanae (Stierle et al. 1993; Stierle and Stobel 1995). Paclitaxel has also been
extracted from Seimatoantlerium tepuiense, Seimatoantlerium nepalense (Bashyal
et al. 1999), Tubercularia sp. strain TF5 (Wang et al. 2000), Metarhizium anisopliae
(Liu et al. 2009), Periconia sp., Bartalinia robillardoides and Colletotrichum
gloeosporioides (Gangadevi and Muthumary 2008a, b).
Vincristine, another anticancer drug that was originally obtained from
Catharanthus roseus (Apocynaceae), was recently detected in cultures of endo-
phytic Fusarium oxysporum (Zhang et al. 2000). This anticancer compound arrests
mitosis by binding to tubulin dimers and inhibiting their assembly to microtubule
structures.
Podophyllotoxin, produced by podophyllum species, is known to be the precursor
to be the anticancer drugs teniposide and etoposide. Both drugs cause apoptosis and
cell death by inhibiting topoisomerase II, which in turn blocks the ligation step of the
cell cycle, harming the integrity of the genome. Recently, endophytic Fusarium
oxysporum, isolated from medicinal plant Juniperus recurva, was reported to pro-
duce approximately 28 μg/g dry mass of podophyllotoxin (Kour et al. 2008). Many
other such bioactive compounds have been extracted from endophytes which are
outlined in Table 19.4.
Former studies repeatedly indicated that endophytes are capable of
biosynthesizing plant secondary metabolites in vitro, but it is not to be believed
that they alone perform the production in the plant. A plausible explanation might be
a horizontal gene transfer at some stage during co-evolution, thus importing the
respective pathways from fungi into the host plant (Kusari et al. 2008) or vice versa.
Nevertheless, it would be of great interest to determine the degree and contribution
of endophytic biosynthetic pathways to the secondary metabolite profiles of plants as
this would offer an additional explanation for the patchy distribution of certain
natural products, such as certain alkaloids, cardiac glycosides and anthraquinones,
in the plant kingdom (Wink 2008). Endophytes have a great potential in
bioprospecting, indicating that many other important and interesting examples of
endophyte secondary metabolism are yet to be discovered.
19.8 Biodegradation Effects of Endophytes
Even though it has not been widely recognised, endophytes possess a number of
extracellular enzymes such as ligninases, pectinases, cellulases, phenoloxidase,
proteinase and lignin catabolic enzymes (Oses et al. 2006; Bischoff et al. 2009).
All these enzymes are instrumental in penetration and colonisation of the host plant.
Later, when the plant dies, the endophytes utilise the plant litter as a source of carbon
such as glucose, hemicellulose, cellulose, keratin, lignin, lipids, pectin and protein
(Kudanga and Mwenje 2005; Urairuj et al. 2003).
19
Table 19.4 Natural products derived or produced from various endophytes

Organism Plant association Active agent Activity References
Taxomyces andreanae Taxus brevifolia Taxol Anticancer Strobel et al. (1993)
Pseudomonas viridiflava Grass Ecomycins B and C Antimicrobial Miller et al. (1998)
Streptomyces griseus Kandelia candel p-Aminoacetophenonic acids Antimicrobial Guan et al. (2005)
Streptomyces NRRL 30562 Kennedia nigricans Munumbicins Munumbicin D Antibiotic antimalarial Castillo et al. (2002)
Streptomyces NRRL 30566 Grevillea pteridifolia Kakadumycins Antibiotic Castillo et al. (2003)
Serratia marcescens Rhyncholacis penicillata Oocydin A Antifungal Strobel et al. (2004)
Paenibacillus polymyxa Wheat Fusaricidin A–D Antifungal Beck et al. (2003)
Role of Endophytes in Plant Disease Management
Cytonaema sp. Quercus sp. 103 Cytonic acids A and D Antiviral Guo et al. (2000)
Streptomyces sp. Monstera sp. Coronamycin Antimalarial antifungal Ezra et al. (2004)
Fusarium solani Camptotheca acuminata Camptothecin Anticancer Kusari et al. (2011)
Eupenicillium parvum Azadirachta indica Azadirachtin A and B Insecticidal Kusari et al. (2012)
Streptosporangium oxazolinicum Unspecified orchid Spoxazomicins A and B Antiprotozoal Inahashi et al. (2011)
Penicillium sp. Garcinia nobilis Penialidin C and citromycetin Antibacterial Jouda et al. (2016)
413
Three endophytic fungi, Alternaria, Phoma and Phomopsis, were isolated from
surface-sterilised pods of Colophospermum mopane and showed lignocellulolytic
enzyme activity. Scanning electron microscope (SEM) studies revealed that
Alternaria and Phomopsis had the ability of degrading heavily lignified fibres,
while Phoma was able to degrade those mesophyll cells that were moderately
lignified. The lignocellulolytic abilities displayed by the endophytes considerably
accelerate the decay of pods, resulting in effective germination of seeds in an arid
environment under favourable conditions (Jordaan et al. 2006).
19.9 Formulations
Large-scale use of endophytes necessitates the availability of environmental friendly

alternative approach, and hence various formulations of these beneficial
microorganisms as bioinoculants are being extensively studied to find the most
efficient formulation. Various experiments have been conducted on different types
of inorganic and organic carriers along with their evaluation for shelf life and storage
conditions, finding suitable formulation of bioinoculants (Vidhyasekaran et al. 1997;
Sallam et al. 2013) that would help in increasing microbial population as well as their
survival in soil (Bashan 1998). For the purpose of commercialisation of endophytes,
it is important that the bioinoculants must remain viable in the prescribed formula-
tion for a certain period of storage (Bazilah et al. 2011). The bioinoculant
formulations that have been developed so far have utilised inert carrier materials
(Vyas et al. 2010), which act only as carrier media while being neutral to microbial
population in every aspect.
Studies have shown that viability counts of plant growth-promoting rhizobacteria
(PGPR) bioinoculants decreased in a regular pattern until 210 days, when sawdust
was used as a carrier (Arora et al. 2008), while talcum-based formulations exhibited
better results in terms of storage as well as management of various plant pathogens
(Sah et al. 2011; Shanmugam et al. 2011; Prathuangwong et al. 2013). Kumar et al.
(2012) conducted an experiment to assess the application of talcum-based
formulations of Piriformospora indica and other PGPRs and reported the overall
growth promotion in Vigna mungo. Then that talcum powder–based formulation
exhibited the best results with regard to long shelf life as well as plant growth
response. Due to the application of talc-based powder formulations of Trichoderma
at the time of planting, an increase in the survival percentage of cantaloupe plants
was observed under greenhouse condition (Sallam Nashwa et al. 2014). They found
that peat- and talc-based formulations were the most helpful carriers in sustaining the
microbial population at the time of storage, while Pseudomonas fluorescens sur-
vived on the chickpea seeds for at least 180 days, when applied as talc-based
formulation.
Table 19.5 A list of pollutants that have been associated with phytoremediation strategies using
bacterial endophyte
Compound Organism Plant association References
Mono- and Pseudomonas aeruginosa Wild rye (Elymus Siciliano et al.
dichlorinated strain R75 and Pseudomonas dauricus) (1998)
benzoic acids savastanoi strain CB35
TCP and PCB Herbaspirillum sp. K1 Wheat Mannisto et al.
(2001)
Volatile Burkholderia cepacia G4 Yellow lupine Barac et al. (2004)
organic (Lupinus luteus
compounds L.)
and toluene
MTBE, BTEX, Pseudomonas sp. Populus Germaine et al.
TCE cv. Hazendans (2004), Porteous-
and Moore et al. (2006)
cv. Hoogvorst
Methane Methylobacterium populi Poplar tissues Van Aken et al.
BJ001 (Populus (2004)
deltoides nigra
DN34)
Toluene Bacillus cepacia Bu61(pTOM- Poplar (Populus) Taghavi et al. (2005)
Bu61)
2,4-D Pseudomonas putida VM1450 Poplar (Populus) Germaine et al.
and willow (2006)
(Salix)
Arsenic Staphylococcus arlettae Brassica juncea Srivastava et al.
(2013)
Zinc, cadmium, Pseudomonas koreensis AGB-1 Miscanthus Babu et al. (2015)
arsenic and sinensis
lead
19.10 Endophytes in Phytoremediation
Over the years, extensive studies on various endophytes have suggested that they
have the ability to break down complex and toxic pollutants in the soil, which would
render them harmless to the plant. Table 19.5 outlines various studies that demon-
strate the role of endophytes in phytoremediation.
It was observed by Van Aken et al., in 2004, that Methylobacterium, isolated
from hybrid poplar trees (Populus deltoides x nigra), had the ability of biodegrading
numerous nitroaromatic compounds such as 2,4,6-trinitrotoluene. An application of
bacterial endophytes with considerable biotechnological potential was described by
Barac et al. (2004) who reported an increase of plant tolerance to toluene and
decrease the transpiration of toluene to the atmosphere, when engineered
Burkholderia cepacia G4 endophytes were applied to the plants. Pseudomonas
endophytes, present in pea, have the ability of degrading organochlorine herbicide,
2, 4-dichlorophenoxyacetic acid (2, 4-D), as reported by Germaine et al. (2006). This

was further proved when pea plants not inoculated with Pseudomonas endophytes
showed considerable 2,4-D toxicity with advanced visual symptoms such as reduced
leaf biomass, callus development and leaf abscission.
Phytoremediation enhancement might be the outcome of increased nutrient
uptake and increased pathogen protection or due to a higher degradation of heavy
metals. Although few phytoremediation studies have been conducted on fungal
endophytes, their utility in bioremediation clearly indicates a huge potential that is
yet to be utilised.
19.11 Conclusion
Recently many scientists are focusing their research on endophytes as biocontrol

agents for plant disease management. In this chapter, we addressed that the potential
mechanisms of endophytes against inhibition of plant pathogen are by direct and
indirect effects; also added attentions are given on its ecological effects. Due to high
production cost by the use of chemical fertilizers and pesticides and its negative
effect on environment, the use of endophytes may have an advantageous role in
sustainable agriculture if the added inoculants are potential.
While endophyte research still has a lot of lack and is limited, only a few teams
are working on it. We strongly believe that several endophytes with unique modes of
action exist in our ecosystem, and only strong research can find out about them. We
also need to assess whether it could be possible to affordably promote endophytes in
farmers’ field. Further researches are required on host–endophyte interactions, which
could be a strong evidence for its implementation in the farmers field.
References
Ait BE, Nowak J, Clement C (2006) Enhancement of chilling resistance of inoculated grapevine
plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain
PsJN. Appl Environ Microbiol 72:7246–7252
Araujo WL, Marcon J, Maccheroni W Jr, Van Elsas JD, Van Vuurde JWL, Azevedo JL (2002)
Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in
citrus plants. Appl Environ Microbiol 68:4906–4914
Arora NK, Khare E, Naraian R, Maheshwari DK (2008) Sawdust as a superior carrier for production
of multipurpose bioinoculant using plant growth promoting rhizobial and pseudomonad strains
and their impact on productivity of Trifolium repense. Curr Sci 95:90
Atmosukarto I, Castillo U, Hess WM, Sears J, Strobel G (2005) Isolation and characterization of
Muscodor albus I-41.3s, a volatile antibiotic producing fungus. Plant Sci 169:854–861
Azevedo JL, Maccheroni W Jr, Pereira JO, de Araújo WL (2000) Endophytic microorganisms: a
review on insect control and recent advances on tropical plants. Electron J Biotechnol 3:15–16
Babu GA, Shea PJ, Sudhakar D, Jung IB, Oh BT (2015) Potential use of Pseudomonas koreensis
AGB-1 in association with Miscanthus sinensis to remediate heavy metal(loid)-contaminated
mining site soil. J Environ Manag 151:160–166
Barac T, Taghavi S, Borremans B, Provoost A, Oeyen L, Colpaert JV, Vangronsveld J, van der
Lelie D (2004) Engineered endophytic bacteria improve phyto-remediation of water-soluble,
volatile, organic pollutants. Nat Biotechnol 22:583–588
Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Klenk HP, Clément C,
Ouhdouch Y, van Wezel GP (2016) Taxonomy, physiology, and natural products of
Actinobacteria. Microbiol Mol Biol Rev 80:1–43
Barrow JR (2003) A typical morphology of dark septate fungal root endophytes of Bouteloua in arid
south western USA range lands. Mycorrhiza 3:239–247
Bary A (1866) Morphologie und physiologie der pilze, flechten und myxomyceten. Leipzig
Bashan Y (1998) Inoculants of plant growth promoting bacteria for use in agriculture. Biotechnol
Adv 16:729–770
Bashyal B, Li JY, Strobel GA, Hess WM (1999) Seimatoantlerium nepalense, an endophytic taxol
producing coelomycete from Himalayan yew (Taxus wallachiana). Mycotaxon 72:33–42
Bazilah ABI, Sariah M, Zainal Abidin MA, Yasmeen S (2011) Effect of carrier and temperature on
the viability of Burkholderia sp. (UPMB3) and Pseudomonas sp. (UPMP3) during storage. Int J
Agric Biol 13:198–202
Beck HC, Hansen AM, Lauritsen FR (2003) Novel pyrazine metabolites found in polymyxin
biosynthesis by Paenibacillus polymyxa. FEMS Microbiol Lett 220:67–73
Benhamou N, Chet I (1997) Cellular and molecular mechanisms involved in the interaction
between Trichoderma harzianum and Pythium ultimum. Appl Environ Microbiol 63:2095–2099
Bérdy J (2012) Thoughts and facts about antibiotics: where we are now and where we are heading. J
Antibiot 65:385
Bischoff KM, Wicklow DT, Jordan DB, de Rezende ST, Liu SQ, Hughes SR, Rich JO (2009)
Extracellular hemicellulolytic enzymes from the maize endophyte Acremonium zeae. Curr
Bolton MD (2009) Primary metabolism and plant defense fuel for the fire. Mol Plant-Microbe
Interact 22:487–497
Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a
historical perspective. Plant Sci 161:839851
Campbell BG, Thompson JA (1996) 1-Aminocyclopropane1-carboxylate deaminase genes from
Pseudomonas strains. FEMS Microbiol Lett 138:207–210
Castillo UF, Strobel GA, Ford EJ, Hess WM, Porter H, Jensen JB, Albert H, Robison R, Condron
MA, Teplow DB, Stevens D (2002) Munumbicins, wide-spectrum antibiotics produced by
Streptomyces NRRL 30562, endophytic on Kennedia nigriscansa. Microbiology
148:2675–2685
Castillo U, Harper JK, Strobel GA, Sears J, Alesi K, Ford E, Lin J, Hunter M, Maranta M, Ge H,
Yaver D (2003) Kakadumycins, novel antibiotics from Streptomyces sp. NRRL 30566, an
endophyte of Grevillea pteridifolia. FEMS Microbiol Lett 224:183–190
Chaudhary HS, Soni B, Shrivastava AR, Shrivastava S (2013) Diversity and versatility of
actinomycetes and its role in antibiotic production. J Appl Pharm Sci 3:S83–S94
Cohen AC, Travaglia CN, Bottini R, Piccoli PN (2009) Participation of abscisic acid and
gibberellins produced by endophytic Azospirillum in the alleviation of drought effects in
maize. Botany 87:455–462
Czarny JC, Grichko VP, Glick BR (2006) Genetic modulation of ethylene biosynthesis and
signaling in plants. Biotechnol Adv 24:410–419
Daft GC, Leben C (1972) Bacterial blight of soybeans: epidemiology of blight outbreaks. Phytopa-
thology 62
Dai CC, Yu BY, Li X (2008) Screening of endophytic fungi that promote the growth of Euphorbia
pekinensis. Afr J Biotechnol 7:3505–3509
Dingle J, Mcgee PA (2003) Some endophytic fungi reduce the density of pustules of Puccinia
recondita f. sp. tritici in wheat. Mycol Res 107:310–316
Ezra D, Castillo UF, Strobel GA, Hess WM, Porter H, Jensen JB, Condron MA, Teplow DB,
Sears J, Maranta M, Hunter M (2004) Coronamycins, peptide antibiotics produced by a
verticillate Streptomyces sp. (MSU-2110) endophytic on Monstera sp. Microbiology

150:785–793
Faeth SH, Fagan WF (2002) Fungal endophytes: common host plant symbionts but uncommon
mutualists. Integr Comp Biol 42:360–368
Fernandez O, Theocharis A, Bordiec S, Feil R, Jacquens L, Clement C, Fontaine F, Barka EA
(2012) Burkholderia phytofirmans PsJN acclimates grapevine to cold by modulating carbohy-
drate metabolism. Mol Plant-Microbe Interact 25:496–504
Foley JA, Defries R, Asner GP, Barford C, Bonan G, Carpenter SR, Chapin FS, Coe MT, Daily GC,
Gibbs HK, Helkowski JH, Holloway T, Howard EA, Kucharik CJ, Monfreda C, Patz JA,
Prentice IC, Ramankutty N, Snyder PK (2005) Global consequences of land use. Science
309:570–574
Fravel DR. (1988) Role of antibiosis in the biocontrol of plant diseases. Annu Rev Phytopathol
75–91
Gagné S, Richard C, Rousseau H, Antoun H (1987) Xylem-residing bacteria in alfalfa roots. Can J
Gangadevi V, Muthumary J (2008a) Taxol, an anticancer drug produced by an endophytic fungus
Bartalinia robillardoides Tassi, isolated from a medicinal plant, Aegle marmelos Correa ex
Roxb. World J Microbiol Biotechnol 24:717–724
Gangadevi V, Muthumary J (2008b) Isolation of Colletotrichum gloeosporioides, a novel endo-
phytic taxol-producing fungus from the leaves of a medicinal plant, Justicia gendarussa. Mycol
Balc 5:1–4
Gangwar M, Dogra S, Gupta UP, Kharwar RN (2014) Diversity and biopotential of endophytic
actinomycetes from three medicinal plants in India. Afr J Microbiol Res 8:184–191
Gayathri P, Muralikrishnan V (2013) Isolation and characterization of endophytic actinomycetes
from mangrove plant for antimicrobial activity. Int J Curr Microbiol Appl Sci 2:78–89
Germaine K, Keogh E, Garcia-Cabellos G, Borremans B, Lelie D, Barac T, Oeyen L,
Vangronsveld J, Moore FP, Moore ERB, Campbell CD, Ryan D, Dowling DN (2004)
Colonisation of poplar trees by gfp expressing bacterial endophytes. FEMS Microbiol Ecol
48:109–118
Germaine K, Liu X, Cabellos G, Hogan J, Ryan D, Dowling DN (2006) Bacterial endophyte-
enhanced phyto-remediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid.
FEMS Microbiol Ecol 57:302–310
Giménez C, Cabrera R, Reina M, González Coloma A (2007) Fungal endophytes and their role in
plant protection. Curr Org Chem 11:707–720
Glick BR (2014) Bacteria with ACC deaminase can promote plant growth and help to feed the
world. Microbiol Res 169:30–39
Golinska P, Wypij M, Agarkar G, Rathod D, Dahm H, Rai M (2015) Endophytic actinobacteria of
medicinal plants: diversity and bioactivity. Antonie Van Leeuwenhoek 108:267–289
Griffin MR (2007) Beauveria bassiana, a cotton endophyte with biocontrol activity against seedling
disease. PhD Dissertation. The University of Tennessee, Knoxville, TN, USA
Guan SH, Sattler I, Lin WH, Guo DA, Grabley S (2005) P-Aminoacetophenonic acids produced by
a mangrove endophyte: Streptomyces griseus subspecies. J Nat Prod 68:1198–1200
Gunatilaka AAL (2006) Natural products from plant-associated microorganisms: distribution,
structural diversity, bioactivity, and implications of their occurrence. J Nat Prod 69:509–526
Guo B, Dai JR, Ng S, Huang Y, Leong C, Ong W, Carte BK (2000) Cytonic acids A and B: novel
tridepside inhibitors of hCMV protease from the endophytic fungus Cytonaema species. J Nat
Prod 63:602–604
Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in
agricultural crops. Can J Microbiol 43:895–914
Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species – opportunistic,
avirulent plant symbionts. Nat Rev Microbiol 2:43
Hellwig V, Grothe T, Mayer-Bartschmid A, Endermann R, Geschke FU, Henkel T, Stadler MA

(2002) Altersetin, a new antibiotic from cultures of endophytic Alternaria spp. taxonomy,
fermentation, isolation, structure elucidation and biological activities. J Antibiot 55:881–892
Herrera-Carillo Z, Torres MS, Singh AP, Vorsa N, Gianfagna T, Meyer W, White Jr JF. (2009)
Phenolic, flavonoid and antioxidant profiling for cool-season grasses with and without endo-
phyte. In: Proceedings of the 18th annual Rutgers Turfgrass symposium, vol 12
Hoffman MT, Gunatilaka MK, Wijeratne K, Gunatilaka L, Arnold AE (2013) Endohyphal bacte-
rium enhances production of indole-3-acetic acid by a foliar fungal endophyte. PLoS One 8:
e73132
Hurek T, Reinhold-Hurek B, Van Montagu M, Kellenberger E (1994) Root colonization and
systemic spreading of Azoarcus sp. strain BH72 in grasses. J Bacteriol 176:1913–1923
Ibrahim SR, Elkhayat ES, Mohamed GA, Khedr AI, Fouad MA, Kotb MH, Ross SA (2015)
Aspernolides F and G, new butyrolactones from the endophytic fungus Aspergillus terreus.
Phytochem Lett 14:84–90
Inahashi Y, Iwatsuki M, Ishiyama A, Namatame M, Nishihara-Tsukashima A, Matsumoto A,
Hirose T, Sunazuka T, Yamada H, Otoguro K, Takahashi Y (2011) Spoxazomicins A–C,
novel antitrypanosomal alkaloids produced by an endophytic actinomycete, Streptosporangium
oxazolinicum K07-0460 T. J Antibiot 64:303
Istifadah N, Mcgee PA (2006) Endophytic Chaetomium globosum reduces development of tan spot
in wheat caused by Pyrenophora tritici-repentis. Australas Plant Pathol 35:411–418
Jalgaonwala RE, Mohite BV, Mahajan RT (2011) A review: natural products from plant associated
endophytic fungi. J Microbiol Biotechnol Res 1:21–32
Jha Y, Subramanian RB, Patel S (2011) Combination of endophytic and rhizospheric plant growth
promoting rhizobacteria in Oryza sativa shows higher accumulation of osmoprotectant against
saline stress. Acta Physiol Plant 33:797–802
Jha PN, Gupta G, Jha P, Mehrotra R (2013) Association of rhizospheric/endophytic bacteria with
plants: a potential gateway to sustainable agriculture. Greener J Agric Sci 3:73–84
Jogawat A, Vadassery J, Verma N, Oelmüller R, Dua M, Nevo E, Johri AK (2016) PiHOG1, a
stress regulator MAP kinase from the root endophyte fungus Piriformospora indica, confers
salinity stress tolerance in rice plants. Sci Rep 6:36765
Jordaan A, Taylor JE, Rossenkhan R (2006) Occurrence and possible role of endophytic fungi
associated with seed pods of Colophospermum mopane (Fabaceae) in Botswana. S Afr J Bot
72:245–255
Jouda JB, Mawabo IK, Notedji A, Mbazoa CD, Nkenfou J, Wandji J, Nkenfou CN (2016) Anti-
mycobacterial activity of polyketides from Penicillium sp. endophyte isolated from Garcinia
nobilis against Mycobacterium smegmatis. Int J Mycobacteriol 5:192–196
Jumpponen ARI, Trappe JM (1998) Dark septate endophytes: a review of facultative biotrophic
root-colonizing fungi. New Phytol 140:295–310
Karthikeyan B, Joe MM, Islam R, Sa T (2012) ACC deaminase containing diazotrophic endophytic
bacteria ameliorate salt stress in Catharanthus roseus through reduced ethylene levels and
induction of antioxidative defense systems. Symbiosis 56:77–86
Kavroulakis NS, Zervakis GI, Ehaliotis C, Haralampidis K, Papadopoulou KK (2007) Role of
ethylene in the protection of tomato plants against soil-borne fungal pathogens conferred by an
endophytic Fusarium solani strain. J Exp Bot 58:3853–3864
Kim S, Shin DS, Lee T, Oh KB (2004) Periconicins, two new fusicoccane diterpenes produced by
an endophytic fungus Periconia sp. with antibacterial activity. J Nat Prod 67:448–450
Kloepper JW, Ryu CM (2006) Bacterial endophytes as elicitors of induced systemic resistance. In:
Boyle CJ, Schulz B, Sieber T (eds) Microbial root endophytes. Springer, Berlin/Heidelberg, pp
33–52
Kobayashi DY, Palumbo JD (2000) Bacterial endophytes and their effects on plants and uses in
agriculture. In: Bacon CW, White JF (eds) Microbial endophytes. Dekker, New York, pp
199–236
Kour A, Shawl AS, Rehman S, Qazi PH, Sudan P, Khajuria RK, Sultan P, Verma V (2008) Isolation
and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin
from Juniperus recurva. World J Microbiol Biotechnol 24:1115–1121
Krings M, Taylor TN, Hass H, Kerp H, Dotzler N, Hermsen EJ (2007) Fungal endophytes in a
400-million-yr-old land plant: infection pathways, spatial distribution, and host responses. New
Phytol 174:648–657
Kudanga T, Mwenje E (2005) Extracellular cellulase production by tropical isolates of
Aureobasidium pullulans. Can J Microbiol 51:773–776
Kuldau G, Bacon C (2008) Clavicipitaceous endophytes: their ability to enhance resistance of
grasses to multiple stresses. Biol Control 46:57–71
Kumar V, Sarma MVRK, Saharan K, Srivastava R, Kumar L, Sahai V, Bisaria VS, Sharma AK
(2012) Effect of formulated root endophytic fungus Piriformospora indica and plant growth
promoting rhizobacteria fluorescent pseudomonads R62 and R81 on Vigna mungo. World J
Kusari S, Lamshöft M, Zühlke S, Spiteller M (2008) An endophytic fungus from Hypericum
perforatum that produces hypericin. J Nat Prod 71:159–162
Kusari S, Zühlke S, Spiteller M (2011) Effect of artificial reconstitution of the interaction between
the plant Camptotheca acuminata and the fungal endophyte Fusarium solani on camptothecin
biosynthesis. J Nat Prod 74:764–775
Kusari S, Verma VC, Lamshoefft M, Spiteller M (2012) An endophytic fungus from Azadirachta
indica A. Juss. That produces azadirachtin. World J Microbiol Biotechhnol 28(3):1287–1294
Lazarovits G, Nowak J (1997) Rhizobacteria for improvement of plant growth and establishment.
HortScience 32:188–192
Lee K, Pan JJ, May G (2009) Endophytic Fusarium verticillioides reduces disease severity caused
by Ustilago maydis on maize. FEMS Microbiol Lett 299:31–37
Lehtonen PT, Helander M, Siddiqui SA, Lehto K, Saikkonen K (2006) Endophytic fungus
decreases plant virus infections in meadow ryegrass (Lolium pratense). Biol Lett 2:620–623
Liu CH, Zou WX, Lu H, Tan RX (2001) Antifungal activity of Artemisia annua endophyte cultures
against phytopathogenic fungi. J Biotechnol 88:277–282
Liu L, Liu SC, Chen XL, Guo LD, Che YS (2009) Pestalofones A-E, bioactive cyclohexanone
derivatives from the plant endophytic fungus Pestalotiopsis fici. Bioorg Med Chem 17:606–613
Lu H, Zou WX, Meng JC, Hu J, Tan RX (2000) New bioactive metabolites produced by
Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant Sci 151:67–73
Ma Y, Rajkumar M, Freitas H (2009) Inoculation of plant growth promoting bacterium
Achromobacter xylosoxidans strain Ax10 for the improvement of copper phytoextraction by
Brassica juncea. J Environ Manag 90:831–837
Macagnan D, Romeiro RS, Pomella AWV, deSouza JT (2008) Production of lytic enzymes and
siderophores, and inhibition of germination of basidiospores of Moniliophthora (ex Crinipellis)
perniciosa by phylloplane actinomycetes. Biol Control 47:309–314
Macia-Vicente JG, Jansson HB, Talbot NJ, Lopez-Llorca LV (2009) Real-time PCR quantification
and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusar-
ium equiseti and Pochonia chlamydosporia. New Phytol 182:213–228
Mahapatra S, Rayanoothala P, Solanki MK, Das S (2020) Wheat microbiome: present status and
future perspective. In: Solanki M, Kashyap P, Kumari B (eds) Phytobiomes: current insights and
future vistas. Springer, Singapore. https://doi.org/10.1007/978-981-15-3151-4_8
Malfanova N, Lugtenberg BJ, Berg G (2013) Bacterial endophytes: who and where, and what are
they doing there. Mol Microbial Ecol Rhizosphere 1:2
Malinowski DP, Zuo H, Belesky DP, Alloush GA (2004) Evidence for copper binding by
extracellular root exudates of tall fescue but not perennial ryegrass infected with Neotyphodium
spp. endophytes. Plant Soil 267:1–12
Mannisto MK, Tiirola MA, Puhakka JA (2001) Degradation of 2,3,4,6-tetrachlorophenol at low
temperature and low dioxygen concentrations by phylogenetically different groundwater and
bioreactor bacteria. Biodegradation 12:291–301
Márquez LM, Redman RS, Rodriguez RJ, Roossinck MJ (2007) A virus in a fungus in a plant:
three-way symbiosis required for thermal tolerance. Science 315:513–515
Marshall D, Tunali B, Nelson LR (1999) Occurrence of fungal endophytes in species of wild
Triticum. Crop Sci 39:1507–1512
Mejıa LC, Rojas EI, Maynard Z, Bael SV, Elizabeth Arnold A, Hebbar P, Samuels GJ, Robbins N,
Herre EA (2008) Endophytic fungi as biocontrol agents of Theobroma cacao pathogens. Biol
Control 46:4–14
Miller CM, Miller RV, Garton-Kenny D, Redgrave B, Sears J, Condron MM, Teplow DB, Strobel
GA (1998) Ecomycins, unique antimycotics from Pseudomonas viridiflava. J Appl Microbiol
84:937–944
Naveed M, Hussain MB, Zahir ZA, Mitter B, Sessitsch A (2014) Drought stress amelioration in
wheat through inoculation with Burkholderia phytofirmans strain PsJN. Plant Growth Regul
73:121–131
Nuangmek W, McKenzie EHC, Lumyong S (2008) Endophytic fungi from wild banana (Musa
acuminata Colla.) works against anthracnose disease caused by Colletotrichum musae. Res J
Oses R, Valenzuela S, Freer J, Baeza J, Rodriguez J (2006) Evaluation of fungal endophytes for
lignocellulolytic enzyme production and wood biodegradation. Int Biodeterior Biodegrad
57:129–135
Pal KK, Gardener BM. (2006) Biological control of plant pathogens
Pandey V, Ansari MW, Tula S, Yadav S, Sahoo RK, Shukla N, Bains G, Badal S, Chandra S, Gaur
AK, Kumar A (2016) Dose-dependent response of Trichoderma harzianum in improving
drought tolerance in rice genotypes. Planta 243:1251–1264
Petrini O (1991) Fungal endophytes of tree leaves. In: Microbial ecology of leaves. Springer,
Petrini O, Dreyfuss MM (1981) Endophytische Pilze in epiphyischen Araceae, Bromeliaceae und
Orchidaceae. Sydowia 34:135–148
Pirttilä AM, Joensuu P, Pospiech H, Jalonen J, Hohtola A (2004) Bud endophytes of scots pine
produce adenine derivatives and other compounds that affect morphology and mitigate
browning of callus cultures. Physiol Plant 121:305–312
Porteous-Moore F, Barac T, Borremans B, Oeyen L, Vangronsveld J, van der Lelie D, Campbell D,
Moore ERB (2006) Endophytic bacterial diversity in poplar trees growing on a BTEX-
contaminated site: the characterisation of isolates with potential to enhance phytoremediation.
Syst Appl Microbiol 29:539–556
Prathuangwong S, Dusit A, Wilawan C, Tiyakhon C, Natthiya B (2013) Bioformulation Pseudo-
monas fluorescens SP007s against dirty panicle disease of rice. Afr J Microbiol Res
7:5274–5283
Priti V, Ramesha BT, Singh S, Ravikanth G, Ganeshaiah KN, Suryanarayanan TS, Shaanker RU
(2009) How promising are endophytic fungi as alternative sources of plant secondary
metabolites. Curr Sci 97:477–478
Redman RS, Freeman S, Clifton DR, Morrel J, Brown G, Rodriguez RJ (1999) Biochemical
analysis of plant protection afforded by a nonpathogenic endophytic mutant of Colletotrichum
magna. Plant Physiol 119:795–804
Redman RS, Sheehan KB, Stout RG, Rodriguez RJ, Henson JM (2002) Thermotolerance generated
by plant/fungal symbiosis. Science 298:1581–1581
Ren Y, Strobel GA, Graff JC, Jutila M, Park SG, Gosh S, Teplow D, Condron M, Pang E, Hess
WM, Moore E (2008) Colutellin A, an immunosuppressive peptide from Colletotrichum
dematium. Microbiology 154:1973–1979
Rivera-Varas VV, Freeman TA, Gudmestad NC, Secor GA (2007) Mycoparasitism of
Helminthosporium solani by Acremonium strictum. Phytopathology 97:1331–1337
Rodriguez RJ, Henson J, Van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim Y, Redman RS
(2008) Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2:404–416
Rodriguez RJ, White JF Jr, Arnold AE, Redman ARA (2009) Fungal endophytes: diversity and
functional roles. New Phytol 182:314–330
Romo M, Leuchtmann A, García B, Zabalgogeazcoa I (2007) A totivirus infecting the mutualistic
fungal endophyte Epichloë festucae. Virus Res 124:38–43
Roos IM, Hattingh MJ (1983) Scanning electron microscopy of Pseudomonas syringae pv,
morsprunorum on sweet cherry leaves. J Phytopathol 108:18–25
Rosen HR (1936) Mode of penetration and of progressive invasion of fire-blight bacteria into apple
and pear blossoms. Arkansas Agric Exp Sta Bull 331:1–68
Rosenblueth M, Martínez-Romero E (2004) Rhizobium etli maize populations and their competi-
tiveness for root colonization. Arch Microbiol 181:337–344
Ryu CM, Farag MA, Hu CH, Reddy MS, Wei HX, Pare PW, Kloepper JW (2003) Bacterial
volatiles promote growth in Arabidopsis. Proc Natl Acad Sci USA 100:4927–4932
Saar DE, Polans NO, Sørensen PD, Duvall MR (2001) Angiosperm DNA contamination by
endophytic fungi: detection and methods of avoidance. Plant Mol Biol Rep 19:249–260
Sah A, Negi H, Kapri A, Anwar S, Goel R (2011) Comparative shelf life and efficacy of LDPE and
PVC degrading bacterial consortia under bioformulations. Ekologija 57:55–61
Saikkonen K, Saari S, Helander M (2010) Defensive mutualism between plants and endophytic
fungi? Fungal Divers 41:101–113
Sallam Nashwa M, Riad Shaimaa N, Mohamed MS, Seef Eleslam A (2013) Formulations of
Bacillus spp and Pseudomonas fluorescens for biocontrol of cantaloupe root rot caused by
Fusarium solani. J Plant Prot Res 53:275–300
Sallam Nashwa M, Riad Shaimaa N, Mohamed MS, Seef EA (2014) Biocontrol of cantaloupe
damping-off disease caused by Fusarium semitectum by using formulations of antagonistic
fungi. J Phytopathol Pest Manag 1:5–15
Sánchez Márquez M, Bills GF, Zabalgogeazcoa I (2007) The endophytic mycobiota of the grass
Dactylis glomerata
Schulz B, Boyle C (2006) What are endophytes? In: Microbial root endophytes. Springer, Berlin/
Heidelberg, pp 1–13
Serfling A, Wirsel SGR, Lind V, Deising HB (2007) Performance of the biocontrol fungus
Piriformospora indica on wheat under greenhouse and field conditions. Phytopathology
97:523–531
Shanmugam V, Kanoujia N, Singh M, Singh S, Prasad R (2011) Biocontrol of vascular wilt and
corm rot of gladiolus caused by Fusarium oxysporum f. sp. gladioli using plant growth
promoting rhizobacterial mixture. Crop Prot 30:807–813
Siciliano SD, Goldie H, Germida JJ (1998) Enzymatic activity in root exudates of dahurian wild rye
(Elymus dauricus) that degrades 2-chlorobenzoic acid. J Agric Food Chem 46:5–7
Silva GH, Teles HL, Zanardi LM, Marx Young MC, Eberlin MN, Hadad R, Pfenning LH, Costa-
Neto CM, Castro-Gamboa I, Bolzani YS, Araújo AR (2006) Cadinane sesquiterpenoids of
Phomopsis cassiae, an endophytic fungus associated with Cassia spectabilis (Leguminosae).
Phytochemistry 67:1964–1969
Singh R, Dubey AK (2015) Endophytic actinomycetes as emerging source for therapeutic
compounds. Indo Glob J Pharm Sci 5:106–116
Singh JS, Pandey VC, Singh DP (2011) Efficient soil microorganisms: a new dimension for
sustainable agriculture and environmental development. Agric Ecosyst Environ 140:339–353
Smith CJ (1996) Accumulation of phytoalexins: defense mechanism and stimulus response system.
New Phytol 32:1–45
Smith JA, Métraux JP (1991) Pseudomonas syringae pv. syringae induces systemic resistance to
Pyricularia oryzae in rice. Physiol Mol Plant Pathol 39:451–461
Srivastava S, Verma PC, Chaudhry V, Singh N, Abhilash PC, Kumar KV, Sharma N, Singh N
(2013) Influence of inoculation of arsenic-resistant Staphylococcus arlettae on growth and
arsenic uptake in Brassica juncea (L.) Czern. Var. R-46. J Hazard Mater 262:1039–1047
Stierle A, Stobel G (1995) The search for a taxol-producing microorganism among the endophytic
fungi of the pacific yew, Taxus brevifolia. J Nat Prod 58:1315–1324
Stierle A, Strobel GA, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an
endophytic fungus of Pacific yew. Science 260:214–216
Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorganisms.
J Nat Prod 67:257–268
Sturz AV, Nowak J (2000) Endophytic communities of rhizobacteria and the strategies required to
create yield enhancing associations with crops. Appl Soil Ecol 15:183–190
Su F, Jacquard C, Villaume S, Michel J, Rabenoelina F, Clément C, Barka EA, Dhondt-Cordelier S,
Vaillant-Gaveau N (2015) Burkholderia phytofirmans PsJN reduces impact of freezing
temperatures on photosynthesis in Arabidopsis thaliana. Front Plant Sci 6:810
Sun X, Guo LD (2012) Endophytic fungal diversity: review of traditional and molecular techniques.
Mycology 3:65–76
Sun C, Johnson JM, Cai D, Sherameti I, Oelmüller R, Lou B (2010) Piriformospora indica confers
drought tolerance in Chinese cabbage leaves by stimulating antioxidant enzymes, the expression
of drought-related genes and the plastid-localized CAS protein. J Plant Physiol 167:1009–1017
Sun H, He Y, Xiao Q, Ye R, Tian Y (2013) Isolation, characterization, and antimicrobial activity of
endophytic bacteria from Polygonum cuspidatum. Afr J Microbiol Res 7:1496–1504
Supong K, Thawai C, Choowong W, Kittiwongwattana C, Thanaboripat D, Laosinwattana C,
Koohakan P, Parinthawong N, Pittayakhajonwut P (2016) Antimicrobial compounds from
endophytic Streptomyces sp. BCC72023 isolated from rice (Oryza sativa L.). Res Microbiol
167:290–298
Sziderics AH, Rasche F, Trognitz F, Sessitsch A, Wilhelm E (2007) Bacterial endophytes contrib-
ute to abiotic stress adaptation in pepper plants (Capsicum annum L). Can J Microbiol
53:1195–1202
Taghavi S, Barac T, Greenberg B, Borremans B, Vangronsveld J, van der Lelie D (2005) Horizontal
gene transfer to endogenous endophytic bacteria from poplar improved phyto-remediation of
toluene. Appl Environ Microbiol 71:8500–8505
Tian P, Nan Z, Li C (2008) Effect of the endophyte Neotyphodium lolii on susceptibility and host
physiological response of perennial ryegrass to fungal pathogens. Eur J Plant Pathol
122:593–602
Tripathi S, Kamal S, Sheramati I, Oelmuller R, Varma A (2008) Mycorrhizal fungi and other root
endophytes as biocontrol agents against root pathogens. Mycorrhiza 3:281–306
Tuteja N (2007) Abscisic acid and abiotic stress signaling. Plant Signal Behav 2:135–138
Urairuj C, Khanongnuch C, Lumyong S (2003) Ligninolytic enzymes from tropical endophytic
Xylariaceae. Fungal Divers 13:209–219
Vallad GE, Goodman RM (2004) Systemic acquired resistance and induced systemic resistance in
conventional agriculture. Crop Sci 44:1920–1934
Van Aken B, Peres C, Doty S, Yoon J, Schnoor J (2004) Methylobacterium populi sp. nov., a novel
aerobic, pink pigmented, facultatively methylotrophic, methane-utilising bacterium isolated
from poplar trees (Populus deltoides x nigra DN34). Evol Microbiol 54:1191–1196
Vandermeer J, Perfecto I, Liere H (2009) Evidence for hyperparasitism of coffee rust (Hemileia
vastatrix) by the entomogenous fungus, Lecanicillium lecanii, through a complex ecological
web. Plant Pathol 58:636–641
Vidhyasekaran P, Sethuraman K, Rajappan K, Vasumathi K (1997) Powder formulation of
Pseudomonas fluorescens to control pigeonpea wilt. Biol Control 8:166–171
Vyas P, Robin J, Sharma KC, Rahi P, Gulati A, Gulati A (2010) Cold adapted and rhizosphere
competent strain of Rahnella sp. with broad-spectrum plant growth-promotion potential. J
Wang J, Li G, Lu H, Zheng Z, Huang Y, Su W (2000) Taxol rom Tubercularia sp. strain TF5, an
endophytic fungus of Taxus Mairei. FEMS Microbiol Lett 193(2):249–253
Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M, Heier T, Hückelhoven R,
Neumann C, Von Wettstein D, Franken P, Kogel KH (2005) The endophytic fungus
Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher
yield. Proc Natl Acad Sci USA 102:13386–13391
Wei G, Kloepper JW, Tuzan S (1991) Induction of systemic resistance of cucumber to

Colletotrichum orbiculare by select strains of plant growth-promoting rhizobacteria.
Phytopathol J 81:1508–1512
White JF Jr, Torres MS (2010) Is plant endophyte-mediated defensive mutualism the result of
oxidative stress protection. Physiol Plant 138:440–446
Wicklow DT, Roth S, Deyrup ST, Gloer JB (2005) A protective endophyte of maize: Acremonium
zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. Mycol Res
109:610–618
Wink M (2008) Plant secondary metabolism: diversity, function and its evolution. Nat Prod
Commun 3:1205–1216
Yong YH, Dai CC, Gao FK, Yang QY, Zhao M (2009) Effects of endophytic fungi on growth and
two kinds of terpenoids for Euphorbia pekinensis. Chin Tradit Herb Drugs 40:18–22
You F, Han T, Wu JZ, Huang BK, Qin LP (2009) Antifungal secondary metabolites from
endophytic Verticillium sp. Biochem Syst Ecol 37:162–165
Yuan ZL, Lin FC, Zhang CL, Kubicek CP (2010) A new species of Harpophora (Magnaporthaceae)
recovered from healthy wild rice (Oryza granulata) roots, representing a novel member of a
beneficial dark septate endophyte. FEMS Microbiol Lett 307:94–101
Zabalgogeazcoa I (2008) Fungal endophytes and their interaction with plant pathogens: a review.
Span J Agric Res 6:138–146
Zhang L, Guo B, Li H, Zeng S, Shao H, Gu S, Wei R (2000) Preliminary study on the isolation of
endophytic fungus of Catharanthus roseus and its fermentation to produce products of thera-
peutic value. Chin Tradit Herb Drugs 31:805–807
Zhao K, Penttinen P, Guan T, Xiao J, Chen Q, Xu J, Lindström K, Zhang L, Zhang X, Strobel GA
(2011) The diversity and anti-microbial activity of endophytic actinomycetes isolated from
medicinal plants in Panxi plateau. China Curr Microbiol 62:182–190
Bioprospecting of Diseases of Horticultural
Crops in India 20
V. Devappa, C. G. Sangeetha, and N. Jhansirani
Abstract
Biological management of plant diseases is the need of the day as it is the most
feasible alternative to the present scenario of environmental pollution leading to
pesticide residue in horticultural produce affecting the health of the consumers.
Bioprospecting is searching for new sources of compounds, genes,
microorganisms, macroorganisms, plants and other natural sources. Botanical
fungicides may be effective, selective, biodegradable and less toxic to the envi-
ronment. The biocontrol efficacy of antagonistic microorganisms like Bacillus,
Trichoderma, Pseudomonas and some endophytic bacteria has been exploited for
the management of the diseases of horticultural crops. Bioprospecting has also
been done for the suppression of nematodes by various biological agents. Char-
acterization of the metabolites which play a role in the suppression is also very
much needed. The biocontrol efficacy of antagonistic microorganisms depends
on a combination of factors such as the characteristics of the antagonistic micro-
organism, the epidemiology of the target pathogen and the environmental
conditions in which the relationship between the pathogen and the antagonist
(s) is taking place. Hence, efforts are needed to develop systems by integrating
several strategies taking into consideration pathogen biology, cultivar resistance
and epidemiology.
Keywords
Bioagents · Bacillus · Trichoderma · Pseudomonas · Ecofriendly · Safe
V. Devappa (*) · C. G. Sangeetha · N. Jhansirani

Department of Plant Pathology, College of Horticulture, UHS Campus, Bengaluru, India
e-mail: devappa.v@uhsbagalkot.edu.in; sangeetha.cg@uhsbagalkot.edu.in

https://doi.org/10.1007/978-981-15-6275-4_20
426 V. Devappa et al.
20.1 Introduction
Biological management of diseases of crops deals mainly with the identification of

various ecofriendly approaches to mitigate the ill effects of plant disease by using
antagonistic microorganisms, naturally derived materials of plant and animal origin
and inorganic and organic compounds, to reduce or replace the use of synthetic
chemicals and to integrate the compatible and synergistic strategies for enhancing
the effectiveness of disease suppression. These approaches will protect the environ-
ment, increase the yield of crops and provide pesticide-free products to the
consumers. It is essential that disease management strategies are compatible with
the present cultural practices adopted in a given geographical location to possess the
acceptance of the farmers. Various concepts of biological management of diseases
have evolved from time to time, supported by the knowledge and techniques to
assess the interactions between pathogens, other microorganisms and the plants. The
term “biological control” or “biocontrol” was earlier applied to indicate the control
of “one organism by another organism.” This term now has been used in a wider
sense as the “use of natural or modified organisms, genes, or gene product to reduce
the effects of pathogenic organisms and to favor the development of the desirable
organisms such as crops, trees, animals, beneficial insects and microorganisms.”
Biological management is hence defined as the utilization of biotic and abiotic
agents which act through one or more mechanisms to reduce the potential of the
pathogen directly or indirectly by activating host defense systems and to reduce the
disease incidence and also intensity.
Bioprospecting is prospecting for the new sources of chemical compounds,
genes, microorganisms, macroorganisms and other valuable products from nature
which could be economically valuable genetic and biochemical resources. It also
involves discovering useful products derived from bioresources including plants,
microorganisms, animals, etc. that can be developed further for commercialization
and overall benefit of the society. Horticultural crops which are grown both under
natural conditions and greenhouse are exposed to different environment and nutrient
regimes. The disease problems are likely to vary widely in the crops. Hence, the
extent of losses due to diseases caused by microbial pathogens depends on the
susceptibility or resistance levels of cultivars and available inoculum potential.
Vegetable and fruit crops are high-value crops and hence, greater attention has to
be bestowed on them to protect them against diseases.
20.2 Botanical Fungicides
Botanical fungicides are the most important aspect in the recent situation which will
help us to decrease the negative impacts of synthetic agents, such as residues,
resistance and environmental pollution. In this aspect, botanical fungicides may be
effective, selective, biodegradable, and less toxic to the environment. Some of the
botanical fungicides which have been tried by various workers are as below:
20 Bioprospecting of Diseases of Horticultural Crops in India 427
Cinnamaldehyde Cinnamaldehyde isolated from cinnamon oil, it is effective

against dry bubble caused by Verticillium fungicola, dollar spot caused by
Sclerotinia homoeocarpa and canker caused by Fusarium moniliforme var.
subglutinans (Copping 2004). According to Wang et al. (2005), the leaves of
indigenous cinnamon (Cinnamomum osmophloeum) exhibited strong antifungal
activity against wood decay fungi. Cinnamaldehyde showed 100% efficacy against
both Coriolus versicolor and Laetiporus sulphureus. Cheng et al. (2008) reported
the antifungal activity of cinnamaldehyde and eugenol against wood-rot fungi.
L-Glutamic Acid + Gamma-Aminobutyric Acid These two ingredients enhance

the expansion of plants like almond, broccoli and onions, prevent development of
mildew on grapes and suppress certain other plant diseases caused by Monilia
sp. (Copping 2004).
Jojoba Oil Jojoba oil obtained from the jojoba bean, it has been found to be very
effective and capable of controlling white flies and mildew on ornamental plants and
grapes.
Laminarin Laminarin is a storage glucan (a polysaccharide of glucose) found in

the blue green algae Laminaria digitata. Laminarin efficiently elicits defense
responses in grapevine against gray mold and downy mildew caused by Botrytis
cinerea and Plasmopara viticola, respectively. Laminarin application reduced
B. cinerea and P. viticola infection by 55% and 75%, respectively (Copping
2004). Hu et al. (2012) reported inhibitory effects of laminarin on growth and
toxin production of Aspergillus flavus.
Milsana The ethanolic extract from the plant Reynoutria sachalinensis decreased
the mildew incidence on tomato by a mixture of induced resistance and direct
antifungal activity. A single spray reduced mildew infection on young seedlings of
tomato by 97% (Reglinski 2009). The active ingredient is reported to act as an
elicitor of phytoalexins, which induces resistance (Copping 2004).
20.3 Fruit Crops
20.3.1 Banana
Fusarium wilt is the most devastating disease of banana occurring worldwide and
causing lot of economic losses. Plant extracts have shown antifungal activity and
reduced mycelium growth of Fusarium under both greenhouse and field conditions
(Akila et al. 2011). Gnanasekaran et al. (2015) investigated the in vitro biological
control of Fusarium oxysporum f. sp. cubense by some Indian medicinal plants.
Among the five plants tested, P. betle L. plant extracts exhibited maximum antifun-
gal activity against the tested Foc followed by V. negundo, C. gigantea, C. asiatica,
and O. sanctum plant extracts.
Pre-inoculation with beneficial endophytes through banana plantlets might be an

effective strategy for both biocontrol and growth promotion of banana (Ho et al.
2015). Priming the tissue-cultured banana plantlets led to a substantial reduction in
the infection and severity of wilt disease. It also led to an increase in plant growth
parameters (Jie et al. 2009). The combined application of endophytic and
rhizospheric bacterial strains was effective in suppressing Fusarium wilt, in both
pot and field trials. Two non-pathogenic F. oxysporum isolates CAV 255 and CAV
241 reduced the Fusarium wilt disease incidence by 87.4 and 75.0%, respectively.
The known Fo47 isolate did not suppress the disease significantly. P. fluorescens
strain WCS417 reduced the disease incidence by 87.4% under greenhouse
conditions (Nel et al. 2006). The bacterial bioagents Serratia marcescens effectively
suppressed the development of F. oxysporum f. sp. cubense race 4 (FocR4). The
bacteria S. marcescens was formulated using montmorillonite clay (carrier), nonfat
skimmed milk (NFSM), and sucrose (enrichment materials), which improved the
bacterial cell viability significantly. Bioformulation of S. marcescens with the above
materials was found to be useful for both storage and field application (Ting et al.
2009).
20.3.1.1 Leaf Spot Disease

The Bacillus subtilis strain B106 isolated from the rhizosphere soil of banana field in
China, exhibited effective suppression of Pseudocercospora musae (teleomorph:
Mycosphaerella musicola), causing banana leaf spot disease and Colletotrichum
musae causing postharvest anthracnose disease of banana fruits during storage.
Development of banana leaf spot was suppressed by 72.3% in the greenhouse
experiment at 10 days after pathogen inoculation. The efficacies of strain B106
(1 106 CFU/ml) for controlling both the banana leaf spot diseases in the field and
the anthracnose disease at postharvest stage were 48.3 and 48.6%, respectively,
under optimized cultural conditions for the BCA to express its antagonistic potential.
20.3.2 Citrus Diseases
20.3.2.1 Phytophthora Root Rot Disease

Many workers have reported biological agents for Phytophthora management in
citrus. Gade (2012) reported significant suppression of P. parasitica by Trichoderma
harzianum and T. virens. There was a continuous reduction in pathogen population
from 41 to 8 propagules/g soil with reduction in root rot/collar rot in Citrus jambhiri.
Biochemically efficient strain of P. fluorescens PfIV was found effective to arrest the
percent mycelial growth (55.20%) of P. parasitica. Gade and Koche (2012) reported
that P. fluorescens in combination with fungicides and organic amendments was
effective in the management of root rot and gummosis in Nagpur mandarin. Signifi-
cant decrease in intensity of both root rot and gummosis was observed. Root rot
intensity reduced from 36.18% to 16.70%. Similarly, significant reduction in gum-
mosis up to 54.76% was recorded. T. viride inhibited the highest mycelial growth of
P. parasitica (75.33%) in vitro, whereas, under glasshouse experiment, combined
application of T. viride at 4 g/Kg + garlic clove extract at 5% significantly reduced

percent root rot incidence (11.32%) as compared to untreated control (44.99%)
(Pente et al. 2015). Lende et al. (2015) found significant increase in shoot length
(23.55 cm) and canopy volume of tree (10.89%) in Nagpur mandarin when bioagent
T. harzianum was incorporated in combination with chemicals and organic
amendments and also found significant reduction in root rot intensity. Greenhouse
studies conducted by Abraham (2005) showed that the Bacillus and some
Trichoderma isolates suppressed Phytophthora root rot disease.
20.3.2.2 Citrus Canker

Some strains of bacteria, viz., P. syringae, E. herbicola, B. subtilis, and
P. fluorescens, isolated from citrus phylloplane are reported to be antagonistic
in vitro to the canker pathogen (Ota 1983; Goto et al. 1979; Unnimalai and
Gnanamanickam 1984).
Kalita et al. (1996) reported that three bacteria, viz., B. subtilis, B. polymyxa, and
P. fluorescens, and three fungi, viz., Aspergillus terreus, T. viride, and T. harzianum,
isolated from the phylloplane of citrus variety Assam lemon (Citrus limon) inhibited
the growth of Xanthomonas citri subsp. citri in vitro. When the antagonists were
tested for their efficacy under field condition by applying them over crop foliage,
they also reduced citrus canker incidence. B. subtilis was found to be the most
effective antagonist exhibiting maximum (14.70 mm) inhibition of the pathogen and
reducing the disease incidence to an extent of 61.90%.
Five collections of P. citrinum (P1, P2, P3, P4, and P5) and three collections of
Trichoderma spp., i.e., T. virens (TD1), T. harzianum (TD2), and T. virens (TD3),
were tested for biocontrol activity against Fusarium solani (F1), F. oxysporum
(F10), and E. chlamydosporum (F6) (Misra 2008). All the Penicillium isolates
were able to suppress all the three Fusarium spp. A very clear zone of inhibition
was formed with T. virens (TD1 and TD3) with all the three Fusarium spp. However,
T. harzianum (TD2) made very clear zone with F. solani and F. oxysporum.
20.3.3 Guava
Guava wilt is one of the major diseases affecting guava. The filterate of the cultures
and released volatile compounds of eight bioagents, comprising three isolates of
Aspergillus niger, three isolates of Trichoderma spp., and two isolates of Penicillium
citrinum, were evaluated against ten isolates of Fusarium spp. (five isolates each of
F. oxysporum f. sp. psidii and F. solani) causing wilt disease of guava. It was found
that all the fungal bioagents significantly reduced the growth of F. oxysporum f. sp.
psidii and F. solani (Misra 2008). Shruthi et al. (2019) studied volatile compound
production by indigenous Trichoderma isolates against C. fimbriata. Eleven isolates
of Trichoderma (PT-I to PT-11) were positive for volatile compound production
indicating their biocontrol activity. Higher concentrations of volatile metabolites
were produced in isolates PT-6 (78.84%) followed by PT-11 (78.67%) and PT-10
(71.84%), and lowest concentration of volatile metabolites was produced by PT-8
(50.00%).
20.3.4 Grapes
20.3.4.1 Powdery Mildew

Powdery mildew disease caused by Uncinula necator is one of the most economical
diseases infecting both leaves and the berries. The basidiomycetous yeast
Pseudozyma flocculosa (syn. Sporothrix flocculosa) was reported to be an efficient
natural antagonist of U. necator (Belagner and Labbe 2002). Sawant et al. (2015)
identified T. afroharzianum NAIMCC-F-01938, which provided about 50% reduc-
tion in disease severity. The workers also reported that replacing two late-season
sulfur applications with that of T. afroharzianum, in a fungicide spray schedule,
enhanced powdery mildew control by 31% as compared to the only fungicide
schedule. Melidossia et al. (2005) reported management of powdery mildew using
mycophagous studied mite, Orthotydeus lambi. The mites applied at 30 mites per
leaf could suppress disease when applied pre-bloom or 1 week after bloom. In
grapes, B. subtilis KSI isolated from grape berry skin could reduce downy mildew
on berries and leaves (Furuya et al. 2011).
20.3.4.2 Downy Mildew Disease

Downy mildew disease of grapes is of historical and economic importance. The
effect of Pen, an aqueous extract of the dry mycelium of Penicillium chrysogenum,
was investigated on the suppression of grapevine downy and powdery mildew
disease under greenhouse and field conditions. Pen extract had no direct inhibitory
effect on the grapevine pathogens. Hence, it is considered that Pen might act
indirectly by inducing resistance in treated plants.
20.3.4.3 Gray Mold Disease

Biocontrol methods for the suppression of the gray mold pathogen Botrytis cinerea
can be successful only with the understanding of the ecology and epidemiology of
B. cinerea in the vineyards. An isolate of Ulocladium oudemansii effectively
suppressed the development of B. cinerea on necrotic grape tissues (Elmer et al.
2003; Shorten et al. 2003). A commercial formulation of U. oudemansii (BOTRY-
Zen®) was found to be as effective as the fungicide program (Elmer et al. 2003). The
efficacy of the chemical elicitor 5-chlorosalicylic acid and the fungal antagonist
U. oudemansii in suppressing the development of Botrytis cinerea infecting
grapevines was compared, when applied alone and in combination under greenhouse
conditions. Mochizuki et al. (2012) isolated B. amyloliquefaciens strain S13-3,
which showed good inhibitory activity against C. gloeosporioides in vitro and
could decrease ripe rot caused by C. gloeosporioides in vineyard.
20.3.4.4 Crown Gall Disease

Crown gall disease caused by Agrobacterium vitis is one of the most destructive
diseases of grapevines occurring in several countries. Agrobacterium rhizogenes
strain K84 was effective against A. tumefaciens infecting peach, but not against
A. vitis (Kerr 1980). Rahnella aquatilis isolated from vineyard soils in Beijing was
evaluated for its biocontrol potential against A. vitis. R. aquatilis strain HX2
exhibited significant suppressive effect on the development of crown galls in
grapevines. Under field conditions, immersion of the basal ends of grape cuttings
with HX2 cell suspensions inhibited or completely prevented crown gall formation
caused by A. vitis K308 in the roots of the plants growing from the cuttings.
20.3.4.5 Anthracnose
Liang et al. (2016) obtained a highly antagonistic strain of Streptomyces atratus
PY-1, which could reduce disease severity by 92.13% in the detached leaf assay and
by 83% in a field assay. Sawant et al. (2015) evaluated 34 Trichoderma isolates
against anthracnose caused by C. gloeosporioides hyphae in dual culture studies.
The isolates produced volatile and non-volatile metabolites which inhibited the
radial growth of C. gloeosporioides, but their efficacies varied. Sawant et al.
(2016) isolated and screened 293 bacteria from the grape ecosystem of 43 spatially
distant vineyards in the Peninsular India and identified 7 Bacillus isolates with
significant biocontrol abilities. Narkar et al. (2017) evaluated 87 endophytic bacteria
isolated from the mature shoots of Thompson Seedless for their antagonistic activity
against carbendazim-resistant C. gloeosporioides isolates. They could identify three
Bacillus amyloliquefaciens strains which gave good control of anthracnose under
natural field conditions. Mochizuki et al. (2012) isolated B. amyloliquefaciens strain
S13-3 which showed good inhibitory activity against C. gloeosporioides in vitro and
could control ripe rot caused by C. gloeosporioides in vineyard. They attributed the
antagonistic activity of S13-3 toward C. gloeosporioides on iturin A production by
the strain.
20.4 Vegetables
20.4.1 Tomato
20.4.1.1 Fusarium Wilt Disease

Application of soil amendments to encourage the antagonistic microorganisms has
been followed for the reduction of soilborne diseases. Chitosan has been applied as a
soil amendment to suppress the development of Fusarium oxysporum f. sp. radicis-
lycopersici. The efficacy of strains of non-pathogenic Fusarium oxysporum in
suppressing the Fusarium wilt of tomato was reported by Larkin and Fravel
(1998). The BCA strain CS-20 reduced the Fusarium wilt disease at all temperature
tested and four different field soils varying in texture and organic matter content.
This strain was also effective against all three races and reduced the disease
incidence by 48–66%. All formulations (FOR1 to FOR8) were applied to seedlings
in seedbeds at 7 days before transplanting. The percent disease reduction varied from
22 to 64% with all formulations. Pseudomonas fluorescens suppressed the develop-
ment of tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The Pf1
formulation at 10 ml/kg seeds and 150 ml/ha were found to be optimum for seed
treatment and seedling root dip treatments to achieve effective control of tomato wilt
disease (Manikandan et al. 2010). The bio-efficacy assays showed that addition of
glycerol in the production medium reduced the tomato Fusarium wilt disease by
44–50% (Sriram et al. 2011).
Srivastava et al. (2010) observed that the combination of the bacterial Pseudomo-
nas spp. and Trichoderma harzianum and mycorrhizal bioagents protected the
tomato plants in a highly effective manner, resulting in a reduction of disease
incidence by 74 and 67%, respectively, under greenhouse and field conditions.
Yield of tomato fruits due to treatment was also increased by 20%. Addition of
cow dung compost still further reduced disease incidence and improved the yield in
all treated plots. Application of carbendazim at a low concentration (1 mg/ml) in
combination with B. cepacia or B. megaterium reduced the disease symptoms by
46 and 84%, respectively, compared with carbendazim alone (77%) and untreated
control. The antagonistic potential and compatibility with fungicides of Bacillus
megaterium (strain 96) and Burkholderia cepacia (strain 91) were investigated for
the control of tomato crown and root rot caused by F. oxysporum f. sp. radicis-
lycopersici (Omar et al. 2006).
20.4.1.2 Damping-Off
Bacterial bioagents like Pseudomonas fluorescens, P. putida, P. marginalis,
P. corrugata, and P. viridiflava reduced the incidence of damping-off caused by
both P. aphanidermatum and P. ultimum (Gravel et al. 2005). Treatment of tomato
seeds with Bacillus subtilis AUBS-1 formulations in lignite, lignite+ fly ash, and
bentonite paste resulted in the effective suppression of the damping-off disease
caused by P. aphanidermatum and enhancement of plant biomass under glasshouse
and field conditions (Jayaraj et al. 2005). Soil application of Bacillus subtilis RB14-
C protected the tomato seedlings against the damping-off disease caused by Rhizoc-
tonia solani (Szezech and Shoda 2006). Pseudomonas fluorescens isolate CW2 in
combination with fungicides azoxystrobin, metalaxyl-M, or pyraclosporin was
assessed for their efficacy. The fungicides were fungitoxic to P. ultimum, but did
not inhibit the development of P. fluorescens in in vitro assays (Salman and
Abuamsha 2012).
20.4.1.3 Late Blight

Tran et al. (2007) showed that the compound mass A produced by P. fluorescens was
effective in preventing tomato late blight infection and the further spread of the late
blight lesions also reduced significantly. The study determined that induced systemic
resistance was responsible for their results (Tran et al. 2007). Zakharchenko et al.
(2011) demonstrated increased protection against phytopathogens including
P. infestans when plants were colonized with a strain of P. aureofaciens. Various
naturally occurring microorganisms, like T. viride, Penicillium viridicatum,
P. aurantiogriseum, Chaetomium brasiliense (Gupta et al. 2004), Acremonium

strictum, Myrothecium verrucaria, and P. aurantiogriseum (Roy et al. 1991),
showed antagonistic effect against P. infestans. The antagonistic activities of
P. fluorescens, Pseudomonas sp., Aspergillus flavus, A. niger, Penicillium sp.,
T. virens, and T. harzianum showed positive inhibition of mycelial growth of
P. infestans. Bacillus pumilus SE34 and Pseudomonas fluorescens 89B61 were
employed to elicit systemic resistance in tomato against late blight disease.
20.4.1.4 Early Blight

T. viride was found to be effective against early blight of potato for reducing disease
intensity under field conditions (Yadav and Pathak 2011). The bioagents
T. harzianum and P. fluorescens (seed treatment + foliar spray) were effective in
reducing the disease intensity of early blight of tomato and also increasing the yield
(Mane et al. 2014).
20.4.1.5 Bacterial Wilt

Biocontrol of bacterial wilt by the use of antagonists such as P. fluorescens, Bacillus
spp., avirulent P. solanacearum, and actinomycetes has been found to be effective in
some countries (Mclaughlin and Sequerira 1988; Aspiras et al. 1986). Vesicular
arbuscular mycorrhizae (VAM) increased growth and yield of tomatoes and reduced
infection by R. solanacearum. This may be due to competition or the mechanical
barrier in the form of VAM vesicles and hyphae that inhibit the bacterial pathogen
from deeper penetration into host tissues (Halos and Zorilla 1979). Treatment of
tubers with avirulent strain of R. solanacearum and strain of P. fluorescens caused a
significant reduction in disease severity of bacterial wilt of potato (Kempe and
Sequeira 1983).
Biological control of R. solanacearum has been reported in tomato (Guo et al.
2004) and in brinjal (Ramesh et al. 2009). Pseudomonas fluorescens induced ISR in
tomato plants and prevented bacterial wilt (Kempe and Sequeira 1983). Xue et al.
(2009) reported soil drenching with antagonistic strains of Acinetobacter sp. and
Enterobacter sp. which reduced bacterial wilt incidence in tomato plants. Bacillus
species are very effective in reducing bacterial wilt incidence in tomato (Li et al.
2004).
Fujiwara et al. (2011) reported effectiveness of R. solanacearum specific phage
ORSL1, a jumbo phage belonging to Myoviridae, in preventing bacterial wilt in
tomato. Infective phage particles were detected in plant rhizosphere and bulk soil
highlighting its usefulness as BCA. Other phages with less effectiveness in bacterial
wilt control include phage ORSAI (Myoviridae) with a very wide host range (infects
race 1, 3, or 4 and biovar 1, N2, 3, 4 of R. solanacearum) and ORSB1, with T7-like
morphology belonging to Podoviridae (infects race 1, 3, or 4 of R. solanacearum
strains) (Fujiwara et al. 2011; Kawasaki et al. 2009). Phage PE226, a long lytic
flexible filamentous (Inoviridae) with circular (+) sense single-strand DNA genome,
has been isolated from tomato, potato, and pepper rhizosphere soil and infects nine
different R. solanacearum including GMIl000 (Murugaiyan et al. 2010). A new
phage ORS138 (Siphoviridae) has been isolated from tomato field soil lysed strains
of R. solanacearum (Thi et al. 2016). Co-inoculation of phage PE204 (Podoviridae)

and R. solanacearum in tomato rhizosphere completely inhibited bacterial wilt
infection. However, pretreatment with phage PE204 was less effective than post-
treatment in bacterial wilt prevention (Bae et al. 2012). Addy et al. (2012a) reported
loss of major virulence factors EPS, endoglucanase, and extracellular hydrolytic
enzymes in R. solanacearum cells infected with phage ORSM3 (Inoviridae). Con-
trastingly, phage ORSSl (Inoviridae) enhanced virulence of R. solanacearum by
inducing early expression of phcA (Addy et al. 2012b).
Frey et al. (1994) reported bacteriocin involved in bacterial wilt prevention by
Hrp mutants of R. solanacearum. Avirulent R. solanacearum strains in combination
with Pseudomonas fluorescens induced ISR in tomato plants and prevented bacterial
wilt (Kempe and Sequeira 1983). Etchebar et al. (1998) studied xylem colonization
by HrcV mutant of R. solanacearum and reported its efficacy in the prevention of
bacterial wilt in tomato.
20.4.1.6 Bacterial Spot

In the field trials conducted in Alabama and Florida, Pseudomonas syringae strain
Cit7 was found to be the most effective in suppressing bacterial spot disease in two
of three trials. Bacillus pumilus SE34 suppressed the development of bacterial leaf
spot in two field trials. Combined application of these two strains was effective
against bacterial spot and bacterial speck of Pseudomonas syringae pv. tomato in all
the trials. Both bacterial strains appear to enhance the level of resistance in treated
tomato plants (Ji et al. 2006). Pseudomonas fluorescens and benzothiadiazole (BTH)
were applied as seed treatment or foliar spray for the control of bacterial spot disease
under field conditions. All treatments effectively reduced the severity of bacterial
spot disease, compared with untreated control plants. Foliar application of
P. fluorescens was the most effective treatment in reducing disease severity. The
combined application of P. fluorescens and BTH reduced the pathogen population
effectively and also promoted plant growth (Abo-Elyousr and El-Hendawy 2008).
Phosphorus acid salts (PASs) were evaluated for their ability to suppress the
development of tomato bacterial spot disease under field conditions for a period of
3 years.
20.4.1.7 Bacterial Speck

Non-pathogenic strains of P. syringae Cit7 most effectively reduced the disease
intensity in the greenhouse conditions, when P. syringae strain TLP2, Pseudomonas
fluorescens strain A506, and P. syringae pv. syringae DC3000 hrp mutants were
also tested for their efficacy. The strain Cit7 provided a mean level of disease
reduction of 78%, and hence, this strain was tested under field conditions at different
locations in Alabama and Florida, USA, and Ontario, Canada. P. syringae Cit7 was
the most effective in reducing disease severity. The mean level of disease reduction
was 28% over ten different field experiments. P. fluorescens A506 available com-
mercially as BlightBan provided a mean level of disease reduction of 18% over nine
different field experiments. Commercially available plant activators like
benzothiadiazole (BTH) (inducer of SAR) and plant growth-promoting rhizobacteria
have been shown to be effective, when applied individually. Application of BTH on

greenhouse-grown tomatoes effectively reduced bacterial speck disease incidence
and severity, both alone and in combination with the ISR-inducing bacterial product.
20.4.1.8 Bacterial Canker

The effectiveness of Bacillus subtilis, Trichoderma harzianum, and Rhodosporidium
diobovatum in suppressing the development of tomato bacterial canker disease was
indicated by Utkhede and Koch (2004). Pretreatment of tomato plants with ASM
reduced the severity of the canker disease. Development of resistance to canker
disease required an interval of 1–7 days between inducer application and challenge
inoculation. Highest level of protection could be obtained, when plants were
inoculated at 3 days after ASM application (Soylu et al. 2003; Baysal et al. 2003).
Foliar sprays at 500 mg/ml of DL-ß-aminobutyric acid (BABA) suppressed canker
disease development up to 54% 14 days after inoculation. Bacterial populations were
reduced by 84% in planta treated with BABA (Baysal et al. 2003). In another
investigation, the effect of BABA application alone or in combination with Pseudo-
monas fluorescens isolate CW2 in suppressing the development of tomato canker
disease was assessed. Soil treatment with BABA or isolate CW2 significantly
reduced the incidence of bacterial canker. Combined sequential treatments with
BABA and isolate CW2 were found to be more effective in reducing the disease
severity, compared to treatment with either BABA or isolate CW2. The combined
application was effective not only for protecting the tomato plants against canker
disease but also for promoting the growth of plants (Hassan and Buchenauer 2008).
20.4.1.9 Tomato Spotted Wilt Disease

Lecanicillium lecanii, Metarhizium anisopliae, and Beauveria bassiana were
reported to be pathogenic on F. occidentalis (Vestergaard et al. 1995; Sengonca
et al. 2006). A strain of L. lecanii originally isolated from glasshouse whitefly was
also effective against thrip species Frankliniella occidentalis (Van der Schaaf et al.
1991). Paecilomyces lilacinus, a soil-inhabiting nematophagous fungus, produces
chitinases and proteases capable of breaking down egg shell, facilitating penetration
into insect body. This mechanism was demonstrated to operate effectively against
F. occidentalis (Fiedler and Sosnowska 2007). In another investigation, five strains
of Beauveria bassiana were evaluated for their efficacy against F. occidentalis. The
strain RSB of B. bassiana was the most virulent causing 69–96% mortality at
concentrations of 1 104 to 1 107 conidia/ml, at 10 days after inoculation of
first instar larvae. In greenhouse evaluation, RSB strain applied to broccoli foliage
significantly reduced adult and larval populations of F. occidentalis (Gao et al.
2012).
20.4.2 Potato
20.4.2.1 Late Blight

The antagonist Bacillus subtilis B5 was found effective in inhibiting the growth of
P. infestans (Ajay and Sunaina 2005). Certain phyllosphere microorganisms like
Sporobolomyces spp., Acetobacter spp., Pseudomonas spp., and Bacillus spp. were
antagonistic to P. infestans (Ramos et al. 1993; Sanchez et al. 1998). Bacillus
sp. inhibited mycelial growth of P. infestans both in vitro and in vivo (Sadlers 1996).
Streptomyces violaceusniger strain YCED-9 was strongly antagonistic to isolates
of P. infestans in vitro. Rhamnolipid-based formulation (0.25%) from Pseudomonas
spp. was tested under field trials at three different locations. The terminal disease
severity in rhamnolipid formulation was 45% (compared to 100% in control). Ramos
et al. (1993) and Sanchez et al. (1998) reported that certain microorganisms in the
phyllosphere are antagonistic to P. infestans. These include the yeast
Sporobolomyces spp. and isolates of Pseudomonas spp.
20.4.2.2 Verticillium Wilt

The efficacy of sweet corn varieties (Jubilee Sweet Corn and Jubilee Super Sweet
Corn) as green manure was assessed for suppressing the Verticillium wilt disease of
potato caused by Verticillium dahliae. The sweet corn varieties suppressed the
disease incidence by 60–70%. These treatments did not influence the pathogen
populations directly, but the colonization of V. dahliae on potato feeder roots and
in potato tissues of stem pieces was reduced. Feeder root colonization was positively
correlated with Verticillium wilt disease incidence (P < 0.05) and negatively
corrected with yield. In addition, corn green manures increased the populations of
several soil fungi such as Ulocladium and Fusarium equiseti. When potato was
grown consecutively for 2 years, the beneficial effects of sweet corn green manures
were almost entirely lost. But following two consecutive years of potato, a single
sweet corn crop was enough to restore the original benefit of disease suppression and
enhanced yields, although the pathogen populations had increased by fourfold. The
results indicated the effectiveness of growing green manure crops that could reduce
disease incidence and increase the yield as well (Davis et al. 2010a). Austrian peas,
Sudan grass, rape, oats, and rye also exerted similar beneficial effects by reducing
disease incidence and enhancing potato yields (Davis et al. 2010b). Bacterial isolate
DF37 could reduce the Verticillium wilt disease by 29–43% and increased the yield
of the cultivar Russet Burbank by 24% (Uppal et al. 2008).
20.4.2.3 Stem Rot

Sixteen isolates belonging to 11 species of Trichoderma were evaluated for their
potential to suppress the development of stem rot disease. In addition, one isolate of
Talaromyces flavus was also included in the evaluation. Spore suspensions of these
fungi were sprayed on the foliage in the greenhouse assays. T. koningii, T. virens,
T. ceramicum, and T. viridescens were more effective in reducing disease severity,
while T. flavus was the least effective against the stem rot pathogen. T. viridescens
followed by T. ceramicum provided the best protection to the potato plants against
S. sclerotiorum (Ojaghian 2011).
20.4.3 Brinjal
20.4.3.1 Damping-Off
Seed coating and soil application of Trichoderma formulation reduced the damping-
off disease caused by Pythium spp. in many vegetables. Seed priming with slurry of
Trichoderma resulted in 70% more plant stand in nursery bed as compared to
non-treated plot in tomato and brinjal (Harmann and Taylor 1989). Seed treatment
with talc-based formulation of T. viride @ 4 g/kg showed 7.0 and 12.5% pre- and
post-emergence damping-off of chili, respectively, against 27.50 and 75% in control
with reduction of pathogen population (Manoranjitham et al. 2000).
20.4.3.2 Verticillium Wilt

B. subtilis strain could effectively reduce the incidence and severity of wilt under
greenhouse and field condition (Luo et al. 2010). Application of bioorganic fertilizer
containing B. subtilis at the beginning of nursery and during transplanting control the
Verticillium wilt by significantly changing fungal community structure and reducing
pathogen population in rhizosphere (Lang et al. 2012). Karagiannidis et al. (2002)
inoculated mycorrhizal fungi Glomus mosseae to control Verticillium wilt of egg-
plant and found very low disease incidence due to enhanced root colonization,
growth, and nutrient uptake in plant. A root- and rhizosphere-colonizing fungal
strain QLP12 with broad-spectrum antifungal activity was identified in China as
Purpureocillium lilacinum which showed excellent growth-promoting effect on
eggplant seed germination (76.70%), bud growth (79.40%), chlorophyll content
(47.83%), and root activity (182.02%) and reduced the incidence of Verticillium
wilt by 83.82% in greenhouse (Lan et al. 2017). Mixing the transplant soil plug with
Paenibacillus alvei Kl65 or non-pathogenic F. oxysporum F2, at a rate of 10 and
20% (v/V), respectively, reduced Verticillium wilt symptom development with PRI
and PR4 expression in the roots of brinjal (Angelopoulou et al. 2014).
20.4.3.3 Collar Rot

The soil and seedling dip treatment with T. viride @4 g/kg of seedling and 50 mg
with 5 g FYM/m, respectively, was found most effective in reducing the percent
disease incidence of collar rot followed by summer plowing and also increased the
growth (Jadon 2009). Elad et al. (1999) proposed that spore germination and germ
tube elongation of S. sclerotiorum were hindered by protease released from
Trichoderma spp. Sporidesmium sclerotivorum was detected in soil sample from
fields which caused a natural decline of S. sclerotiorum in field soils. The increase in
glucanase activity may be stimulated by the production of haustoria by S.
sclerotivorum in the cells of sclerotia of S. sclerotiorum. Talaromyces flavus is
another mycoparasite of S. sclerotiorum (Su and Leu 1980). McLaren et al. (1982)
obtained 92% disease control of sunflower wilt caused by S. sclerotiorum when

T. flavus and sclerotia were buried together in the field.
20.4.3.4 Fusarium Wilt

Several species of Trichoderma and P. fluorescens were widely used for controlling
vascular wilt caused by Fusarium spp. by several workers (Elad and Baker 1985;
Biswas and Das 1999; Vyas and Mathur 2002; Najar et al. 2011).
20.4.3.5 Stem and Root Rot

Several microorganisms including Trichoderma spp. and P. fluorescens are widely
used for controlling Rhizoctonia disease in soil by suppressing the competitive
saprophytic ability. Bunker and Mathur (2001) reported that 51% more disease
control was achieved by seed treatment and soil application of Trichoderma formu-
lation against capsicum root rot caused by R. solani. Plant root colonized by VAM
fungi tolerates the infection of R. solani to a great extent (Zambolim and Schenck
1983). Combined seed bacterization with B. subtilis CA32 and soil application of
compatible T. harzianum RUO1 significantly enhanced protection of brinjal from
R. solani (Abeysinghe 2009).
20.4.3.6 Phomopsis Blight

T. virens, T. harzianum, and T. viride are effective in antagonizing the mycelial
growth of P. vexans on brinjal in vitro (Muneeshwar et al. 2011). Antimicrobial
actions of B. subtilis, Streptomyces griseus, and fungal species such as Aspergillus,
Penicillium, Trichoderma, and Periconia have been tested against Phomopsis
vexans (Varma and Bhale 2010). Antagonistic P. fluorescens and T. harzianum
seed treatment and foliar applications are effective against P. vexans (Srinivasa
et al. 2005; Das et al. 2014). Sowing of apparently healthy seeds treated with garlic
bulb extract and soil treatment with T. harzianum completely controlled damping-
off, tip over, and seedling blight in the nursery bed with 48.83% increased seed
germination over control in Bangladesh (Islam and Meah 201). Rohini et al. (2016)
reported that combined application of rhizosphere-colonizing bacteria P. putida
Has-1/c and phylloplane-colonizing bacteria B. subtilis Br/ph-11 significantly
reduced the disease incidence (18.00%) and severity (0.54) in comparison with
distilled water-treated control (91.00% and 6.00%).
20.4.3.7 Bacterial Wilt

Bacteria of the genera Pseudomonas, Bacillus, Paenibacillus, and Sphingomonas
isolated from bacterial wilt-resistant plants were reported to be highly antagonistic to
R. solanacearum (Feng et al. 2013). Very high biocontrol effect against bacterial wilt
and growth promotion in tomato was exhibited by P. fluorescens (Seleim et al.
2011). Endophytic strains of Burkholderia sp., Bacillus sp., and DAPG-producing
Pseudomonas sp. from brinjal were found inhibitory to R. solanacearum (Ramesh
et al. 2009).
20.4.4 Cucurbitaceous Crops
20.4.4.1 Pythium Root Diseases

The bacterial strains Bacillus subtilis, Pseudomonas fluorescens, and P. corrugata
and two fungal strains Trichoderma viride and T. (Gliocladium) virens were
evaluated for their ability to suppress the damping-off disease of cucumber caused
by P. ultimum. Among the antagonists, Pseudomonas spp. were superior to Bacillus
subtilis in reducing the incidence of damping-off disease in cucumber. Combination
of antagonists did not show any additive effect. The effectiveness of disease sup-
pression was greater, when the bacterial antagonists were applied by drenching or by
coating the cucumber seeds with bacteria in a peat carrier (Georgakopoulos et al.
2002).
20.4.4.2 Fusarium Wilt Disease

The strains of Pseudomonas aeruginosa isolated from the composts exhibited the
greatest antagonistic activity against FORC. Further, internal stem colonization of
FORC was significantly reduced by P. aeruginosa (Bradley and Punja 2010).
Application of compost reduced the incidence of Fusarium wilt disease of melon.
Aspergillus sp. isolated from the compost was the most effective one. The suppres-
siveness of the compost was associated with the population of Aspergillus spp.
(Suárez-Estrella et al. 2007). The efficacy of two citrus composts composed of
40% citrus wastes, 20% sludge obtained from citrus industry wastewater treatment
facility, and 40% green residues and C2 composed of 60% citrus wastes amended
with Trichoderma harzianum T-78 was evaluated for the control of Fusarium
oxysporum f. sp. melonis (FOM). Incidence of Fusarium wilt disease on melon
and growth promotion effect of the treatments was recorded. Disease incidence was
significantly reduced in C2Th (extract amended with T-78), while C1Th was not
effective. Population of T-78 significantly decreased at first sampling time compared
to the initial level, but later recovered over time. The results indicated that combina-
tion of citrus compost and T. harzianum T-78 could become a viable alternative to
peat and also adoption of this strategy could minimize the chemical use for the
management of Fusarium wilt disease in greenhouse nurseries for melon seedling
production (Lopez-Mondejar et al. 2010). The bioorganic fertilizer containing an
organic fertilizer and Paenibacillus polymyxa (3 107 CFU/g) and Trichoderma
harzianum (5 107 CFU/g) was evaluated for its efficacy in suppressing the
development of watermelon Fusarium wilt disease caused by F. oxysporum f. sp.
niveum. The incidence of Fusarium wilt disease was reduced by 84.9 and 75.0% at
27 and 63 days after treatment with bioorganic fertilizer (0.5%) under greenhouse
conditions (Wu et al. 2009). The incidence of Fusarium wilt disease was reduced by
60–100% in the greenhouse and by 59–73% under field conditions. Nursery appli-
cation of B10 reduced the pathogen population in the soil significantly. The bacterial
BCA Paenibacillus polymyxa present in the product effectively colonized the
rhizosphere of watermelon and proliferated along the extending plant roots (Ling
et al. 2010).
20.4.4.3 Anthracnose Disease

Plant growth-promoting rhizobacterial strains Bacillus pumilus strain INR7, Bacillus
subtilis GB03, and Curtobacterium flaccumfaciens strain ME1 either alone or in
combination of strains reduced the severity of anthracnose disease caused by
Colletotrichum orbiculare and bacterial angular leaf spot caused by Pseudomonas
syringae pv. lachrymans. Serratia marcescens suppressed the development of
anthracnose disease caused by C. orbiculare in cucumber through induced systemic
resistance (ISR). Bacillus mycoides isolate BmJ and B. mojavensis isolate 203-7
were tested for their ability to induce systemic resistance in treated plants against the
anthracnose disease. The isolates BmJ and 203-7 delayed disease onset and reduced
total (43 and 56%) and live spore production (38 and 49%) per mm2 of lesion area by
inducing systemic acquired resistance in cucumber. Field experiments (2004 and
2005) were conducted to evaluate the efficacy of applications of BmJ and fungicides
for the control of anthracnose in cucumber cv. General Lee and cantaloupe. BmJ
applied 7 days before inoculation with the pathogen reduced disease severity by 41%
in cucumber in 2004 and by 21–14% in cantaloupe for both years, compared with
water controls which were in equivalence to fungicides azoxystrobin and
chlorothalonil. BmJ applied 1 week prior to inoculation with the pathogen signifi-
cantly reduced the AUDPC values (P ¼ 0.05) in cucumber, compared with control
plots (Neher et al. 2009).
20.4.4.4 Powdery Mildew Disease

Verticillium (¼ Lecanicillium) lecanii is reported to effectively suppress the cucum-
ber powdery mildew development. In addition, V. lecanii was also pathogenic to the
aphid Macrosiphum euphorbiae which is an efficient vector of Cucumber mosaic
virus infecting cucumber and other cucurbitaceous crops. The biocontrol efficacy of
two mycoparasite-based products AQ10® containing Ampelomyces quisqualis and
Mycotal® containing Lecanicillium lecanii as well as three strains of Bacillus
subtilis was assessed for the control of the melon powdery mildew disease caused
by Podosphaera fusca. The mycoparasites were more effective, when the relative
humidity values in the greenhouse were 90–95%. The effectiveness of the
mycoparasites A. quisqualis and L. lecanii was absolutely dependent on mineral
oil. The mycoparasites were most effective only in combination with the mineral oil
ADDIT, showing a disease reduction of 80–95%. On the other hand, the strains of
Bacillus subtilis without any complementary additive were very effective in
providing disease reduction similar to mycoparasites with mineral oil or the fungi-
cide azoxystrobin. The results revealed the effectiveness of these BCAs for
suppressing the development of powdery mildew disease of greenhouse melons/
cucurbits either as single products or as a component of integrated control programs
(Romero et al. 2007). Three commercially available products Actinovate (Strepto-
myces lydicus WYEC108), Companion (Bacillus subtilis GB03), and Sonata ASO
(Bacillus pumilus QRD2808) were evaluated along with the fungicide quinoxyfen
for their efficacy in suppressing the development of powdery mildew in pumpkin
under field conditions where the disease pressure was high. Penetration of foliar
sprays of the bacterial BCA strains to the lower surfaces appeared to be restricted,
resulting in higher incidence and severity of the disease in the lower surface of
leaves. Disease incidence was lower in B. subtilis-treated foliage than on leaves
treated with S. lydicus or B. pumilus. Disease incidence on the upper surface of
leaves treated with three species in rotation was similar to levels in B. subtilis-treated
plots. The fungicide quinoxyfen was the most effective in reducing the disease
severity and outperformed all three bacterial BCAs tested (Janousek et al. 2009).
20.5 Medicinal Plants
20.5.1 Cumin
Bio-efficacy of different bioagents, viz., T. harzianum, T. viride, Aspergillus niger,

A. flavus, P. fluorescens, Bacillus subtilis, Paecilomyces lilacinus, and
P. chlamydosporia, has been tested under in vitro and in vivo conditions (Deepak
et al. 2009; Shelar 2013). With the aim of sustainable management of cumin blight,
in vitro mycelial growth inhibition of Alternaria burnsii was achieved by using
T. harzianum strain II (85.45%), T. viride (69.8%), P. fluorescens (45.3%), and
B. subtilis (26.7%) separately (Vihol et al. 2009). Similarly it is evident with
bioagents when applied either through seed treatment or as soil/foliar application
under field conditions (Vyas and Mathur 2002; Sharma and Pandey 2011).
20.5.2 Funnel
The different antagonists, viz., T. harzianum, T. viride, and B. subtilis, were used for
coating seed @ 5 g/kg of seeds to protect seedling from damping-off and root rot
diseases (Gebily 2015). Commercial formulation of T. harzianum, T. viride,
P. fluorescens, and B. subtilis is used with Tween 80 @ 0.3% for coating seed
12 h prior to sowing (Ahmed et al. 2016), while for the management of root-knot
nematode (Meloidogyne javanica) organic amendment with neem cake @100 kg/ha
or castor @1000 kg/ha is found to be effective as nematicidal treatment at
Kapadvanj, Gujarat (Patel et al. 2005). Exploitation of nematode antagonists, viz.,
Paecilomyces lilacinus, Verticillium chlamydosporium, and Bacillus sp., will also
provide very good options for managing root-knot nematodes in spices (Ramana and
Eapen 1995).
20.5.3 Coriander
Wilt caused by Fusarium oxysporum f. sp. coriandrii is the most common problem,
which severely affects the crop. T. harzianum was found most effective to inhibit
83.69% of mycelial growth of fungus in vitro, and seed treatment with T. harzianum
@ 4 g kg/seed was recorded to be more effective in reducing the wilt incidence to

20.81% and 61.75% disease control under field condition and maximum 801.33 kg/
ha seed yield (Jat et al. 2017). Similarly, P. fluorescens@ 10 g kg and T. viride (4 g/
kg) as seed treatment + soil application reduce the wilt incidence. This crop also
suffers from stem gall (Protomyces macrosporus), which is a serious problem in
Rajasthan and responsible for heavy losses in seed yield. It can be managed by seed
treatment and subsequent three foliar sprays of T. viride and P. fluorescens (0.4%) at
regular interval (Kumar et al. 2014). Problem of nematodes in coriander is also
increasing day by day. P. fluorescens is proved to be best to reduce the gall index
(Sultan et al. 2011).
20.5.4 Fenugreek
Bioagents like T. viride, P. lilacinus, and P. fluorescens are very effective to manage
these soilborne pathogens. Of them, root rot (Rhizoctonia solani) can be effectively
managed by seed treatment with T. viride and/or P. fluorescens (Reddy 2014). The
efficacy of seed pelleting and soil application with Trichoderma sp. along with neem
cake @150 kg/ha also provides better protection to root rot protection (Reddy 2014).
Similarly, seed treatment with T. viride @4 g/kg followed by soil application @
5 kg/ha along with 150 kg/ha of neem cake suppresses the population of root-knot
nematodes.
20.5.5 Black Pepper
The antagonistic potential of bacterial isolates was also evaluated by dual culture
technique against P. capsici, the foot rot pathogen of black pepper, and the efficient
bacterial strains that showed up to 72% inhibition of P. capsici were shortlisted.
Similarly, out of about 172 rhizobacteria isolated from seed spices, 25 were
shortlisted based on useful agronomic traits like P solubilization, enzyme produc-
tion, etc. for greenhouse trials. Based on growth promotion, two isolates were
selected for large-scale testing in seed spices-growing areas (Bini et al. 2011).
Biocontrol agents Pseudomonas fluorescens and Trichoderma harzianum
isolated from rhizosphere of black pepper, ginger, and cardamom have been
evaluated for their efficiency, and efficient isolates for each crop were identified
and used against the pathogens of respective crops. In this study the efficient isolates
of Trichoderma, namely, IISR-1369 and IISR-1370 from black pepper, ISR-1371
from ginger, and IISR-1292 from cardamom, were made into different combinations
and tested along with P. fluorescens strains from black pepper (IISR-11) and ginger
(IISR-6) for their ability in disease suppression and growth promotion in these crops.
The experiments on black pepper and ginger were performed in the greenhouse, and
that of cardamom was carried out in already-existing field. Out of the 22 different
treatments, 3 treatments were found to be effective in suppressing root rot
(P. capsici) disease in black pepper, soft rot (P. aphanidermatum) in ginger, and
clump rot (P. vexans) in cardamom. The best biocontrol agents included
T. harzianum isolate, IISR-1369, and P. fluorescens strain, IISR-11 or IISR- 6 in
common. The maximum disease suppression obtained by the treatment combination,
P. harzianum isolate, IISR-1369, and P. fluorescens strain, IISR-11, in black pepper
was 63, and for cardamom, it was 36% over control. The same treatment could
impart 66.2% survival of ginger tillers after challenge inoculation with the pathogen.
The combination of T. harzianum isolate, ISR-1369, and P. fluorescens strain,
ISR-11, could improve the vigor of the plant in both black pepper and ginger. The
same treatment combination imparted maximum yield in ginger and cardamom. Our
earlier studies had proved the mutual compatibility between T. harzianum and
P. fluorescens. When these biocontrol agents were applied in combination, there
was synergistic effect for both growth promotion and disease suppression.
The egg parasitic fungus P. chlamydosporium and the obligate bacterial parasite
P. penetrans were able to check the root-knot nematode multiplication in cardamom
nurseries. Significant reduction in nematode population was observed in plots where
P. chlamydosporium was applied. There was a significant increase in the total
biomass of individual cardamom seedlings treated with P. lilacinus, but both
Trichoderma and P. lilacinus, either alone or together, significantly improved the
number of quality seedlings. The rhizome rot incidence was drastically reduced
wherever any of these bioagents was applied. In general, biocontrol agents
performed better in solarized soil than in non-solarized beds (Eapen and Venugopal
1995; Eapen et al. 2005).
Leaf rot (Rhizoctonia solani) and foot rot quick wilt (Phytophthora capsici) are
serious concerns in nursery and main field when warm humid condition prevails.
These soilborne pathogens can be managed by using biocontrol agents such as VAM
@ 110 cc/kg of soil mixture, Trichoderma sp. @ 5 g/kg of soil (cfu 10/g), and
P. fluorescens @1 g/kg of soil (cfu 18/g). Sometimes, T. harzianum enriched
pre-wetted neem cake or FYM @1 kg/100 kg perform very best in controlling of
wilt disease, when applied during pre-monsoon season @5 kg/vine below 10 years
and 10 kg/vine above 10 years (Reddy 2014).
20.5.6 Ginger
Soft rot of ginger (Erwinia sp.) is another serious problem that massively affects the
ginger production and causes significant loss in storage, and its infestation starts
from field. Hence, in situ protection strategy should be followed. To ensure health of
planting materials, rhizomes should be disinfected through solarization, followed by
treatment with bio-inoculant of T. harzianum and rhizobacterial strain in consortia
for seed treatment as well as soil application. T. harzianum in combination with
P. fluorescens showed a synergistic effect in reducing the soft rot infection and
ensured higher yields and soft rot suppression in storage. In addition to this,
application of Glomus spp. helps in better growth promotion.
20.5.7 Turmeric
Rhizome treatment with T. viride + P. fluorescens (@ 4 g/kg seed) and soil applica-
tion of T. viride (2.5 kg/ha) and P. fluorescens (25 kg/ha) along with FYM @10
MT/ha also helps in minimizing rhizome rot (Muthulakshmi and Saveetha 2009).
20.5.8 Cardamom
Sometimes, infestation of root-knot nematode predisposes the cardamom seedlings

to R. solani infection (Ali and Venugopal 1993). Hence, integrated strategy should
be followed up for managing the problem. Solarization of nursery beds and
subsequent soil application of T. harzianum reduce root rot infection and also
nematode infection. P. lilacinus in combination with Trichoderma sp. can also be
applied to suppress nematode-rhizome rot (R. solani) complex when incorporated in
solarized cardamom nursery beds (Eapen and Venugopal 1995).
20.5.9 Coleus
20.5.9.1 Root Rot/Wilt

Management of soilborne plant pathogens in particular, by organic and biological
methods, is being considered because chemical methods can result in accumulation
of harmful residues which may lead to serious ecological and health problems (Singh
et al. 2009). Some workers reported that the root rot/wilt of C. forskohlii could be
reduced by the application of Trichoderma viride, Pseudomonas fluorescens, and
AM fungi like Glomus fasciculatum and G. mosseae (Boby and Bagyaraj 2003;
Singh et al. 2009). T. viride and P. fluorescens reduced the disease incidence by
20–21% (Paramasivan et al. 2007). Combination of T. viride + Neemato (neem-
based product applied at 500 g/ 5 m2) resulted in lowest wilt incidence by 12.76%
(Kulkarni et al. 2007).
Botanical extracts like Eucalyptus citriodora, Ricinus communis, and
Azadirachta indica @ 5% significantly inhibited the growth of bacterial (Ralstonia
solanacearum) and fungal (Fusarium chlamydosporum) growth under in vitro con-
dition (Divya et al. 2010). Application of neem cake also reduced disease up to 40%.
20.5.9.2 Root-Knot Nematode

Biological control agents are gaining importance in the field of nematode manage-
ment. Another importance of these agents is their role as plant growth-promoting
microorganism (Sharon et al. 2001a, b). Trichoderma spp. found in close association
with roots contribute as plant growth stimulators (Ousley et al. 1994). Soil applica-
tion with bioagents like T. viride and P. fluorescens significantly reduced the
nematode population in soil and root and increased the growth and yield of
C. forskohlii crop (Senthamarai et al. 2008). Integration of strategies such as stem
cutting dipping in P. fluorescens + soil application of neem cake @ 400 kg/

ha + growing marigold as intercrop followed by their biomass incorporation during
earthing up increased the yield (22.7–30.0%) and reduced the root-knot nematode
population (71.2–73.8%) superiorly, followed by the integration of P. fluorescens +
marigold intercrop, which were almost equally effective (Seenivasan and Deevrajan
2008).
20.6 Economically Important Nematode Pests

of Horticultural Crops
Economically important genera of phytonematodes associated with various horticul-

tural crops around the globe comprise Meloidogyne (root-knot nematode),
Heterodera (cyst nematode), Ditylenchus (stem and bulb nematode), Globodera
(potato golden cyst nematode), Tylenchulus (citrus nematode), Xiphinema (dagger
nematode), Radopholus (burrowing nematode), Rotylenchulus (reniform nematode),
and Helicotylenchus (spiral nematode).
Kannan and Veeravel (2012) studied the effect of different dose and application
methods of Paecilomyces lilacinus against Meloidogyne incognita on okra at two
different locations. In field trials maximum shoot length (60 and 90 DAS), shoot
weight (90 DAS), and root length (90 DAS) were documented, and they were
positively correlated with fruit yield of okra (Rao et al. 2012). Hallmann and Sikora
(1993) evaluated 200 isolates of endophytic fungi, representing different genera,
isolated from tomato roots. They found a reduction in gall formation by M. incognita
between 52 and 75% after application of four endophytic strains of the fungus
Fusarium oxysporum. They also found that M. incognita attraction and penetration
of tomato seedlings were significantly reduced following treatment with the culture
filtrate of Fusarium oxysporum.
Biocontrol of the root-knot nematodes (Meloidogyne spp.) by different species of
Trichoderma has been reported by several scientists (Sharon et al. 2001a, b, 2007,
2011; Affokpon et al. 2011; Mascarin et al. 2012; Naserinasab et al. 2011;
Al-Shammari et al. 2013).
Hallmann et al. (1995a, b) found some evidence that endophytic bacteria may
contribute to control of plant parasitic nematodes. They evaluated seven isolates of
endophytic bacteria isolated from cucumber and cotton roots against root-knot
nematode, M. incognita, and they found a significant reduction of 50% in the number
of galls on cucumber. Munif et al. (2000) screened the endophytic bacteria isolated
from tomato roots toward M. incognita on tomato under greenhouse conditions.
They showed antagonistic properties in the screening of 21 out of 181 endophytic
bacteria toward M. incognita. Application of these rhizobacteria to sugar beet seed
and potato seed pieces caused significant decreases in early root infection of the
sugar beet cyst nematode Heterodera schachtii and potato cyst nematode Globodera
pallida (Racke and Sikora 1985; Oostendorp and Sikora 1986). Zavaleta-Meija and
Van Gundy (1985) found that rhizobacteria have biocontrol activity toward root-
knot nematode, and they showed that more than 12% of the rhizobacteria tested
reduced the number of galls of M. incognita on cucumber and tomato. Sikora (1992)
reported that 7–10% of the rhizosphere bacteria isolated from potato, sugar beet, or
tomato root systems have antagonistic activity against cyst and root-knot nematodes.
Sikora and Hoffimann-Hergarten (1993) revealed that plant health-promoting
rhizobacteria influence the intimate relationship between the plant parasitic nema-
tode and its host by regulation of nematode behavior during the early root penetra-
tion phase of parasitism which is extremely important for crop yield. Strains of
Pseudomonas chitinolytica were also shown to reduce M. javanica on tomato as
reported by Spiegel et al. (1991). Smith and Grenfell (1994) reported that Bacillus
sp. strain 23a reduced M. javanica densities on tomato and Pseudomonas
fluorescens strain reduced the number of galls and egg masses of M. incognita on
tomato roots (Santhi and Sivakumar 1995).
Khan and Haque (2011) showed that application of P. fluorescens decreased gall
index from 3.0 to 1.33 and improved plant growth of two susceptible tobacco
cultivars by up to 32%. Trichoderma harzianum suppressed gall index from 3 to
2. Similar effects of P. fluorescens and Trichoderma spp. on different crops against
Meloidogyne spp. have been reported (Khan et al. 2007). The bacterium is a
phosphate solubilizer (Khan et al. 2009) but may also suppress pathogens through
antibiosis (Nielsen et al. 1998), induced systemic resistance (Kloepper et al. 1992),
and production of phytohormones (Garcia de Salamone et al. 2001). Bari et al.
(2004) who reported that T. harzianum 1 g/plant reduced root-knot nematode and
enhanced vegetative growth of lady’s finger in the field. Prasad et al. (2014) reported
lowest root-knot nematode M. incognita population and increased plant growth of
carrot with T. harzianum at 25 g per m2 followed by isolated T. harzianum and
commercial T. harzianum at 20 g per m. Chormule et al. (2017) found that the
efficacy of bioagents, viz., P. fluorescens, P. lilacinus, Phule Trichoderma plus,
T. viride, and P. chlamydosporia, @ 20 kg/ha and organic amendment with neem
cake @ 2 t/ha were effective in reducing the root-knot nematode population, number
of root galls, and egg masses and increasing fruit yield at terminations.
Bacterial isolates obtained from spices were screened against nematodes initially
by employing the buffer method to assess their nematode suppressing ability. Except
a few isolates, most of the bacterial isolates caused very less mortality of nematodes
in this test. Based on their efficacy, 30 bacterial isolates were selected for further
in vitro evaluation using different methods like culture filtrate assay, direct assay of
bacterial suspension, and assay of volatile and non-volatile metabolites. Culture
filtrates of 77 bacterial isolates were studied for their nematode toxic activity. Out
of these, 22 isolates caused >90% mortality to root-knot nematodes, while another
40 isolates possessed high (>50% mortality) nematicidal property. Metabolites of
67 bacterial isolates were also tested for their nematicidal activities. Volatile
metabolites play a crucial role in killing the nematodes. Besides, the production of
HCN and H2S by these bacteria was also monitored. Out of the 98 isolates screened,
only 6 isolates produced HCN. H2S production was observed in another 6 isolates
among the 50 tested. The egg parasitic fungus P. chlamydosporium and the obligate
bacterial parasite P. penetrans were able to check the root-knot nematode
multiplication in cardamom nurseries. Significant reduction in nematode population

was observed in plots where P. chlamydosporium was applied.
20.7 Conclusion
The search for alternative ways to manage plant diseases is the most important
concern to protect ourselves from the widespread use of chemicals that contaminate
the soil and water and leave toxic residues that affect the environment. The biocon-
trol efficacy of antagonistic microorganisms depends on a combination of factors
such as the characteristics of the antagonistic microorganism, the epidemiology of
the target pathogen, and the environmental conditions in which the relationship
between the pathogen and the antagonist(s) is taking place. Hence, efforts are needed
to commercialize these novel microbes to bring in a second revolution within the
country. However, it’s clear that the stage is set for biological control agents to play a
greater part in agriculture and horticulture. This approach undoubtedly would
encourage environmentally desirable products that are desired by the general public
to succeed in the marketplace rapidly. Biological disease management systems have
to be developed by integrating several strategies taking into consideration pathogen
biology, cultivar resistance, and epidemiology.
References
Abeysinghe S (2009) Use of nonpathogenic Fusarium oxysporum and rhizobacteria for suppression
of Fusarium root and stem rot of Cucumis sativus caused by Fusarium oxysporum f. sp. radicis-
cucumerinum. Arch Phytopathol Pflanzenschutz 42(1):73–82
Abo-Elyousr KA, El-Hendawy HH (2008) Integration of Pseudomonas fluorescens and
acibenzolar-S-methyl to control bacterial spot disease of tomato. Crop Protect 27(7):1118–1124
Abraham AO (2005) Biological control of Phytophthora root rot of citrus seedlings and cuttings.
Doctoral dissertation
Addy HS, Askora A, Kawasaki T, Fujie M, Yamada T (2012a) The filamentous phage φRSS1
enhances virulence of phytopathogenic Ralstonia solanacearum on tomato. Phytopathology
102:244–251
Addy HS, Askora A, Kawasaki T, Fujie M, Yamada T (2012b) Loss of virulence of the phytopath-
ogen Ralstonia solanacearum through infection by φRSM filamentous phages. Phytopathology
102:469–477
Affokpon A, Coyne DL, Htay CC, Agbede RD, Lawouin L, Coosemans J (2011) Bio control
potential of native Trichoderma isolates against root-knot nematodes in West African vegetable
production systems. Soil Biol Biochem 43:600–608
Ahmed MFA, Zayan AMS, Rashed MS (2016) Evaluation of seed coating with certain bio-agents
against damping off and root rot diseases of fennel under organic farming system. J Phytopathol
Pest Manag 3:11–23
Ajay S, Sunaina V (2005) Direct inhibition of Phytophthora infestans, the causal organism of late
blight of potato by Bacillus antagonists. Am Potato J 32:3–4
Akila R, Rajendran L, Harish S, Saveetha K, Raguchander T, Samiyappan R (2011) Combined
application of botanical formulations and biocontrol agents for the management of Fusarium
oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana. Biol Control 57:175–183
Ali SS, Venugopal MN (1993) Prevalence of damping off and rhizome rot diseases in nematode
infested cardamom nurseries in Karnataka. Curr Nematol 4:19–24
AL-Shammari TA, Bahkali AH, Elgorban AM, El-Kahky MT, Al-Sum BA (2013) The use of
Trichoderma longibrachiatum and Mortierella alpina against root-knot nematode, Meloidogyne
javanica on tomato. J Pure Appl Microbiol 7:199–207
Angelopoulou DJ, Naska EJ, Paplomatas EJ, Tjamos SE (2014) Biological control agents (BCAs)
of verticillium wilt: influence of application rates and delivery method on plant protection,
triggering of host defence mechanisms and rhizosphere populations of BCAs. Plant Pathol 63
(5):1062–1069
Aspiras RB, de la Cruz AR, Parsley GJ (1986) Biocontrol of bacterial wilt in tomato and potato
through pre-emptive colonization using Bacillus polymyxa FU-6, and Pseudomonas
fluorescens. ACIAR Proceedings 13:89–92
Bae JY, Wu J, Lee HJ, Jo EJ, Murugaiyan S, Chung E, Lee SW (2012) Biocontrol potential of a
lytic bacteriophage PE204 against bacterial wilt of tomato. J Microbiol Biotechnol 22
(12):1613–1620
Bari MA, Faruk MI, Rahman ML, Ali MR (2004) Effect of organic soil amendments and nemati-
cide on root-knot nematode of brinjal. Bangladesh J Pl Pathol 20:27–30
Baysal O, Soylu EM, Soylu S (2003) Induction of defence related enzymes and resistance by the
plant activator acibenzolar-S-methyl in tomato seedlings against bacterial canker caused by
Clavibacter michiganensis ssp. michiganensis. Plant Pathol 52:747–753
Belagner RR, Labbe C (2002) Control of powdery mildews: prophylactic and biological alternative
for horticultural crops. In: Belanger RR, Bushnell WR, Dik AJ, Carver TLW (eds) The powdery
mildews – a comprehensive treatise. APS Press, St. Paul, pp 256–267
Bini YK, Anandaraj M, Dinesh R, Silna N, Kumar A (2011) Evaluation of PGPR strains for growth
and disease suppression in ginger (Zingiber officinale Rosc.). In: Plant growth-promoting
rhizobacteria (pgpr) for sustainable agriculture, p 523
Biswas KK, Das ND (1999) Biological control of pigeon pea wilt caused by Fusarium udum with
Trichoderma spp. Plant Prot Sci 7(1):46–50
Boby VU, Bagyaraj DJ (2003) Biological control of root-rot of Coleus forskohlii Briq. Using
microbial inoculants. World J Microbiol Biotechnol 19:175–180
Bradley GG, Punja ZK (2010) Composts containing fluorescent pseudomonads suppress fusarium
root and stem rot development on greenhouse cucumber. Can J Microbiol 56:896–905
Bunker RN, Mathur K (2001) Antagonism of local biocontrol agents to Rhizoctonia solani inciting
dry root-rot of chilli. J Mycol Plant Pathol 31:50–53
Cheng SS, Liu JY, Chang EH, Chang ST (2008) Antifungal activity of cinnamaldehyde and
eugenol congeners against wood-rot fungi. Bio Resour Technol 99:5145–5149
Chormule AJ, Mhase NL, Gurve SS (2017) Management of root-knot nematode, meloidogyne
incognita infesting grape under field conditions. Plant Protect Sci 25(1):181–185
Copping LG (2004) The manual of bio control agents, 3rd edn. BCPC Publications, Alton/Hants,
p 702
Das SN, Sarma TC, Tapadar SA (2014) In vitro evaluation of fungicides and two species of
Trichoderma against Phomopsis vexans causing fruit rot of brinjal (Solanum melongena L.).
Int J Sci Res 4:1–2
Davis JR, Huisman OC, Everson DO, Nolte P, Sorenson LH, Schneider AT (2010a) The suppres-
sion of Verticillium wilt of potato using corn as a green manure crop. Am J Potato Res
87:195–208
Davis JR, Huisman OC, Everson DO, Nolte P, Sorenson LH, Schneider AT (2010b). The suppres-
sion of Verticillium wilt of potato using corn as a green manure crop. Am J Potato Res 87
(2):195–208
Deepak PL, Saran, Lal G (2009) In vitro and in vivo control of Fusarium oxysporum f. sp. cumini
and Alternaria burnsii through various bioagents. Indian J Agric Sci 79:84–87
Divya S, Singh R, Parameswaran TN (2010) In vitro antifungal and antibacterial activity of various
plants extract on growth of pathogens (Fusarium chlamydosporum and Ralstonia
solanacearum) causing root rot/wilt of Coleus forskohlii Briq. In: International symposium on
current status and opportunities in aromatic and medicinal plants (AROMED), CIMAP,
Lucknow. J Med Arom Plant Sci 31: 155
Eapen SJ, Venugopal MN (1995) Field evaluation of Trichoderma spp. and Paecilomyces lilacinus
for control of root knot nematodes and fungal diseases of cardamom nurseries (Abs.). Indian J
Nematol 25:15–16
Eapen SJ, Beena B, Ramana KV (2005) Tropical soil microflora of spice-based cropping systems as
potential antagonists of root-knot nematodes. J Invertebr Pathol 88:218–225
Elad Y, Baker R (1985) Influence of trace amounts of cations and siderophore-producing
pseudomonads on chlamydospore germination of Fusarium oxysporum. Phytopathology 75
(9):1047–1052
Elad Y, Malathrakis NE, Dik AJ (1999) Biological control of botrytis-incited diseases and powdery
mildews in greenhouse crops. Crop Protect 15(3):229–240
Elmer PAG, Reglinski T, Wood PN, Hill RA, Marsden SM, Parry FJ, Taylor JT. (2003). Suppres-
sion of Botrytis in grapes using a combination of elicitors and fungal antagonists. In: 8th
International plant pathology congress, Christchurch, p 43
Etchebar C, Trigalet-Demery D, van Gijsegem F, Vasse J, Trigalet A (1998) Xylem colonization by
an HrcV mutant of Ralstonia solanacearum is a key factor for the efficient biological control of
tomato bacterial wilt. Mol Plant-Microbe Interact 11(9):869–877
Feng H, Li Y, Liu Q (2013) Endophytic bacterial communities in tomato plants with differential
resistance to Ralstonia solanacearum. Afr J Microbiol Res 7:1311–1318
Fiedler Z, Sosnowska D (2007) Nematophagous fungus Paecilomyces lilacinus (Thom) Samson is
also a biological agent for control of greenhouse insects and mite pests. Biol Control
52:547–558
Frey P, Prior P, Marie C, Kotoujansky A, Demery DT, Trigalet A (1994) Hrp-mutants of Pseudo-
monas solanacearum as potential biocontrol agents of tomato bacterial wilt. Appl Environ
Fujiwara A, Fujisawa M, Hamasaki R, Kawasaki T, Fujie M, Yamada T (2011) Biocontrol of
Ralstonia solanacearum by treatment with lytic bacteriophages. Appl Environ Microbiol
77:4155–4162
Furuya S, Mochizuki M, Aoki Y, Kobayashi H, Takayanagi T, Shimizu M, Suzuki S (2011)
Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal
diseases. Biocontrol Sci Technol 21:705–720
Gade RM (2012) Biological and chemical management of Phytophthora root rot/collar rot in citrus
nursery. Bioscan 7:631–635
Gade RM, Koche MD (2012) Integrated disease management for root rot and gummosis in Nagpur
mandarin. Indian Phytopathol 65:272
Gao Y, Reitz SR, Wang J, Xu X, Lei Z. (2012) Potential of a strain of the entomopathogenic fungus
Beauveria bassiana (Hypocreales: Cordycipitaceae) as a biological control agent against.
Biocontrol Sci Technol 22(4)
García de Salamone IE, Hynes RK, Nelson LM (2001) Cytokinin production by plant growth
promoting rhizobacteria and selected mutants. Can J Microbiol 47:404–411
Gebily D (2015) Studies on root rot disease of fennel under organic farming regulations. MSc
thesis. Faculty of Agriculture. Fayoum University, Fayoum, Egypt, p 121
Georgakopoulos DG, Fiddaman P, Leifert C, Malathrakis NE (2002) Biological control of cucum-
ber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal
antagonists. J Appl Microbiol 92:1078–1086
Gnanasekaran P, Salique SM, Panneerselvam A, Umamagheswari K (2015) In vitro biological
control of Fusarium oxysporum f. sp. cubense by using some Indian medicinal plants. Intern J
Curr Res Acad Rev 3:107–116
Goto M, Tadanchi Y, Okabe N (1979) Interaction between Xanthomonas citri and Erwinia
herbicola in vitro and in vivo. Ann Phytopathol Soc Jpn 45:618–624
Gravel V, Martinez C, Antoun H, Tweddell RJ (2005) Antagonistic microorganisms with the ability
to control Pythium damping-off of tomato seeds in rockwool. Biol Control 50:771–786
Guo JH, Qi HY, Guo YH, Ge HL, Gong LY, Zhang LX, Sun PH (2004) Biocontrol of tomato wilt
by plant growth-promoting rhizobacteria. Biol Control 29:66–72
Gupta H, Singh BP, Mohan J (2004) Biocontrol of late blight of potato. Potato J 31:39–42
Hallmann J, Sikora RA (1993) Occurrence of plant parasitic nematodes and non-pathogenic species
of Fusarium in tomato plants in Kenya and their role as mutualistic synergists for biological
control of root knot nematodes. Int J Pest Manag 40:321–325
Hallmann J, Kloepper JW, Rodriguez-Kabana R, Sikora RA (1995a) Endophytic rhizobacteria as
antagonists of Meloidogyne incognita on cucumber. Phytopathology 85:136
Hallmann J, Quadt-Hallmann A, Rodrıguez-Kabana R, Kloepper JW (1995b) Interactions between
Meloidogyne incognita and endophytic bacteria in cotton and cucumber. Soil Biol Biochem
30:925–937
Halos PM, Zorilla RA (1979) Vesicular-arbuscular mycorrhizae increase growth and yield of
tomatoes and reduce infection by Pseudomonas solanacearum. Philipp Agric 62:309–315
Harman GE, Taylor AG (1989) Combining effective strains of Trichoderma harzianum and soil
matrix priming to improved biological seed treatments. Plant Dis 73:631–637
Hassan MAE, Buchenauer H (2008) Enhanced control of bacterial wilt of tomato by DL-3-
aminobutyric acid and the fluorescent Pseudomonas isolate CW2. J Plant Dis Protect 115
(5):199–207
Ho YN, Chiang HM, Chao CP, Su CC, Hsu HF, Guo C, Hsieh JL, Huang CC (2015) In planta
biocontrol of soilborne Fusarium wilt of banana through a plant endophytic bacterium,
Burkholderia cenocepacia 869T2. Plant Soil 387:295–306
Hu LB, Li HB, Sun JL, Zeng J (2012) Effect of laminarin on Aspergillus flavus growth and
aflatoxin production. Adv Mater Res 343:1168–1171
Jadon KS (2009) Eco- friendly management of brinjal collar rot caused by Sclerotium rolfsii Sacc.
Indian Phytopath 62:345–347
Janousek CN, Bay IS, Herche RW, Gubler WD (2009) Powdery mildew control on pumpkin with
organic and synthetic fungicides: 2009 field trial
Jat MK, Ahir RR, Choudhary S, Kakraliya GL (2017) Management of coriander wilt using
biocontrol agents. Int J Chem Stud 5:523–525
Jayaraj J, Radhakrishnan NV, Kannan R, Sakthivel K, Suganya D, Venkatesan S, Velazhahan R
(2005) Development of new formulations of Bacillus subtilis for management of tomato
damping-off caused by Pythium aphanidermatum. Biocontrol Sci Technol 15:55–65
Ji P, Campbell HL, Kloepper JW, Jones JB, Suslow TV, Wilson M (2006) Integrated biological
control of bacterial speck and spot of tomato under field conditions using foliar biological
control agents and plant growth promoting rhizobacteria. Biol Control 36:358–367
Jie L, Zifeng W, Lixiang C, Hongming T, Patrik I, Zide J, Shining Z (2009) Artificial inoculation of
banana tissue culture plantlets with indigenous endophytes originally derived from native
banana plants. Biol Control 51:427–434
Kalita P, Bora LC, Bhagabati KN (1996) Phylloplane microflora of citrus and their role in
management of citrus canker. Indian Phytopath 49:234–237
Kannan R, Veeravel R (2012) Effect of different dose and application methods of Paecilomyces
lilacinus (Thom.) Samson against root-knot nematode, Meloidogyne incognita (Kofoid and
White) Chitwood in okra. J Agric Sci 4:119–127
Karagiannidis N, Bletsos F, Stavropoulos N (2002) Effect of Verticillium wilt (Verticillium dahliae
Kleb.) and mycorrhiza (Glomus mosseae) on root colonization, growth and nutrient uptake in
tomato and eggplant seedlings. Sci Hortic 94:145–156
Kawasaki T, Shimizu M, Satsuma H, Fujiwara A, Fujie M, Usami S (2009) Genomic characteriza-
tion of Ralstonia solanacearum phage φRSB1, a T7-like wide-host-range phage. J Bacteriol
191:422–427
Kempe J, Sequeira L (1983) Biological control of bacterial wilt of potatoes: attempts to induce
resistance by treating tubers with bacteria. Plant Dis 67:499–503
Kerr A (1980) Biological control of crown gall through production of agrocin 84. Plant Dis
87:195–208
Khan MR, Haque Z (2011) Soil application of Pseudomonas fluorescens and Trichoderma
harzianum reduces root-knot nematode, Meloidogyne incognita, on tobacco. Phytopathol
Mediterr 50(2):257–266
Khan MR, Khan SM, Mohiddin FA, Askary TH. (2007) Effect of certain phosphate-solubilizing
bacteria on root-knot nematode disease of mungbean. In: 1st International meeting on microbial
phosphate solubilization. Springer, Dordrecht, pp 341–346
Khan MR, Altaf S, Mohiddin FA, Khan U, Anwer A (2009) Biological control of plant nematodes
with phosphate solubilizing microorganisms. Phosphate solubilizing microbes for crop
improvement” (MS Khan, A. Zaidi, eds.). Nova Science Publishers, New York, pp 395–426
Kloepper JW, Tuzun S, Kuc JA (1992) Proposed definitions related to induced disease resistance.
Biocontrol Sci Technol 2:349–351
Kulkarni MS, Ramprasad S, Hedge Y, Laxminarayan H, Hedge NK (2007) Management of collar
rot complex disease of Coleus forskohlii (wild) Briq. Using bioagents, organic amendments and
chemicals. Biomedicine 2:37–40
Kumar G, Yadav SK, Patel JS, Sarkar A, Awasthi LP (2014) Management of stem gall disease in
coriander using Pseudomonas and Trichoderma (bioagents) and fungicides. J Pure Appl
Lan X, Zhang J, Zong Z, Ma Q, Wang Y (2017) Evaluation of the biocontrol potential of
Purpureocillium lilacinum QLP12 against Verticillium dahliae in eggplant. Biomed Res Int
Lang J, Hu J, Shen Q (2012) Fungal diversity of soils with cotton Verticillium wilt as affected by
application of bio-organic fertilizer using DGGE and real-time PCR. Biol Fertil Soils
48:191–203
Larkin RP, Fravel DR (1998) Efficacy of various fungal and bacterial biocontrol organisms for
control of Fusarium wilt of tomato. Plant Dis 82:1022–1028
Lende AS, Gade RM, Shitole AS (2015) Management of Phytophthora root rot in Nagpur mandarin
by using integrated approach. Ecoscan 8:331–336
Li L, Zhao Y, McCaig BC (2004) The tomato homolog of coronatine-insensitive 1 is required for
the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular
trichome development. Plant Cell 16:126–143
Liang C, Zang C, McDermott MI, Zhao K, Yu S, Huang Y (2016) Two imide substances from a
soil-isolated Streptomyces atratus strain provide effective biocontrol activity against grapevine
downy mildew. Biomed Sci Technol 26(10):1337–1351
Ling N, Xue C, Huang Q, Yang X, Xu Y, Shen Q (2010) Development of a mode of application of
bioorganic fertilizer for improving the biocontrol efficacy to Fusarium wilt. Biol Control
55:673–683
Lopez-Mondejar R, Bernal-Vicente A, Ros M, Tittarelli F, Canali S, Intrigiolo F, Pascual JA (2010)
Utilisation of citrus compost-based growing media amended with Trichoderma harzianum T-78
in Cucumis melo L. seedling production. Bioresour Technol 101:3718–3723
Luo J, Ran W, Hu J, Yang XM, Xu YC, Shen QR (2010) Fungal diversity in the rhizosphere of
cotton: effects of repeated application of Bacillus subtilis enhanced bio-organic fertilizer for
control of Verticillium wilt. Soil Sci Soc Am J 74:2039–2048
Mane MM, Lal A, Zghair NQ, Sobita S (2014) Efficacy of certain bio agents and fungicides against
early blight of potato (Solanum tuberosum L.). Int J Plant Protect 7:433–436
Manikandan R, Saravanakumar D, Rajendran L, Raguchander T, Samiyappan R (2010)
Standardization of liquid formulation of Pseudomonas fluorescens Pf1 for its efficacy against
Fusarium wilt of tomato. Biol Control 54:83–89
Manoranjitham SK, Prakasan V, Rajappan K, Amutha G (2000) Effect of two antagonists on
damping off disease of tomato. Indian Phytopathol 53:441–443
Mascarin GM, Bonfim Junior MF, de Araújo Filho JV (2012) Trichoderma harzianum reduces
population of Meloidogyne incognita in cucumber plants under greenhouse conditions.
Embrapa Arroz e Feijão-Artigo em periódico indexado (ALICE)
McLaren DL, Huang HC, Rimmer SR. (1982) Hyphal interactions occurring between Sclerofiniu
sclerotiorum and Penicillium aermiculatum 4: 308
McLaughlin RJ, Sequeira L (1988) Evaluation of an avirulent strain of Pseudomonas solanacearum

for biological control of bacterial wilt of potato. Am J Potato Res 65:255–268
Melidossia HS, Seem RC, English-Loeb G, Wilcox WF, Gadoury DM (2005) Suppression of
grapevine powdery mildew by a mycophagous mite. Plant Dis 89:1331–1338
Misra AK (2008 November 24–26) Important diseases of guava and their management. Souvenir,
national guava symposium. Improvement, production and utilization. pp 8-14
Mochizuki M, Yamamoto S, Aoki Y, Suzuki S (2012) Isolation and characterization of Bacillus
amyloliquefaciens S13-3 as a biological control agent for anthracnose caused by Colletotrichum
gloeosporioides. Biocontrol Sci Technol 22:697–709
Muneeshwar S, Razdan VK, Sourav G (2011) Occurrence of phomopsis leaf blight and fruit rot of
brinjal caused by Phomopsis vexans in Jammu. Ann Plant Protect Sci 19:396–399
Munif A, Hallmann J, Sikora RA (2000) Evaluation of the biocontrol activity of endophytic bacteria
from tomato against Meloidogyne incognita. Med Fac Landbouww Univ Gent 65:471–480
Murugaiyan S, Bae JY, Wu J, Lee SD, Um HY, Choi HK (2010) Characterization of filamentous
bacteriophage PE226 infecting Ralstonia solanacearum strains. J Appl Microbiol 110:296–303
Muthulakshmir P, Saveetha K (2009) Management of turmeric rhizome rot using eco-friendly
biocontrol consortia. J Biol Control 23:181–184
Najar AG, Anwar A, Masoodi L, Khar MS (2011) Evaluation of native biocontrol agents
against Fusarium solani f. sp. melongenae causing wilt disease of brinjal in Kashmir. J Phytol
3:31–34
Narkar SP, Sawant IS, Shete HG (2017) Isolation and identification of Bacillus amyloliquefaciens
strains for bio-control of grapevine anthracnose. J Eco Agric 12:62–66
Naserinasab F, Sahebani N, Etebarian HR (2011) Biological control of Meloidogyne javanica by
Trichoderma harzianum BI and salicylic acid on tomato. Afr J Food Sci 5:276–280
Neher OT, Johnston MR, Zidack NK, Jacobsen BJ (2009) Evaluation of Bacillus mycoides isolate
BmJ and B. mojavensis isolate 203-7 for the control of anthracnose of cucurbits caused by
Glomerella cingulata var. orbiculare. Biol Control 48:140–146
Nel B, Steinberg C, Labuschagne N, Viljoen A (2006) The potential of nonpathogenic Fusarium
oxysporum and other biological control organisms for suppressing Fusarium wilt of banana.
Nielsen MN, Sørensen J, Fels J, Pedersen HC (1998) Secondary metabolite-and endochitinase-
dependent antagonism toward plant-pathogenic microfungi of Pseudomonas fluorescens
isolates from sugar beet rhizosphere. Appl Environ Microbiol 64:3563–3569
Ojaghian MR (2011) Potential of Trichoderma spp. and Talaromyces flavus for biological control of
potato stem rot caused by Sclerotinia sclerotiorum. Phytoparasitica 39(2):185–193
Omar I, O’Neill TM, Rossall S (2006) Biological control of Fusarium crown and root rot of tomato
with antagonistic bacteria and integrated control when combined with the fungicide
carbendazim. Plant Pathol 55:92–99
Oostendorp M, Sikora RA (1986) Utilization of antagonistic rhizobacteria as a seed treatment for
the biological control of Heterodera schachtii in sugar beet. Rev Nematol 9:304
Ota T (1983) Interaction in vitro and in vivo between Xanthomonas campestris pv. citri and
antagonistic Pseudomonas sp. Ann Phytopath Soc Jpn 49:308
Ousley MA, Lynch JM, Whipps JM (1994) Potential of Trichoderma spp. as consistent plant
growth stimulators. Biol Fertil Soils 17:85–90
Paramasivan M, Mohan S, Muthukrishnan N (2007) Management of Coleus dry root rot pathogen
Macrophomina phaseolina by fungal and bacterial antagonist. Indian J Plant Prot 35:133–135
Patel SK, Patel HV, Patel AD, Patel DJ (2005) Integrated management of root knot nematode
Meloidogyne javanica (Treub) Chitwood in fennel (Foeniculum vulgare mill). J Spices Arom
Crops 14:152–154
Pente R, Gade RM, Belkar YK, Priya S (2015) Evaluation of botanicals and bio-agents against
Phytophthora root rot in citrus. Trends Biosci 8(3):752–758
Prasad GG, Ravichandra NG, Narasimhamurthy TN, Kumar CP, Yadahalli P (2014) Management
of Meloidogyne incognita infecting carrot by using bioagents. J Biopest 7:144
Racke J, Sikora RA (1985) Isolation, formulation and antagonistic activity of rhizobacteria toward
the potato cyst nematode Globodera pallida. Soil Biol Biochem 24:521–526
Ramana KV, Eapen SJ (1995) Parasitic nematodes and their management in major spices. J Spices
Arom Crops 4:1–16
Ramesh R, Joshi A, Ghanekar M (2009) Pseudomonads: major antagonistic endophytic bacteria to
suppress bacterial wilt pathogen, Ralstonia solanacearum in the eggplant (Solanum melongena
L). World J Microbiol Biotechnol 25:47–55
Ramos L, Ciampi L, Gonzales S (1993) Biological control of Phytophthora infestans in potato
plants. Simiente 63:53–54
Rao MS, Sowmya DS, Chaya MK, Kumar RM, Rathnamma K, Gavaskar J, Priti K, Ramachandran
N (2012) Management of nematode induced wilt disease complex in capsicum using Pseudo-
monas fluorescens and Paecilomyces lilacinus. Nematol Mediterr 40:101–105
Reddy PP (2014) Spice crops. In: Fungal diseases and their management in horticultural crops (Eds.
Reddy). Scientific Publishers, Jodhpur, pp 312–347
Reglinski T (2009) Chapter 4: Induced resistance for plant disease control. In: Walters D
(ed) Disease control in crops: biological and environmentally friendly approaches. Wiley-
Blackwell, Oxford
Rohini HG, Hariprasad P, Niranjana S (2016) Cultural, morphological and physiological character-
ization of Phomopsis vexans isolates from different brinjal (Solanum melongena L) growing
regions of Karnataka, India. Int J Pharm Biol Sci 7:70–77
Romero D, De Vicente A, Zeriouh H, Cazorla FM, Fernandez-Ortuno D, Tores JA, Perez-Garcia A
(2007) Evaluation of biological control agents for managing cucurbit powdery mildew on
greenhouse-grown melon. Plant Pathol 56:976–986
Roy S, Singh BP, Bhattacharyya SK (1991) Biocontrol of late blight of potato. Phytophthora Newsl
17:18
Sadlers HM (1996) Use of bacteria in controlling fungal diseases. Gemuse Munchen 32:180–181
Salman M, Abuamsha R (2012) Potential for integrated biological and chemical control of
damping-off disease caused by Pythium ultimum in tomato. Biol Control 57:711–718
Sanchez V, Bustamante E, Shattock R (1998) Selection of antagonists for biological control of
Phytophthora infestans in tomato. Manejo Integrado de Plagas 48:25–34
Santhi A, Sivakumar CV (1995) Biocontrol potential of Pseudomonas fluorescens (Migula) against
root knot nematode, Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 on
tomato. J Biol Control 9:113–115
Sawant IS, Ghule SB, Sawant SD (2015) Molecular analysis reveals that lack of chasmothecia
formation in Erysiphe necator in Maharashtra, India is due to presence of only MAT1-2 mating
type idiomorph. VITIS-J Grapevine Res 54:87–90
Sawant IS, Wadkar PN, Rajguru YR, Mhaske NH, Salunkhe VP, Sawant SD, Upadhyay A (2016)
Biocontrol potential of two novel grapevine associated Bacillus strains for management of
anthracnose disease caused by Colletotrichum gloeosporioides. Biomed Sci Technol
26:964–979
Seenivasan N, Deevrajan K (2008) Integrated approach for the management of root-knot nematode,
Meloidogyne incognita in medicinal coleus. Indian J Nematol 38:154–158
Seleim MAA, Saead FA, Abd-El-Moneem KMH, Abo-Elyousr KAM (2011) Biological control of
bacterial wilt of tomato by plant growth promoting rhizobacteria. Plant Pathol J 10:146–153
Sengonca C, Thungrabeab M, Blaeser P (2006) Potential of different isolates of entomopathogenic
fungi from Thailand as biological control agents against western flower thrips, Frankliniella
occidentalis (Pergande) (Thysanoptera: Thripidae). J Plant Dis Prot 113:74–80
Senthamari K, Poornima K, Subramanian S, Sudheer J (2008) Nematode-fungal disease complex
involving Meloidogyne incognita and Macrophomina phaseolina on medicinal coleus, Coleus
forskohlii Briq. Indian J Nematol 38:30–33
Sharma S, Pandey RN (2011) Evaluation of bacterial and fungicidal bioagent against Alternaria
burnsii causing blight of Cumin. J Plant Dis Sci 6:191–192
Sharon E, Bar-Eyal M, Chet I, Herrera-Estrella A, Kleifeld O, Spiegel Y (2001a) Biological control
of root knot nematode Meloidogyne javanica by Trichoderma harzianum. Phytopathology
91:687–693
Sharon E, Bar-Eyal M, Chet I, Herrera-Estrella A, Kleifeld O, Spiegel Y (2001b) Biological control

of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum. Phytopathology
91:687–693
Sharon E, Chet I, Viterbo A, Bar-Eyal M, Nagan H, Samuels GJ, Spiegel Y (2007) Parasitism of
Trichoderma on Meloidogyne javanica and role of the gelatinous matrix. Euro J Plant Pathol
118:247–258
Sharon E, Chet I, Spiegel Y (2011) Trichoderma as a biological control agent. Biol Control Plant
Parasitic Nematodes:183–201
Shelar KT (2013) Studies on Seed, Rhizosphere Mycoflora and Blight of Cumin (Cuminum
cyminum L.) caused by Alternaria burnsii. Doctoral dissertation, JNKVV
Shorten PR, Elmer PAG, Soboleva TK (2003) A dynamic model to describe the interaction between
Botrytis cinerea and the biological control agent, Ulocladium oudemansii. In: 8th international
plant pathology congress, Christchurch, p 23
Shruthi TH, Sunkad G, Mallesh SB, Yenjerappa ST (2019) Isolation and bio-efficacy screening of
native Trichoderma species as a potential biocontrol agent against pomegranate wilt caused by
Ceratocystis fimbriata Ellis and Halst. J Pharmacogn Phytochem 8(5):1581–1585
Sikora RA (1992) Management of the antagonistic potential in agricultural ecosystems for the
biological control of plant-parasitic nematodes. Annu Rev Phytopathol 30:245–270
Sikora RA, Hoffmann-Hergarten S (1993) Biological control of plant parasitic nematodes with
plant-health-promoting rhizobacteria. In: Lumsden RD, Vaughn JL (eds) Pest management:
biotechnology based technologies. American Chemical of Society, Washington, pp 166–172
Singh R, Parameswaran TN, Prakasa Rao EVS, Puttanna K, Kalra A, Srinivas KVNS, Bagyaraj DJ,
Divya S (2009) Effect of arbuscular mycorrhizal fungi and Pseudomonas fluorescens on root-rot
and wilt, growth and yield of Coleus forskohlii. Biocontrol Sci Technol 19:835–841
Smith G, Grenfell BT (1994) Modelling of parasite populations: gastrointestinal nematode models.
Vet Parasitol 54(1–3):127–143
Soylu S, Baysal O, Soylu EM (2003) Induction of disease resistance by the plant activator,
acibenzolar-S-methyl (ASM), against bacterial canker (Clavibacter michiganensis ssp.
michiganensis) in tomato seedlings. Plant Sci 165:169–175
Spiegel Y, Cohn E, Galper S, Sharon E, Chet I (1991) Evaluation of newly isolated bacterium,
Pseudomonas chitinolytica for controlling the root-knot nematode Meloidogyne javanica.
Biocontrol Sci Technol 1:115–125
Srinivas C, Niranjana SR, Shetty HS (2005) Effect of bioagents and fungicides against Phomopsis
vexans and on seed quality of brinjal. Crop Improv 32:95–101
Sriram S, Roopa KP, Savitha MT (2011) Extended shelf-life of liquid fermentation derived talc
formulations of Trichoderma harzianum with the addition of glycerol in the production
medium. Crop Prot 30:1334–1339
Srivastava R, Khalid A, Singh US, Sharma AK (2010) Evaluation of arbuscular mycorrhizal
fungus, fluorescent Pseudomonas and Trichoderma harzianum formulations against Fusarium
oxysporum f. sp. lycopersici for the management of tomato wilt. Biol Control 53:24–31
Su SJ, Leu LS (1980) Three parasitic fungi on Sclerotinia sclerotiorum (Lib.) de Bary. Plant Prot
Bull Taiwan 22:253–262
Suarez-Estrella F, Vargas-García C, Lopez MJ, Capel C, Moreno J (2007) Antagonistic activity of
bacteria and fungi from horticultural compost against Fusarium oxysporum f. sp. melonis. Crop
Prot 46–53
Sultan MS, Sharma SK, Dhillon NK (2011) Evaluation of biopesticides for the management of root
knot nematode in fennel, coriander and fenugreek. Plant Dis Res 26:193–194
Szezech M, Shoda M (2006) The effect of mode of application of Bacillus subtilis RB14-C on its
efficacy as a biocontrol agent against Rhizoctonia solani. J Phytopathol 154:370–377
Thi BVT, Khanh NHP, Namikawa R, Miki K, Kondo A, Thi PTD, Kamei K (2016) Genomic
characterization of Ralstonia solanacearum phage ϕRS138 of the family Siphoviridae. Arch
Virol 161:483–486
Ting ASY, Meon S, Kadir J, Radu S, Singh G (2009) Induced host resistance by non-pathogenic
Fusarium endophyte as a potential defense mechanism in Fusarium wilt management of banana.
Pest Technol 3:67–72
Tran H, Ficke A, Asiimwe T, Hofte M, Raaijmakers JM (2007) Role of the cyclic lipopeptide
massetolide A in biological control of Phytophthora infestans and in colonization of tomato
plants by Pseudomonas fluorescens. New Phytol 175:731–742
Unnamalai N, Gnanamanikam SS (1984) Pseudomonas fluorescence is an antagonist to
Xanthomonas citri, the incitant of citrus canker. Curr Sci 53:703–704
Uppal AK, El Hadrami A, Adam LR, Tenuta M, Daayf F (2008) Biological control of potato
Verticillium wilt under controlled and field conditions using selected bacterial antagonists and
plant extracts. Biol Control 44:90–100
Utkhede R, Koch C (2004) Biological treatments to control bacterial canker of greenhouse
tomatoes. Biol Control 49:305–313
Van der Schaaf DA, Ravenberg WJ, Malais M (1991) Verticillium lecanii as a microbial insecticide
against white fly. IOBC/WPRS Bull 14:120–123
Van Gundy SD (1985) Ecology of Meloidogyne spp.-emphasis on environmental factors affecting
survival and pathogenicity. Bio Control 178–182
Varma RK, Bhale MS (2010) Microorganisms for the management of phomopsis blight and fruit rot
of eggplant caused by Phomopsis vexans (Sacc. & Syd.) Harter. J Mycopathol Res 48:245–249
Vestergaard S, Gillespie AT, Butt TM, Schreiter G, Eilenberg J (1995) Pathogenicity of the
hyphomycete fungi Verticillium lecanii and Metarhizium anisopliae to the western flower
thrips, Frankliniella occidentalis. Bio Sci Technol 5:185–192
Vihol JB, Patel KD, Jaiman RK, Patel NR (2009) Efficacy of plant extracts, biological agents and
fungicides against Alternaria blight of cumin. J Mycol Plant Pathol 39:516–519
Vyas RK, Mathur K (2002) Distribution of Trichoderma spp. in cumin rhizosphere and their
potential in suppression of wilt. Indian Phytopath 55(4):451–457
Wang SY, Chen PF, Chang ST (2005) Antifungal activities of essential oils and their constituents
from indigenous cinnamon (Cinnamomum osmophloeum) leaves against wood
Wu HS, Yang XN, Fan JQ, Miao WG, Ling N, Xu YC, Huang QW, Shen Q (2009) Suppression of
Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic
microorganisms. Biol Control 54(2):287
Xue QY, Chen Y, Li SM, Chen LF, Ding GC, Guo DW, Guo JH (2009) Evaluation of the strains of
Acinetobacter and Enterobacter as potential biocontrol agents against Ralstonia wilt of tomato.
Bio Control 48:252–258
Yadav R, Pathak SP (2011) Management of early blight of potato through fungicides and botanicals
and bio agents. Plant Arch 11:1143–1145
Zakharchenko NS, Kochetkov VV, Buryanov YI, Boronin AM (2011) Effect of rhizosphere
bacteria Pseudomonas aureofaciens on the resistance of micro propagated plants to
phytopathogens. Appl Biochem Microbiol 47:661
Zambolim L, Schenck NC (1983) Reduction of the effects of pathogenic, root-infecting fungi on
soybean by the mycorrhizal fungus, Glomus mosseae. Phytopathology 73:1402–1405
Transgenerational Plant Immunity in Plant
Disease Management 21
Md Mahtab Rashid, Raina Bajpai, Basavaraj Teli, Ankita Sarkar,
and Birinchi Kumar Sarma
Abstract
Plants have the potentiality to transfer the message of threat to their offspring.
Plants adopt such mechanisms probably due to the fact that the infants and the
younger ones are particularly viewed to be vulnerable to the detrimental effects of
the environment. Plants can pass on such messages to the next generation through
their seeds. Parents use mostly three mechanisms that are present at disposal to
the higher organisms to start and sustain the epigenetic gene regulation such as
DNA methylation, histone modification, and RNA interference. Plants may be
induced to bring out epigenetic modifications for their signature stress memories
through a process known as “priming.” Priming can induce epigenetic
modifications in plants to face both biotic and abiotic stresses and the same can
be passed on to their modifications. Therefore, transgenerational epigenetics is
seen as a future strategy to combat both biotic and abiotic stresses in plants.
Keywords
Plant immunity · Transgeneration · Epigenetics · DNA methylation · Histone
modifications
21.1 Introduction
All living organisms on this planet, that is, humans, animals, plants, microbes, along
with the soil and environment are suffering from the casualties posed by the
pesticidal regime that is being followed currently, and we are in search of much-
needed advancement in the present agricultural practices. Infants and the younger
M. M. Rashid · R. Bajpai · B. Teli · A. Sarkar · B. K. Sarma (*)

Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu
University, Varanasi, India

https://doi.org/10.1007/978-981-15-6275-4_21
458 M. M. Rashid et al.
ones are particularly viewed to be vulnerable to the detrimental effects of the

pesticides (Eskenazi et al. 1999). Apart from that, the change in climatic condition
is being expected to increase the predominance of extreme environmental conditions
which poses an undeniable threat to the loss in crop yield in near future (Lobell et al.
2011). On the account of such conditions, improved tolerance to various stress and
substitutes to synthetic pesticides is the need of the hour (Sarma et al. 2007; Singh
et al. 2013a). Although the responses of plants to distinct stresses are relatively well
understood, however, the natural occurrence of stress is incessant or recurrent and
reactions to such stresses are limitedly understood. The recent studies have
suggested that plants carry a stress memory which helps in adapting to the recurrent
stress (Bruce et al. 2007; Crisp et al. 2016). A highly promising measure to improve
tolerance to stress and to curtail the use of pesticides in agricultural field is by
reinforcing the host innate immunity (Singh et al. 2013b) through improvements in
the stress memory either by activating priming responses or via alteration(s) in
specific regions of the epigenome.
The gene accessibility for the transcriptional process is regulated by the chroma-
tin structure, and so it is an indispensable component of regulated expression of the
gene in responses to stress and development (Struhl and Segal 2013; Zentner and
Henikoff 2013). The access to separate regulatory elements and the overall packing
is affected essentially by methylation of the DNA along with the positioning and
spacing of nucleosomes. The chromatin consists of nucleosomes as their basic unit,
which is made up of two molecules, namely, histone octamer having histones H2A,
H2B, H3, and H4; wrapped around in almost two turns with 147 bp of DNA. The
overall packaging is contributed by the variation in length of three unpackaged
linker-DNA sections present in between two nucleosomes and the bond of linker
histone H1. Posttranslational modifications of histone tail such as acetylation,
methylation, phosphorylation, and ubiquitination; the decisive positioning and ten-
ancy of nucleosomes; and inclusion of the variants of histones replacing the canoni-
cal histones lead to an alteration in the chromatin structure. Additionally, DNA
modification is also achieved by methylation of cytosine which affects the DNA
sequence accessibility without any change in base pairing or the genetic code.
Cytosine methylation in plants take place at CpG, CHG, or CHH sequences and,
in this context DNA methylation is differentiated into asymmetrical and symmetrical
(Matzke and Mosher 2014; Du et al. 2015). The mechanism of inheritance of CpG or
symmetrical DNA methylation is straightforward by DNA replication, resulting two
daughter strands which are hemi-methylated, and the missing methylation mark can
be filled by recruiting a DNA methyltransferase on newly replicated daughter
strands. This symmetrical DNA methylation is a faithful model of inheritance
mitotically and is often referred to as a mark of epigenetics (Lämke and Bäurle
2017).
Epigenetics is defined as the study of all the stable, reversible, heritable changes
in the gene expression of an organism without any change in the DNA sequence, that
is change in the phenotype of the organism without any change in their genotype.
Basically, these changes or modification leads to change in pattern in how the cells
read the genes. These epigenetic changes occur regularly and naturally as well as
21 Transgenerational Plant Immunity in Plant Disease Management 459
several factors influence it including age, environment, and disease. The term
“epigenetics” was coined originally by Conrad Hal Waddington in 1942. Epigenetic
modification, in general, is a term used for the changes in the structure of nucleo-
some through modifications in histone, histone variants, or DNA modification,
although, all the epigenetic modifications are not necessarily of the epigenetic
phenomenon. These epigenetic modifications that are caused due to abiotic or biotic
stresses lead to a stress memory which is described as the phenomenon by which
message about a past stress clue is retained and as a result, a modified response is
seen when the stress occurs recurrently. This stress memory can be intergenerational
or transgenerational. The intergenerational stress memory can be defined as the
stress memory which is passed on to just the first stress-free offspring generation
of the organisms from one stressed generation, while the transgenerational stress
memory can be defined as the stress memory which is noticeable after at least two
stress-free generations of organisms.
21.2 Epigenetic Mechanisms in Plants
Plants use all the three mechanisms that are present at disposal to the higher
organisms to start and sustain the epigenetic gene regulation – DNA methylation,
histone modification, and RNA interference. In DNA methylation process, the
methylation of cytosine is the sole method for epigenetic regulation, but histone
modifications include numerous methods which involve acetylation, methylation,
phosphorylation, ribosylation, sumoylation, and ubiquitination. RNA interference,
on the other hand, is a feature of both DNA methylation and histone modifications.
The three mechanisms of epigenetics have various effects and implications on the
plants. The mechanism is discussed as below:
(i) DNA Methylation: This is a method for the epigenetic inheritance, that is,
transfer of expression state of the gene from the mother to daughter cells. It is
also a very effective regulatory system of gene expression (Tchurikov 2005).
The process involves the addition of a methyl group in the cyclic carbon-5 of
cytosine ring in DNA. The levels of methylation greatly vary among the
organisms as the methylated cytosines (5mC) percentage ranges from 0 to
35 in insects to more than 30% in some plants (Adams 1996). The DNA
methylation in plants is controlled by plant hormones and also influenced by
various phytopathogens, and it is also specific to species, tissue, organelle, and
age (Vanyushin 2005). The sites of DNA methylation in plants are CpG
(cytosine–phosphate–guanine) islands, CpNpG (N ¼ A, C, or T), and
CpNpN. The methylation is restricted to symmetrical sites (CpG) in mammals,
but it occurs in asymmetrical sites (CpNpN) in plants. The catalyst in the DNA
methylation process is a family of enzyme which is conserved and known as
DNA methyltransferases (DNA MTases). There are three types of DNA
MTases, namely, maintenance methylases, de novo methylases, and domain-
rearranged methylases (DRMs).
The proposed prominent roles of DNA methylation process are as follows:

• To direct the developmental process of an organism by providing a heritable
epigenetic mark (Holliday and Pugh 1975; Regev et al. 1998; Wolffe and
Matzke 1999)
• To implement the genomic defense responses against the parasitic mobile
organism (Yoder et al. 1997)
• To inhibit the transcriptional chaos in organisms having a large number of
genes (Bird 1995)
• To remember the activity pattern of the gene by stabilizing gene silencing
that occurs through other mechanisms (Bird 2002)
Despite the above-mentioned roles, we can expect the methylation process
to also serve diverse functions and to perform distinct task both within and
among the organisms, as it is an evolutionary tool (Colot and Rossignol
1999).
The DNA methylation is associated closely with silencing of the gene,
although it is not the cause but the consequence. The silencing occurs by
methylation in the specified genes or in the promoter region which results in
suppression of the transcription process. Evidences to support the role of
methylation in gene suppression have been shown by studies on transposable
elements regulation (Fedoroff 1996; Martienssen 1996) and silencing by trans-
gene in genetically engineered plants (Morel et al. 2000; Paszkowski and
Whitam 2001; Fojtova et al. 2003; Matzke et al. 2004).
(ii) Histone Modification: The modifications in the histone protein have come out
as a critical epigenetic modifier which leads to regulation of DNA-encoded
information. As described earlier, the nucleosome is the basic unit of chromatin
made up of histone octamer and 147 bp of DNA. Within the nucleosome, each
core histone protein has a globular domain-mediating histone–histone
interactions and a highly dynamic terminal tail of around 20–35 amino acid
residues of which mainly are basic in nature. H2A histone additionally has
approximately 37 amino acid carboxy-terminal domain which protrudes from
the nucleosome. The modifications of all the histones occur inside the cell
nucleus, although by far only some of the modifications have been studied. Till
date, 200 and more distinct posttranslational modifications (PTMs) have been
identified and the numbers are still growing. Posttranslational modifications of
histones take place by an array of processes which include lysine (K) and
arginase (R) acetylation and methylation; serine (S) and threonine
(T) phosphorylation; lysine ubiquitination, sumoylation, and biotinylation;
and ribosylation of ADP (Munshi et al. 2015).
Of the above-mentioned processes of posttranslational modifications, histone
acetylation causes transcriptional activation (Jacobson and Pillus 2004), lysine
methylation in histones (H1, H3–K9, H3–K27, H4–K20) causes silencing, in
histones (H3–K4, H3–K79) causes transcriptional activation (Bastow et al.
2004; Kirmizis et al. 2004; Schotta et al. 2004; Schneider et al. 2004), and
histone phosphorylation causes transcriptional activation (Cheng and Shearn
2004). The enormous diversity makes a “histone code” (Strahl and Allis 2000),
which arises due to the multiple combinations of different modifications and is

read and interpreted by various factors of the cell. These modifications in
histones lead to remodeling in chromatin which in turn regulates responses to
abiotic stress (Luo et al. 2012).
(iii) RNA Interference (RNAi): Epigenetic gene regulation is also influenced by the
presence of non-protein-coding RNAs. After the discovery of double-stranded
RNAs (dsRNAs) as a robust means of gene silencers in Caenorhabditis elegans
and plants, RNA interference (RNAi) has evolved as a novel approach to
understand gene expression regulation. RNAi also brings about gene
downregulation by small RNAs (about 21–24 nucleotides) which directs
proteins of Argonaute protein family to a nucleotide target sequence through
complementary base pairings. Based on the effector complexes
(or RNA-induced silencing complexes, RISC) protein composition and the
target sequence’s nature, the downregulation or silencing of genes can be
through mRNA degradation, posttranscriptional gene silencing (PTGS) by
repression of translation and alternative splicing of mRNA, or transcriptional
gene silencing (TGS) by genome modification (Munshi et al. 2015). The role of
RNA-mediated gene silencing pathways is essential in the development of
plants, the structure of the chromosome, and resistance to virus (Tsaftaris
et al. 2005).
21.3 Mechanism and Regulation of DNA Methylation
Methylation of cytosine occurs by the transfer of methyl group from S-adenosyl

methionine to the 50 position of cytosine in the presence of a covalent enzyme
catalyst that transcends into the formation of 5-methylcytosine (5mC). There are
comparatively high levels of 5mC in plants, that is, 6–25% of total cytosines as per
the species (Steward et al. 2000). Due to the symmetrical nature of CpG and CpNpG
methylation, they are copied simply after replication of DNA, although after each
subsequent DNA replication cycle the non-symmetrical CpNpN methylation has to
be established de novo (Karlsson et al. 2011). During the vegetative phase of plants,
this epigenetic memory gets accumulated under the influence of environment and is
passed to the subsequent generation by germline cells, that later gets established
during development. As described earlier also, methylation of DNA takes place at
promoter as well as in body regions of the gene which leads to the existence of genes
in a repressed state. So, there is likely to be an increased gene expression when the
level of methylation declines (Finnegan et al. 1998).
The enzymes which take part in cytosine methylation are categorized into three
groups – methyltransferase1 (MET1), chromomethylase3 (CMT3), and domains
rearranged methylase (DRM). MET1 is responsible for the CpG methylation and
the defective plants which do not possess the enzyme lack CpG methylation
(Lindroth et al. 2001). The enzyme CMT3 is responsible for CpNpG methylation
at transposons and at centromeric repeats (Lindroth et al. 2001 and Tompa et al.
2002). Two of these methylation processes imprint symmetric methylation on the
parental DNA (Chan et al. 2005). There are two methyltransferases in DRM, that is,
DRM1 and DRM2 which act as a catalyst in de novo methylation at CpNpNp sites
and is of asymmetrical type (Ramsahoye et al. 2000; Gowher and Jeltsch 2002; Cao
and Jacobsen 2002). Apart from the above-specified catalysis of enzymes, there are
also reports of various functional redundancies of CMT3 and MET1 in methylation
at CpNpG sites (Cao et al. 2003). There is also the implication that there exists a
functional redundancy of CMT3 for methylation of CpG and CpNpG sites, like
single and double mutants of met1 and cmt3 lead to the activation of CACTA
transposon which is normally silenced (Kato et al. 2003).
In plants, the status of DNA methylation is regulated by various developmental
processes, physiological processes, and various abiotic and biotic stresses. The
process of histone and DNA methylation are interdependent as there is a loss of
H3K9 methylation due the result of losing CpG methylation in met1 (Soppe et al.
2002; Tariq et al. 2003), although, there was no effect on CpG methylation due to the
loss of H3K9 methylation in kyp (Kryptonite) histone methyltransferase
(Jasencakova et al. 2003). As from the reports, we can conclude that the methylation
of H3K9 acts later than CpG methylation which fortifies the foundation of hetero-
chromatin. In contrast, the CpNpG methylation is reported to be reliant partially on
the kyp activity (Jackson et al. 2002). DNA methyltransferase and DNA demethyla-
tion enzymes both are responsible for the status of overall DNA methylation. The
demethylation occurs in either an active or passive way. The active demethylation
may take place through the activity of glycolyase by the removal of methylcytosines
from DNA (Zhu et al. 2000, 2007; Agius et al. 2006; Morales-Ruiz et al. 2006),
rather than this the passive demethylation may take place through hinderance in de
novo methylation or incompetency to preserve the paternal imprints after the repli-
cation of DNA (Kankel et al. 2003). These processes may play vital functions in the
prevention of the formation of epialleles with stable hypermethylation in the genome
of a plant (Penterman et al. 2007).
Additionally, small RNAs also assume an imperative job in the regulation of
epigenetics through the RNA-directed DNA methylation (RdDM) in reverberation
to the various stresses, growth, and development by means of transcriptional gene
silencing. The mechanism starts with producing the transcripts for biogenesis of
siRNA after mediation through RNA Pol II and RNA Pol IV by the pathway of RNA
interference. In the initial phase, the RNA-dependent RNA polymerase 2 (RDR2)
converts the single-stranded RNAs (ssRNAs) into double-stranded RNAs (dsRNAs)
which are previously produced from transcription of methylated DNA through RNA
Pol IV; while the inverted repeat regions are targeted by RNA Pol II for a dsRNAs
generation. The advanced processing of these dsRNAs is done by Dicer-like
3 (DCL3), HUA enhancer1 (HEN1), and finally loading of this processed product
on Argonaut 4 (AGO4). The complex thus formed interfaces with the Pol V’s largest
subunit via WG/GW repeats of Nuclear RNA polymerase (NRPE1) at C-terminal
domain (El-Shami et al. 2007). The DNA methylation of the homologous DNA
sequence is inducted by this machinery of siRNA and affiliated proteins by DRM2.
Apart from that, the binding of AGO4 to specific gene promoters also takes place
with the assistance of Pol V-derived long non-coding RNAs (lncRNAs). The
avocation of this complex escorts asymmetrical CpNpN-type methylation of DNA in

promoter regions that sequentially modulates the expression of the target gene
(Zheng et al. 2013).
21.4 Priming: A Method to Induce DNA Methylation-Mediated

Stress Memory
Endangering the plants to meagerly virulent necrotrophic pathogens/beneficial

organisms or to any compound that induces resistance leads to a peculiar state of
defense in plants which enables them to acquire intensified defense responses
whenever they are challenged further by biotic agents or abiotic factors. The state
of embellished capability to stimulate stress-induced defense responses has been
named as the “primed” state of the plant (Conrath et al. 2002) (Fig. 21.1). Priming is
defined as the phenomenon by which a transitory biotic or abiotic stress action on the
plants leads to an altered defense response which may be typically accelerated and
vigorous upon the exposure to recurring stress (Conrath et al. 2015). The word
“priming” was formerly conceived in the background of plant’s immunity to the
biotic agents (pathogens), although later it was started as being used for the
responses to abiotic stresses also (Lämke and Bäurle 2017).
The unfavorable growth conditions which repress the natural plant growth and
development is defined as the stress that in extreme cases can be lethal also. These
conditions can arise due to flooding, drought, excessive salt in soil or irrigation
water, attack by a phytopathogen, and/or herbivore. The plants which are in primed
state respond to the triggering stress cue in a modified manner in comparison to those
plants which are in the unprimed (naïve) state. The mechanism of priming functions
at the phenotypic level and does not alter the sequence of DNA, which makes it
eventually reversible (Conrath et al. 2015; Hilker et al. 2016). In general, priming
leads to a faster and stronger pattern of response, which is evident by the modified
activation dynamics of expression of defense genes. The speed of plant defense is
dependent on the time taken by the plant to recognize the attacker which means
sooner the identification, more effective is the defense response.
The stress memory period takes place after the event of priming (Stief et al. 2014)
in which the information is stored about the priming stress clue after its remission.
This memory spans over a period of days to weeks in case of somatic stress memory
and to the offspring (intergenerational or transgenerational). The best possible
mechanisms for memory manifestation can be as follows:
• Modifications in the transcriptional response, that is, transcriptional memory, in

which either sustained alterations in expression of the gene (may be either
activation or repression) is induced by priming stimulant or same by a secondary
stimulant (as in hyperinduction) (Light and Brickner 2013; D’Urso and Brickner
2017).
Fig. 21.1 A detail scheme of seed priming leading to transgenerational immunity via histone
modifications in plants
• Involvement of transcriptional feedback loops (as self-activation of a transcrip-

tion factor) or by posttranslational mechanisms (modifications of proteins)
(Ptashne 2008).
• Mechanisms which are transcription independent, that is, prions transmission or

prion-like proteins transmission (Shorter and Lindquist 2005; Chakrabortee et al.
2016a, b) as notably described in yeast (Tyedmers et al. 2008).
All the cases of stress memory have been confirmed with a possibility of epigenetic
basis and as by the definition it requires the phenomenon to be heritable and stable,
yet change-independent DNA sequence. A true transgenerational stress memory is
mostly expected to be epigenetic, although it would not hold for somatic stress
memory due to its shorter duration. The scientific meaning of the term “epigenetic
mechanisms” contains all the specifications that have an impact on chromatin
structure (may or may not be stably inheritable) including methylation of DNA.
There have been many reports about transgenerational inheritance of DNA methyla-
tion of plants which grows under the stress conditions (Hauser et al. 2011; Feng et al.
2012), and this epigenetic flexibility has a crucial part in the immediate and long-
term adaptation of organisms under stress (Mirouze and Paszkowski 2011). The
phenomenon was reported in distant genotypes of rice (Oryza sativa) when treated
with salt and alkaline stresses and revealed that the level of DNA methylation
persisted in the progenies produced after selfing (Feng et al. 2012). As per Byoko
et al. (2010), while measuring the cytosine methylated DNA level in between the
progenies of treated and untreated plants in Arabidopsis for two generations, the
levels were maintained higher in the treated plant progenies in response to both stress
and control conditions as compared to the untreated plant progenies of the same
generation. The study suggested that there is a decrease in DNA methylation in the
absence of stress. The transgenerational inheritance of stress tolerance is stimulated
even in the untreated progenies of tobacco plants through viral infection and of
Arabidopsis plants through UV-C exposure and flagellin by the means of global
genome methylation.
The progenies of Arabidopsis plants primed either with β-aminobutyric acid
(BABA) or with Pseudomonas syringae pv. tomato (avirulent isolate PstavrRpt2)
responded by showing a quicker and greater transcript accumulation of defense
genes related to salicylic acid signaling pathway (Slaughter et al. 2012). These
progenies also exhibited an embellished resistance to the disease on challenging
with Pseudomonas syringae (virulent isolate) and the oomycetic pathogen
Hyaloperonospora arabidopsidis. In addition to all these, the priming of progenies
of previously primed plants leads to an even greater magnitude of defense responses.
The plants of tomato (Solanum lycopersicon) and Arabidopsis too showed
transgenerational induced resistance which was jasmonic acid–dependent when the
plants at their vegetative growth stage were challenged by herbivory or methyl
jasmonate (Rasmann et al. 2012). These effects were persistent to the second
generation in Arabidopsis and their presence in plants belonging to Solanaceae
and Brassicaceae families proves the resistance to be transgenerational and distantly
dispersed among the plant kingdom (Sarma and Singh 2014). This concept is further
consolidated by the study conducted by Luna et al. (2012) in which the priming of
Arabidopsis plants was done by inoculating them with virulent Pseudomonas
syringae, and it showed the remnants of primed state onto the succeeding generation
and even sustenance of the same over one stress-free generation. A central role is
played by NPR1 in the transgenerational immunity as it is blocked in the SA
signaling nonexpressor of pathogenesis-related genes1 (npr1) mutant and addition-
ally the phenomenon of immunity was shown to be associated with modifications of
chromatin at promoters regions of SA-responsive genes PR-1, WRKY6, and
WRKY53. Apart from this, the transgenerational immunity which was generated
from bacterial infections is transmitted by the means of hypomethylation of genes
that are responsible for directing the priming of SA-dependent genes in the
succeeding generations (Luna et al. 2014).
21.5 Transgenerational Plant Immunity and Abiotic Stresses
Many complex gene regulatory mechanisms have evolved in plants which help in
coping with diverse environmental stresses and among these mechanisms chromatin
remodeling, DNA methylation, and small RNA-based mechanism are the major ones
involved in regulation of the expression of genes responding to climatic stresses
(Subbah et al. 1995; Gravitol et al. 2012). This theory was further consolidated by
the report of the presence of natural epigenetic variations among the mangrove plants
growing at banks of rivers having tall height and thicker stem as compared those
growing at salt-marsh habitat (Lira-Medeiros et al. 2010). The analysis of
methylation-sensitive amplification polymorphism (MSAP) proclaimed DNA
hypermethylation in riverside plants in comparison to the salt-marsh plants, thus
indicating toward the vital function played by the natural epigenetic variations
among the population of plants in adapting to the environment. In another study
consisting of genome-wide MSAP analysis performed for distinct rice genotypes
which have a differential response to salt-stress revealed differing methylation and
expression of salt-related genes and genes related to chromatin modification (Karan
et al. 2012). An investigation consisting of genome-wide analysis of two divergent
rice lines disclosed cytosine variations which are site-specific, recoverable, and
reversible; is regulated epigenetically; and related to drought adaptation (Wang
et al. 2010). Under the water-deficit condition also cytosine hypermethylation has
been seen in rice cultivars that are drought-tolerant and cultivated on lowlands (Suji
and Joel 2010).
There have also been reports of locus-specific changes in methylation in leaf
tissues of plant sustaining N-deficiency (Kou et al. 2011). In forest trees, heat stress
tolerance was discovered as the cork oak (Quercus suber) leaves exhibited interac-
tion of specific methylation of DNA and acetylation of H3 histone as an adaptation to
high temperature (Correia et al. 2013). The DNA methylation gets altered also by the
global warming phenomenon and stresses associated with nitrogen deposition in the
soil, thus offering a molecular basis for adaptation to these stresses in the naturally
occurring plant population as inspected in Leymus chinensis Tzvel. (Yu et al. 2013a).
The hypermethylation of transposable elements was recognized during these envi-
ronmental stresses when compared to other regions of the plant genome. The
physiological processes of plants like photosynthesis and development of
reproductive organs are modulated by the epigenetic mechanisms under the

conditions of stresses leading to its acclimatization (Yaish et al. 2011). Epigenetic
processes have been acknowledged as the functional and elemental factor of abscisic
acid (ABA)-regulated processes and the same phytohormone has been revealed to
operate in the expression of genes which are dependent on DNA methylation/
demethylation through small RNAs (Khraiwesh et al. 2010). The same study
conducted on moss Physcomitrella patens showed that miR1026 accumulation and
PpbHLH gene (miR1026 target gene) hypermethylation at CpG sites are induced by
ABA and eventually lead to decrease in expression of PpbHLH. Apart from these,
numerous researchers have identified ABA as controller of histone modifications
and thus as a regulator of DNA methylation (Chinnusamy et al. 2008; Yaish et al.
2011).
21.6 Transgenerational Plant Immunity and Biotic Stresses
As described earlier, the exposure to biotic stresses activates the plants’ immune
system and basal defense machinery by the process of defense priming
(Muthamilarasan and Prasad 2013). DNA methylation has emerged as a major
approach of defense strategy in plants against the biotic stresses. The progenies
which were obtained from diseased Arabidopsis showed enhancement in resistance
against the downy mildew pathogen and other biotrophs also (Luna and Ton 2012).
Apart from that, there are also reports of DNA methylation playing a role in defense
mechanisms of Arabidopsis against the bacterial pathogens (Dowen et al. 2012; Yu
et al. 2013b). The offspring of Arabidopsis inoculated with Pseudomonas syringae
pv. tomato DC3000 (PstDC3000) exhibited an increased plant immunity when they
were further challenged by the pathogens. The PstDC3000 inoculation activated
salicylic acid (SA)-inducible defense genes and repressed jasmonic acid (JA)-
inducible genes of the plants, and the enhanced resistance of the offspring was not
only limited to PstDC3000 but also to a (semi)biotrophic pathogen
Hyaloperonospora arabidopsidis. As mentioned earlier, it has been shown that
signal transduction is required via the NPR1 gene for transgenerational immunity/
defense response in the plants (Luna et al. 2012).
During the infection of the pathogen, DNA hypomethylation has also been
reported to influence the expression of the defense-related gene as the chemically
demethylated Xa21G (R gene) gene of rice showed heritable resistance against
Xanthomonas oryzae pv. oryzae (Akimoto et al. 2007). A very interesting report
disclosed that regulation of formulation of crown gall tumor is controlled by DNA
methylation through ABA-dependent stress defense in Arabidopsis plants (Gohlke
et al. 2013). Advanced epigenetic researches revealed that the plants use siRNA-
mediated methylation strategy systematically as a mechanism of defense against
viral pathogens through methylation of viral genomic components (Bian et al. 2006;
Tougou et al. 2007; Yadav and Chattopadhyay 2011; Emran et al. 2012; Sharma
et al. 2013) and there is a positive correlation between the high methylation of viral
DNA and recovery of symptoms after infection from virus (Rodríguez-Negrete et al.
2009). A separate study in soybean showing resistance to Mungbean yellow mosaic

India virus (MYMIV) identified a higher level of DNA methylation specific to
Intergenic Region (IR) (Yadav and Chattopadhyay, 2011). In addition to this,
research conducted to study the dynamics of tomato cultivar tolerant to Tomato
leaf curl New Delhi virus (ToLCNDV) also showed substantial methylation at IR
and part of replication-associated protein (rep) gene in the cultivar (Sahu et al. 2010,
2012).
There has been a report of a correlation between genomic DNA hypomethylation
in plants and the abundance of transcripts of genes related to defense (Wada et al.
2004). In tobacco plants infected with Tobacco mosaic virus (TMV), it was found
that 24 hours after the infection of viral pathogen, the transcript accumulation of
NtAlix1 (pathogen-responsive gene) was higher and there were also changes in
methylation level (Sahu et al. 2013). The study suggested that expression of
defense-related factors is regulated by plants through DNA methylation when the
pathogen infestation takes place. A very effective tool for the introduction of DNA
methylation in any gene (endogenous) is virus-induced gene silencing (VIGS) that
relies upon double-stranded transcripts generation. An artificial alteration of DNA
methylation through the Cucumber mosaic virus (CMV)-based gene silencing
system is illustrated as a substitute for epigenetic changeover in plants’ endogenous
gene (Kanazawa et al. 2011). Hence, it can be said that the epigenetics lead to
priming of defense mechanisms in plants, allowing them to secure their succeeding
generations against biotic stresses which occurs repeatedly without any stable
heritable fixation of the trait.
21.7 Conclusions and Future Perspectives
Many reports have consolidated the response of the plant to abiotic and biotic
stresses through DNA methylation, but there are many gaps which pose many
unanswered questions regarding the methylation pathways, activation of adaptive
mechanisms, and sensing of stresses. The study of DNA methylation is much easier
in context of the model plant Arabidopsis thaliana having small genome size and
simple genetics but the true challenge for the plant biologists lies in trying to
understand about the epigenetic mechanisms in response to various stresses and its
feasible utilization in the plants’ genetic manipulation. The transgenerational
epigenetics which are inducible can be further utilized to boost up the production
and protection of agricultural crops. Moreover, it can be seen as a tool for the
development of an easy and convenient technique to bring desirable changes in
plants. Epigenetics can also be seen as promising machinery which can be utilized in
understanding the functional genomics of plants as it has the capability to elucidate
and confer tolerance and/or resistance to stresses among the various species of plants
including the agricultural crops. The transgenerational inducible epigenetic changes
in some of the genes’ expression pattern enable us with the freedom to exploit and to
bring out the desired inheritable changes for better production and protection in the
target crops. A better understanding of the epigenetic phenomenon will aid in
making more developed strategies for regulating plant genes according to the
necessities of agriculture. Apart from the epigenetics, identification and characteri-
zation of promising priming agents (including both abiotic and biotic agents) and
their receptor sites on the host will make the implementation of this compelling
biological mechanism successful in future. Seeds being the primary vehicle for the
production and propagation of plants make the ones harvested from the primed
plants to show a stronger defense potential against the different abiotic and biotic
stresses. Thus, we can conclude that invigorating the plants’ innate immunity by the
transgenerational epigenetics will be significantly beneficial in reducing the applica-
tion of synthetic pesticides against the biotic stresses. The present knowledge and the
future prospects of transgenerational epigenetics in plants have inspired the
researchers to fantasize about a comparatively pesticide-free environment and
which is also the demand to save the ecological balance of our mother earth.
References
Adams RLP (1996) DNA methylation. In: Bittar EE (ed) Principles of medical biology, vol 5. JAI
Press Inc., New York, pp 33–66
Agius F, Kapoor A, Zhu JK (2006) Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active
DNA demethylation. Proc Natl Acad Sci 103(31):11796–11801
Akimoto K, Katakami H, Kim HJ, Ogawa E, Sano CM, Wada Y, Sano H (2007) Epigenetic
inheritance in rice plants. Ann Bot 100(2):205–217
Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C (2004) Vernalization requires
epigenetic silencing of FLC by histone methylation. Nature 427(6970):164
Bian XY, Rasheed MS, Seemanpillai MJ, Rezaian MA (2006) Analysis of silencing escape of
tomato leaf curl virus: an evaluation of the role of DNA methylation. Mol Plant-Microbe
Interact 19(6):614–624
Bird AP (1995) Gene number, noise reduction and biological complexity. Trends Genet 11
(3):94–100
Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16(1):6–21
Boyko A, Blevins T, Yao Y, Golubov A, Bilichak A, Ilnytskyy Y, Hollander J, Meins F Jr,
Kovalchuk I (2010) Transgenerational adaptation of Arabidopsis to stress requires DNA
methylation and the function of Dicer-like proteins. PLoS One 5(3):e9514
Bruce TJ, Matthes MC, Napier JA, Pickett JA (2007) Stressful “memories” of plants: evidence and
possible mechanisms. Plant Sci 173(6):603–608
Cao X, Jacobsen SE (2002) Role of the Arabidopsis DRM methyltransferases in de novo DNA
methylation and gene silencing. Curr Biol 12(13):1138–1144
Cao X, Aufsatz W, Zilberman D, Mette MF, Huang MS, Matzke M, Jacobsen SE (2003) Role of the
DRM and CMT3 methyltransferases in RNA-directed DNA methylation. Curr Biol 13
(24):2212–2217
Chakrabortee S, Byers JS, Jones S, Garcia DM, Bhullar B, Chang A, She R, Lee L, Fremin B,
Lindguist S, Jarosz DF (2016a) Intrinsically disordered proteins drive emergence and inheri-
tance of biological traits. Cell 167(2):369–381
Chakrabortee S, Kayatekin C, Newby GA, Mendillo ML, Lancaster A, Lindquist S (2016b)
Luminidependens (LD) is an Arabidopsis protein with prion behavior. Proc Natl Acad Sci
113(21):6065–6070
Chan SWL, Henderson IR, Jacobsen SE (2005) Gardening the genome: DNA methylation in
Arabidopsis thaliana. Nat Rev Genet 6(5):351
Cheng MK, Shearn A (2004) The direct interaction between ASH2, a Drosophila trithorax group
protein, and SKTL, a nuclear phosphatidylinositol 4-phosphate 5-kinase, implies a role for
phosphatidylinositol 4, 5-bisphosphate in maintaining transcriptionally active chromatin. Genet-
ics 167(3):1213–1223
Chinnusamy V, Gong Z, Zhu JK (2008) Abscisic acid-mediated epigenetic processes in plant
development and stress responses. J Integr Plant Biol 50(10):1187–1195
Colot V, Rossignol JL (1999) Eukaryotic DNA methylation as an evolutionary device. BioEssays
21(5):402–411
Conrath U, Pieterse CM, Mauch-Mani B (2002) Priming in plant–pathogen interactions. Trends
Plant Sci 7(5):210–216
Conrath U, Beckers GJ, Langenbach CJ, Jaskiewicz MR (2015) Priming for enhanced defense.
Correia B, Valledor L, Meijón M, Rodriguez JL, Dias MC, Santos C, Cañal MJ, Rodriguez R, Pinto
G (2013) Is the interplay between epigenetic markers related to the acclimation of cork oak
plants to high temperatures? PLoS One 8(1):e53543
Crisp PA, Ganguly D, Eichten SR, Borevitz JO, Pogson BJ (2016) Reconsidering plant memory:
intersections between stress recovery, RNA turnover, and epigenetics. Sci Adv 2(2):e1501340
D’Urso A, Brickner JH (2017) Epigenetic transcriptional memory. Curr Genet 63(3):435–439
Dowen RH, Pelizzola M, Schmitz RJ, Lister R, Dowen JM, Nery JR, Dixon JE, Ecker JR (2012)
Widespread dynamic DNA methylation in response to biotic stress. Proc Natl Acad Sci 109(32):
E2183–E2191
Du J, Johnson LM, Jacobsen SE, Patel DJ (2015) DNA methylation pathways and their crosstalk
with histone methylation. Nat Rev Mol Cell Biol 16(9):519
El-Shami M, Pontier D, Lahmy S, Braun L, Picart C, Vega D, Hakimi MA, Jacobsen SE, Cooke R,
Lagrange T (2007) Reiterated WG/GW motifs form functionally and evolutionarily conserved
ARGONAUTE-binding platforms in RNAi-related components. Genes Dev 21(20):2539–2544
Emran AM, Tabei Y, Kobayashi K, Yamaoka N, Nishiguchi M (2012) Molecular analysis of
transgenic melon plants showing virus resistance conferred by direct repeat of movement gene
of Cucumber green mottle mosaic virus. Plant Cell Rep 31(8):1371–1377
Eskenazi B, Bradman A, Castorina R (1999) Exposures of children to organophosphate pesticides
and their potential adverse health effects. Environ Health Perspect 107(suppl 3):409–419
Fedoroff N (1996) Epigenetic regulation of the maize Spm transposable element. In: Russo VEA,
Martienssen RA, Riggs AD (eds) Epigenetic mechanisms of gene regulation. Cold Spring
Harbor Laboratory Press, New York, pp 575–592
Feng Q, Yang C, Lin X, Wang J, Ou X, Zhang C, Chen Y, Liu B (2012) Salt and alkaline stress
induced transgenerational alteration in DNA methylation of rice (Oryza sativa). Aust J Crop Sci
6(5):877
Finnegan EJ, Genger R, Kovac K, Peacock WJ, Dennis ES (1998) DNA methylation and the
promotion of flowering by vernalization. Proc Natl Acad Sci 95(10):5824–5829
Fojtova M, Van Houdt H, Depicker A, Kovarik A (2003) Epigenetic switch from posttranscrip-
tional to transcriptional silencing is correlated with promoter hypermethylation. Plant Physiol
133(3):1240–1250
Gohlke J, Scholz CJ, Kneitz S, Weber D, Fuchs J, Hedrich R, Deeken R (2013) DNA methylation
mediated control of gene expression is critical for development of crown gall tumors. PLoS
Genet 9(2):e1003267
Gowher H, Jeltsch A (2002) Molecular enzymology of the catalytic domains of the Dnmt3a and
Dnmt3b DNA methyltransferases. J Biol Chem 277(23):20409–20414
Grativol C, Hemerly AS, Ferreira PCG (2012) Genetic and epigenetic regulation of stress responses
in natural plant populations. Biochim Biophys Acta (BBA) Gene Regul Mech 1819(2):176–185
Hauser MT, Aufsatz W, Jonak C, Luschnig C (2011) Transgenerational epigenetic inheritance in
plants. Biochim Biophys Acta (BBA) Gene Regul Mech 1809(8):459–468
Hilker M, Schwachtje J, Baier M, Balazadeh S, Bäurle I, Geiselhardt S, Hincha DK, Kunze R,
Mueller-Roeber B, Rillig MC, Rolff J, Romeis T, Schmülling T, Steppuhn A, van Dongen J,
Whitcomb SJ, Wurst S, Zuther E, Kopka J (2016) Priming and memory of stress responses in
organisms lacking a nervous system. Biol Rev 91(4):1118–1133
Holliday R, Pugh JE (1975) DNA modification mechanisms and gene activity during development.
Science 187(4173):226–232
Jackson JP, Lindroth AM, Cao X, Jacobsen SE (2002) Control of CpNpG DNA methylation by the
KRYPTONITE histone H3 methyltransferase. Nature 416(6880):556
Jacobson S, Pillus L (2004) Molecular requirements for gene expression mediated by targeted
histone acetyltransferases. Mol Cell Biol 24(13):6029–6039
Jasencakova Z, Soppe WJ, Meister A, Gernand D, Turner BM, Schubert I (2003) Histone
modifications in Arabidopsis–high methylation of H3 lysine 9 is dispensable for constitutive
heterochromatin. Plant J 33(3):471–480
Kanazawa A, Inaba JI, Kasai M, Shimura H, Masuta C (2011) RNA-mediated epigenetic
modifications of an endogenous gene targeted by a viral vector: a potent gene silencing system
to produce a plant that does not carry a transgene but has altered traits. Plant Signal Behav 6
(8):1090–1093
Kankel MW, Ramsey DE, Stokes TL, Flowers SK, Haag JR, Jeddeloh JA, Riddle NC, Verbsky
ML, Richards EJ (2003) Arabidopsis MET1 cytosine methyltransferase mutants. Genetics 163
(3):1109–1122
Karan R, DeLeon T, Biradar H, Subudhi PK (2012) Salt stress induced variation in DNA methyla-
tion pattern and its influence on gene expression in contrasting rice genotypes. PLoS One 7(6):
e40203
Karlsson M, Weber W, Fussenegger M (2011) De novo design and construction of an inducible
gene expression system in mammalian cells. In: Methods in enzymology, vol 497. Academic
Press, pp 239–253
Kato M, Miura A, Bender J, Jacobsen SE, Kakutani T (2003) Role of CG and non-CG methylation
in immobilization of transposons in Arabidopsis. Curr Biol 13(5):421–426
Khraiwesh B, Arif MA, Seumel GI, Ossowski S, Weigel D, Reski R, Frank W (2010) Transcrip-
tional control of gene expression by microRNAs. Cell 140(1):111–122
Kirmizis A, Bartley SM, Kuzmichev A, Margueron R, Reinberg D, Green R, Farnham PJ (2004)
Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27.
Genes Dev 18(13):1592–1605
Kou HP, Li Y, Song XX, Ou XF, Xing SC, Ma J, Von Wettstein D, Liu B (2011) Heritable
alteration in DNA methylation induced by nitrogen-deficiency stress accompanies enhanced
tolerance by progenies to the stress in rice (Oryza sativa L.). J Plant Physiol 168(14):1685–1693
Lämke J, Bäurle I (2017) Epigenetic and chromatin-based mechanisms in environmental stress
adaptation and stress memory in plants. Genome Biol 18(1):124
Light WH, Brickner JH (2013) Nuclear pore proteins regulate chromatin structure and transcrip-
tional memory by a conserved mechanism. Nucleus 4(5):357–360
Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, Jacobsen SE (2001)
Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science
292(5524):2077–2080
Lira-Medeiros CF, Parisod C, Fernandes RA, Mata CS, Cardoso MA, Ferreira PCG (2010)
Epigenetic variation in mangrove plants occurring in contrasting natural environment. PLoS
One 5(4):e10326
Lobell DB, Schlenker W, Costa-Roberts J (2011) Climate trends and global crop production since
1980. Science 1204531
Luna E, Ton J (2012) The epigenetic machinery controlling transgenerational systemic acquired
resistance. Plant Signal Behav 7(6):615–618
Luna E, Bruce TJ, Roberts MR, Flors V, Ton J (2012) Next-generation systemic acquired resis-
tance. Plant Physiol 158(2):844–853
Luna E, López A, Kooiman J, Ton J (2014) Role of NPR1 and KYP in long-lasting induced
resistance by β-aminobutyric acid. Front Plant Sci 5:184
Luo M, Liu X, Singh P, Cui Y, Zimmerli L, Wu K (2012) Chromatin modifications and remodeling
in plant abiotic stress responses. Biochim Biophy Acta (BBA) Gene Regul Mech 1819
(2):129–136
Martienssen R (1996) Epigenetic silencing of Mu transposable elements in maize. In: Epigenetic
mechanisms of gene regulation. Cold Spring Harbor laboratory Press, Plainview, pp 593–608
Matzke MA, Mosher RA (2014) RNA-directed DNA methylation: an epigenetic pathway of
increasing complexity. Nat Rev Genet 15(6):394
Matzke M, Aufsatz W, Kanno T, Daxinger L, Papp I, Mette MF, Matzke AJ (2004) Genetic analysis
of RNA-mediated transcriptional gene silencing. Biochim Biophys Acta (BBA) Gene Struct
Expr 1677(1–3):129–141
Mirouze M, Paszkowski J (2011) Epigenetic contribution to stress adaptation in plants. Curr Opin
Plant Biol 14(3):267–274
Morales-Ruiz T, Ortega-Galisteo AP, Ponferrada-Marín MI, Martínez-Macías MI, Ariza RR,
Roldán-Arjona T (2006) Demeter and repressor of silencing 1 encode 5-methylcytosine DNA
glycosylases. Proc Natl Acad Sci 103(18):6853–6858
Morel JB, Mourrain P, Béclin C, Vaucheret H (2000) DNA methylation and chromatin structure
affect transcriptional and post-transcriptional transgene silencing in Arabidopsis. Curr Biol 10
(24):1591–1594
Munshi A, Ahuja YR, Bahadur B (2015) Epigenetic mechanisms in plants: an overview. In: Plant
biology and biotechnology. Springer, New Delhi, pp 265–278
Muthamilarasan M, Prasad M (2013) Plant innate immunity: an updated insight into defense
mechanism. J Biosci 38(2):433–449
Paszkowski J, Whitham SA (2001) Gene silencing and DNA methylation processes. Curr Opin
Plant Biol 4(2):123–129
Penterman J, Zilberman D, Huh JH, Ballinger T, Henikoff S, Fischer RL (2007) DNA demethyla-
tion in the Arabidopsis genome. Proc Natl Acad Sci 104(16):6752–6757
Ptashne M (2008) Transcription: a mechanism for short-term memory. Curr Biol 18(1):R25–R27.
https://doi.org/10.1016/j.cub.2007.11.017
Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R (2000) Non-CpG
methylation is prevalent in embryonic stem cells and may be mediated by DNA
methyltransferase 3a. Proc Natl Acad Sci 97(10):5237–5242
Rasmann S, De Vos M, Casteel CL, Tian D, Halitschke R, Sun JY, Agarwal AA, Felton GW,
Jander G (2012) Herbivory in the previous generation primes plants for enhanced insect
resistance. Plant Physiol 158(2):854–863
Regev A, Lamb MJ, Jablonka E (1998) The role of DNA methylation in invertebrates: develop-
mental regulation or genome defense? Mol Biol Evol 15(7):880–891
Rodríguez-Negrete EA, Carrillo-Tripp J, Rivera-Bustamante RF (2009) RNA silencing against
geminivirus: complementary action of posttranscriptional gene silencing and transcriptional
gene silencing in host recovery. J Virol 83(3):1332–1340
Sabbah S, Raise M, Tal M (1995) Methylation of DNA in NaCl-adapted cells of potato. Plant Cell
Rep 14(7):467–470
Sahu PP, Rai NK, Chakraborty S, Singh M, Chandrappa PH, Ramesh B, Chattopadhyay D, Prasad
M (2010) Tomato cultivar tolerant to Tomato leaf curl New Delhi virus infection induces virus-
specific short interfering RNA accumulation and defence-associated host gene expression. Mol
Plant Pathol 11(4):531–544
Sahu PP, Rai NK, Puranik S, Roy A, Khan M, Prasad M (2012) Dynamics of defense-related
components in two contrasting genotypes of tomato upon infection with Tomato Leaf Curl New
Delhi Virus. Mol Biotechnol 52(2):140–150
Sahu PP, Pandey G, Sharma N, Puranik S, Muthamilarasan M, Prasad M (2013) Epigenetic
mechanisms of plant stress responses and adaptation. Plant Cell Rep 32(8):1151–1159
Sarma BK, Singh HB (2014) Harnessing transgenerational plant immunity. Curr Sci 107
(12):1941–1942
Sarma BK, Basha SA, Singh DP, Singh UP (2007) Use of non-conventional chemicals as an
alternative approach to protect chickpea (Cicer arietinum) from Sclerotinia stem rot. Crop Prot
26(7):1042–1048
Schneider R, Bannister AJ, Myers FA, Thorne AW, Crane-Robinson C, Kouzarides T (2004)
Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat Cell Biol 6(1):73
Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, Reuter G, Reinberg D, Jenuwein T (2004) A
silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin.
Genes Dev 18(11):1251–1262
Sharma N, Sahu PP, Puranik S, Prasad M (2013) Recent advances in plant–virus interaction with
emphasis on small interfering RNAs (siRNAs). Mol Biotechnol 55(1):63–77
Shorter J, Lindquist S (2005) Prions as adaptive conduits of memory and inheritance. Nat Rev
Genet 6(6):435
Singh A, Jain A, Sarma BK, Upadhyay RS, Singh HB (2013a) Rhizosphere microbes facilitate
redox homeostasis in Cicer arietinum against biotic stress. Ann Appl Biol 163(1):33–46
Singh A, Sarma BK, Upadhyay RS, Singh HB (2013b) Compatible rhizosphere microbes mediated
alleviation of biotic stress in chickpea through enhanced antioxidant and phenylpropanoid
activities. Microbiol Res 168(1):33–40
Slaughter A, Daniel X, Flors V, Luna E, Hohn B, Mauch-Mani B (2012) Descendants of primed
Arabidopsis plants exhibit resistance to biotic stress. Plant Physiol 158(2):835–843
Soppe WJ, Jasencakova Z, Houben A, Kakutani T, Meister A, Huang MS, Jacobsen SE, Schubert I,
Fransz PF (2002) DNA methylation controls histone H3 lysine 9 methylation and heterochro-
matin assembly in Arabidopsis. EMBO J 21(23):6549–6559
Steward N, Kusano T, Sano H (2000) Expression of ZmMET1, a gene encoding a DNA
methyltransferase from maize, is associated not only with DNA replication in actively
proliferating cells, but also with altered DNA methylation status in cold-stressed quiescent
cells. Nucleic Acids Res 28(17):3250–3259
Stief A, Brzezinka K, Lämke J, Bäurle I (2014) Epigenetic responses to heat stress at different time
scales and the involvement of small RNAs. Plant Signal Behav 9(10):e970430
Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403(6765):41
Struhl K, Segal E (2013) Determinants of nucleosome positioning. Nat Struct Mol Biol 20(3):267
Suji KK, Joel AJ (2010) An epigenetic change in rice cultivars under water stress conditions.
Electronic J Plant Breeding 1:1142–1143
Tariq M, Saze H, Probst AV, Lichota J, Habu Y, Paszkowski J (2003) Erasure of CpG methylation
in Arabidopsis alters patterns of histone H3 methylation in heterochromatin. Proc Natl Acad Sci
100(15):8823–8827
Tchurikov NA (2005) Molecular mechanisms of epigenetics. Biochem Mosc 70(4):406–423
Tompa R, McCallum CM, Delrow J, Henikoff JG, van Steensel B, Henikoff S (2002) Genome-wide
profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3. Curr
Biol 12(1):65–68
Tougou M, Yamagishi N, Furutani N, Shizukawa Y, Takahata Y, Hidaka S (2007) Soybean dwarf
virus-resistant transgenic soybeans with the sense coat protein gene. Plant Cell Rep 26
(11):1967–1975
Tsaftaris AS, Polidoros AN, Koumproglou RACHEL, Tani ELENI, Kovacevic NIVEC, Abatzidou
ELENI (2005) Epigenetic mechanisms in plants and their implications in plant breeding. In the
wake of the double helix: from the green revolution to the gene revolution. Avenue Media,
Bologna, pp 157–172
Tyedmers J, Madariaga ML, Lindquist S (2008) Prion switching in response to environmental
stress. PLoS Biol 6(11):e294
Vanyushin BF (2005) Enzymatic DNA methylation is an epigenetic control for genetic functions of
the cell. Biochem Mosc 70(5):488–499
Wada Y, Miyamoto K, Kusano T, Sano H (2004) Association between up-regulation of stress-
responsive genes and hypomethylation of genomic DNA in tobacco plants. Mol Gen Genomics
271(6):658–666
Wang WS, Pan YJ, Zhao XQ, Dwivedi D, Zhu LH, Ali J, Fu BY, Li ZK (2010) Drought-induced
site-specific DNA methylation and its association with drought tolerance in rice (Oryza sativa
L.). J Exp Bot 62(6):1951–1960
Wolffe AP, Matzke MA (1999) Epigenetics: regulation through repression. Science 286
(5439):481–486
Yadav RK, Chattopadhyay D (2011) Enhanced viral intergenic region–specific short interfering
RNA accumulation and DNA methylation correlates with resistance against a Geminivirus. Mol
Plant-Microbe Interact 24(10):1189–1197
Yaish MW, Colasanti J, Rothstein SJ (2011) The role of epigenetic processes in controlling
flowering time in plants exposed to stress. J Exp Bot 62(11):3727–3735
Yoder JA, Walsh CP, Bestor TH (1997) Cytosine methylation and the ecology of intragenomic
parasites. Trends Genet 13(8):335–340
Yu Y, Yang X, Wang H, Shi F, Liu Y, Liu J, Li L, Wang D, Liu B (2013a) Cytosine methylation
alteration in natural populations of Leymus chinensis induced by multiple abiotic stresses. PLoS
One 8(2):e55772
Yu A, Lepère G, Jay F, Wang J, Bapaume L, Wang Y, Abraham AL, Penterman J, Fischer RL,
Vionnet O, Navarro L (2013b) Dynamics and biological relevance of DNA demethylation in
Arabidopsis antibacterial defense. Proc Natl Acad Sci 110(6):2389–2394
Zentner GE, Henikoff S (2013) Regulation of nucleosome dynamics by histone modifications. Nat
Struct Mol Biol 20(3):259
Zheng Q, Rowley MJ, Böhmdorfer G, Sandhu D, Gregory BD, Wierzbicki AT (2013) RNA
polymerase V targets transcriptional silencing components to promoters of protein-coding
genes. Plant J 73(2):179–189
Zhu B, Zheng Y, Angliker H, Schwarz S, Thiry S, Siegmann M, Jost JP (2000) 5-Methylcytosine
DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and
in a related avian sequence. Nucleic Acids Res 28(21):4157–4165
Zhu J, Kapoor A, Sridhar VV, Agius F, Zhu JK (2007) The DNA glycosylase/lyase ROS1 functions
in pruning DNA methylation patterns in Arabidopsis. Curr Biol 17(1):54–59
Concept of Effectors and Receptors
in Improving Plant Immunity 22
C. S. Karibasappa, Yogendra Singh, T. Aravind, and K. P. Singh
Abstract
Plants employ two distinct layers of immunity to encounter pathogen invasion.

The first layer PAMP-triggered immunity (PTI) involves the perception of evo-
lutionarily conserved pathogen structures, termed pathogen-associated molecular
patterns (PAMPs), at the plasma membrane through PRRs, to evade this PTI
pathogens evolved to secrete effector molecules. In response to pathogen
effectors, plants have acquired additional receptors that specifically recognize
the effectors, establishing a second layer of immunity known as the effector-
triggered immunity (ETI). Various strategies have been developed to effectively
integrate ETI into crop improvement programs in different ways. ‘Effectoromics’
is a large-scale screening approach that uses effector candidates to identify host
resistance (R) genes. Stacking multiple NLRs confers resistance for durable
resistance. Engineering new NLR-mediated resistance specificities can be carried
out by altering either NLRs’ domains or host proteins which are guarded by
NLRs. Synthetic TALE nucleases and CRISPR/Cas9 (clustered regularly
interspaced short palindrome repeats) mediated genome editing of host suscepti-
ble genes which are TALE targets were also used to engineer resistance against
plant SWEET sugar transporters. Resistant genotypes are developed, in which
TAL effectors are recognized by plant cells through trap promoters, which are
coupled to an executor-type resistance gene (E-gene). This recognition triggers
hypersensitive (HR) reaction, and this limits the further growth of the pathogen.
Keywords
PAMP-triggered immunity · Effectors · Effector-triggered immunity · CRISPR/
Cas9 · Hypersensitive reaction (HR)
C. S. Karibasappa (*) · Y. Singh · T. Aravind · K. P. Singh

Department of Plant Pathology, GB Pant University of Agriculture and Technology, Pantnagar,
Udam Singh Nagar, Uttarakhand, India

https://doi.org/10.1007/978-981-15-6275-4_22
476 C. S. Karibasappa et al.
22.1 Introduction
Plant pathogens, be it bacteria, fungi or viruses, have different lifestyles and infec-
tion strategies, but one similarity is that they try to colonize and live at the expense of
their host. Essentially, all of these pathogens either evade or suppress the immune
system or modify host physiological processes in the process of infection. To
counteract them, plants evolved to employ two distinct layers of immunity, viz.
PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones and
Dangl 2006). The first, evolutionarily ancient, layer involves the recognition of
evolutionarily conserved pathogen structures, termed pathogen-associated molecular
patterns (PAMPs), at the plasma membrane through conserved and ubiquitous
receptors generally defined as pattern recognition receptors (PRRs). Binding to
these receptors to PAMPs initiates an active defence response, the so-called
PAMP-triggered immunity (PTI), in both host and nonhost plants. In the next
round of host-pathogen conflict, several adapted pathogens escape from detection
or suppress PTI, by passing effector proteins inside the host cells to incite disease,
known as effector-triggered susceptibility (ETS). In response to these pathogen
effectors, plants have acquired additional receptors that precisely recognize the
effectors, establishing a second layer of immunity known as the effector-triggered
immunity (ETI).
22.2 From PTI to ETI: An Overview of Plant Defence Response
In fact, pathogens approaching to enter the cellular cytosol must initially overcome
the first layer of plant immune system called PAMP-triggered immunity (PTI), also
known as surface immunity, in which PAMPs are recognized by PRRs at the cell
membrane to activate PTI to prevent further entry and colonization of the host cells
(Ionis and de Witt 2009). But these basal defences are only partially effective at
restricting pathogens. Once pathogen evolves itself to detect and suppress PTI, then
it can transfer its effector proteins inside the host cells and cause disease. In the
process of evolution, plants also develop mechanisms to overcome the effect of
effector molecules of pathogens; this forms the second layer of plant immunity
called as effector-triggered immunity (ETI). ETI is often associated with a hyper-
sensitive response (HR), at the infection sites; in many cases, it is followed by
systemic acquired resistance (Fig. 22.1).
22.3 The Following Are Some of the Terms Which We Need

to Understand Before Going Through This Chapter
PAMPs (Pattern-Associated Molecular Patterns) are the highly indispensable

molecules for the survivability of the pathogens; impair the viability of pathogens, if
they are lost, since these are evolutionarily stable; form a core component of the
microorganism; and are conserved across larger groups of pathogens, which are not
22 Concept of Effectors and Receptors in Improving Plant Immunity 477
Fig. 22.1 Overview of PTI and ETI
found in the host. Some of the examples include bacterial flagellins,

lipopolysaccharides or elongation factor Tu, fungal chitin or oomycete Pep-13
(Boller and Felix 2009).
Pattern Recognition Receptors (PRRs) like FLS2, ERF, CEBiP etc. are the
plasma membrane-localized receptors, which recognize the presence of PAMPs in
the extracellular environment located in the plasma membrane.
PTI It is the situation in which a cascade of responses are commenced leading to

development of immunity in host plants upon detection of PAMPs (conserved
pathogens molecules important for their reproduction and survival) that elicit a
physiological changes in the host cell activated by the Pattern Recognition Receptors
(PRRs).
Effectors Effectors are any regulatory pathogen-secreted molecule that can modify
the host cell structure and function, thereby facilitating infection or triggering
defence responses (Kamoun 2006). Mainly, effectors perform three functions: First
is the structural role, for example, Avrblb2 of fungi, which is secreted as
extrahaustorial molecules. The second function is to cause nutrient leakage, for
example, P. syringae HoPM effector protein. The third role is to act in pathogenicity
process, for example, HopA1 dephosphorylates MAP kinase which results in the
inhibition of PTI. These include PWL2, pep1, Avr4, AVR2, P6 protein of Cauli-
flower mosaic virus (CaMV), and cyst nematode (G. rostochiensis) expansins
(Leisner and Schoelz 2018).
ETS (Effector-Triggered Susceptibility) is the situation in which susceptibility/

disease is induced in the plant due to the action of effectors on the host system. The
outcome of the deployment of effector molecules favours pathogen virulence.
Effector-Triggered Immunity (ETI) is the situation in which plant defence

response or the immunity is elicited by effector recognition in the host cell. The
effector molecules are recognized by R protein which are governed by R genes:
NB-LRR (nucleotide-binding leucine-rich repeats) and Ser/Thr kinases. ETI occurs
when there is highly specific, direct or indirect interaction of pathogen effectors and
the products of plant R genes. Localized ETI can also induce systemic acquired
resistance (SAR).
When bacterial pathogen comes in contact with plant surface, these cells detect
bacterial molecules (PAMPs) by receptors called PRRs; then, a signalling pathway
named PTI/basal defence is activated. Later, molecular events occur and stop
bacterial growth. If this pathogen has the ability to overcome this PTI effect, then
it can inject effectors directly into the plant cells through type III secretion system,
which often bacterial pathogens possess. Once these effectors enter inside the host
cells, they manipulate the host physiological conditions, which may favour for
disease development. If host plants are resistant, then plant cells have got proteins
that can recognize effectors; they are called resistance proteins and are specific to one
particular effector, and then signalling pathway named ETI is activated. If ETI is
activated, then hypersensitive response (HR) occurs and stops bacterial growth;
ultimately, plant becomes resistant to the bacterium (Fig. 22.2).
22.4 Phases of Plant-Pathogen Interaction
There exists always a coevolutionary arms race between pathogens and plants,
during which pathogens respond by mutating effectors or by developing new
effectors that can avoid or subdue ETI, whereas plants also develop novel R proteins,
facilitating recognition of novel effectors; therefore, new effectors and receptors
keep on evolving in plant-pathogen interaction. This is explained through the
following zigzag model of different phases of the plant-pathogen interaction
(Fig. 22.3).
22.5 Apoplastic Effectors Target Host Defences
The apoplastic space lies between the plant cell wall and the plasma membrane,
constituting an important space where plant and pathogen interact. To overcome
such harsh conditions in the plant apoplast, pathogens have evolved various immune
responses. During infection, plant pathogens secrete a large number of effector
proteins into the apoplastic space, some of which are recognized by the plant
surveillance system, and, thus, plant innate immunity will be activated. Some of
the effectors, which evade plant perception, act in modifying plant apoplast
Fig. 22.2 Main steps involved in plant immunity

Fig. 22.3 Zigzag model depicting different phases of plant-pathogen interaction
Fig. 22.4 Microbial effectors modify plant immunity in the plant apoplast. Modes of action of
several apoplastic effectors with their corresponding plant proteins during interaction are depicted
immunity and favour effective pathogen infection. The concerted actions of

apoplastic effectors often define the consequences of plant-pathogen interactions
(Fig. 22.4). Some of the apoplastic effectors include protease inhibitors, peroxidase
inhibitor of U. maydis and chitin-binding effectors like LysM and Ecp6 effectors of
C. fulvum.
Protease Inhibitors Papain-like cysteine proteases (PLCPs) are essential

constituents of the plant immune response in the apoplast (Doehlemann and
Hemetsberger 2013), and various pathogens secrete PLCP inhibitors. Tomato
plant-derived cysteine protease RCR3 is inhibited by the apoplastic AVR effector
Avr2 of C. fulvum. Indeed, the activation of hypersensitive response occurs when
Avr2-RCR3 complex is recognized by the CF-2 resistance protein in tomato plants
(Rooney et al. 2005).
Chitin-Binding Effectors Fungi secrete apoplastic effectors to block chitin-induced

immunity. Various pathogens secrete apoplastic effectors that either avoid the
release of chitin oligosaccharides from fungal cell walls or sequester these released
oligosaccharides to avoid recognition by the plant surveillance system. For example,
apoplastic effectors LysM and Ecp6 secreted by C. fulvum, which sequester chitin
oligosaccharides which are released from the fungal cell wall (de Jonge et al. 2010),
and one more apoplastic effector of C. fulvum, i.e. Avr4, can bind to plant chitinases
with its different chitin-binding domain and functions to protect the fungal cell wall
from degradation by plant chitinases (van den Burg et al. 2004)
Peroxidase Inhibitor The apoplastic effector Pep1 of U. maydis protects fungal

hyphae from reactive oxygen species (ROS), which constitute the major component
of the plant immune response by surrounding the hyphae in the apoplast and
concentrating as rings around hyphae at cell-to-cell passage sites (Hemetsberger
et al. 2012).
22.6 Damage-Associated Molecular Patterns (DAMPs)
In addition to biotic attack, plants also need to cope up with a range of abiotic
assaults too such as mechanical or cellular damage, as well as environmental stresses
like drought and salinity. Some endogenous molecules activate the innate plant
immune system when they are released into the extracellular space (including
plant apoplast) from their normal location due to damage; these molecules are
referred to as DAMPs or damage-associated molecular patterns (Bianchi 2007).
DAMPs are the endogenous biomolecules that are passively released by the host
upon external damage or infection-induced necrosis. While MAMPs are derived
from microbes and activate the innate immune system, DAMPs are host cell–
derived, and both initiate and perpetuate innate immune responses. It is understood
that these defences help to protect the damaged tissue by preventing microbial
ingress, which is otherwise vulnerable to infection due to the disruption of physical
barriers.
DAMPs in plants are mainly cytosolic proteins, nucleotides, peptides and amino
acids, which are released from damaged cells or secreted by intact cells, which are
undergoing pathogen invasion. In addition, DAMPs also include the oligomeric
fragments of plant cell wall polysaccharides released when tissues are disrupted by
physical injuries or attacks of pathogens and herbivores. As the case of PAMPs,
DAMPs also initiate PRR-mediated immune responses in local sites surrounding a

wound and pathogen invasion and regulate systemic immune signalling (Fig. 22.5).
Examples: Systemin, hydroxyproline-rich systemin, oligogalacturonides (OGs),
extracellular ATP (eATP) and plant elicitor peptides (Peps), i.e. Arabidopsis
AtPep1, a peptide upon recognition by its receptor PEPR1; the plants get alerted
and activate immune responses.
22.7 Plant Defence Responses Associated with PTI
PTI is associated with various plant defence responses, like calcium influx, callose
deposition, oxidative burst and activation of a mitogen-activated protein kinase
(MAPK) cascade, to induce defence gene expression (Nicaise et al. 2009). Cellular
and physiological responses are elicited by patterns in plants. Plant cell surface-
resident pattern recognition receptors (PRRs) perceive microbe-associated molecular
patterns (MAMPs) or DAMPs and recruit the coreceptors, leading to a series of
intertwined cellular and physiological responses. PRR complex formation is
followed by a rapid transphosphorylation in the complex as well as phosphorylation
of receptor-like cytoplasmic kinases (RLCKs). The activation of PRR complexes
also leads to the activation of mitogen-activated protein kinase (MAPK) cascades
and calcium-dependent protein kinases (CDPKs), which regulate gene transcrip-
tional changes and other cellular responses. The hallmarks of PTI responses include
ion efflux, calcium influx, actin filament remodelling, callose deposition,
plasmodesmata (PD) and stomatal closure and production of reactive oxygen species
(ROS), phosphatidic acid (PA), nitride oxide (NO), phytoalexins and
phytohormones. Collectively, these responses contribute to plant resistance against
a variety of pathogens (Fig. 22.6).
22.8 Plant Defence Responses Associated with ETI
ETI is also associated with various plant defence responses, like localized
programmed cell death, autophagy and transcriptional reprogramming of defence-
responsive genes. The striking characteristic of ETI is the HR, which exhibits a rapid
induction of programmed localized cell death at the infection site. The primary
purpose of this cell death is against biotrophic pathogens, which derive nutrients
from living cells. Localized PCD is regulated by salicylic acid concentration gradient
and NPR proteins. In Arabidopsis, PCD is regulated by SA, NPR1 and SA receptors,
viz. NPR3 and NPR4; in this case, low level of SA suppresses cell death, while over-
accumulation of SA leads to cell death. The finding that PCD is regulated by
autophagy is one of the important discoveries related to ETI. Liu et al. (2005)
showed that in Nicotiana benthamiana downregulation of ATG6 and ATG7
(autophagy genes) leads to an extended cell death in TMV-infected plants. Catalase
2 (CAT2) and no catalase activity 1 (NCA1), which are involved in catalase
activities in plants, should, in theory, prevent PCD. However, both CAT2 and
22
Concept of Effectors and Receptors in Improving Plant Immunity
Fig. 22.5 DAMP-triggered immunity in plants is depicted. Pathogen invasion as well as environmental stresses disrupt plant cell wall and plasma membrane,
leading to the release of DAMPs, including fragments of cell walls and apoplastic proteins, and cytoplasmic components. Perception of these DAMPs as well as
483
PAMPs by PRRs in cells surrounding of the damaged cells also promotes the production and release of new DAMPs. These DAMPs join together with PAMPs
to modulate immune responses locally as well as systemically
484
Fig. 22.6 Defence responses developed in plant due to PTI. Abbreviations: DGKs diacylglycerol kinase, JA jasmonic acid, ET ethylene, PLC, PLD
phospholipase D, phospholipase C; TF transcription factor, SA salicylic acid
C. S. Karibasappa et al.
NCA1 contribute to autophagy-dependent PCD, which shows that PCD is regulated

by autophagy (Fig. 22.7).
ETI is a strong plant immune response associated with extensive transcriptional
reprogramming that involves numerous transcriptional regulators (Caplan et al.
2008; Bhattacharjee et al. 2013). WRKY transcription factors reprogramme tran-
scription of defence-responsive genes. During ETI, active recruitment of transcrip-
tional regulators into R protein-mediated signalling pathways is as an important
signalling event just after the recognition of Avr effectors (Rivas 2012; Rivas and
Deslandes 2013) Rapid transcriptional changes in ETI can be extensively influenced
by chromatin modifications using different mechanisms, viz. methylation of cytosine
residues located in DNA, ATP-dependent chromatin remodelling and post-
translational histone modifications.
22.9 Modes of Pathogen Effector Recognition by Receptors or

Modes of Pathogen Effector and Receptor Interaction
There are two ways by which pathogen effectors are recognized by plant receptors.
1. Direct perception of pathogen effectors by R proteins. In this mechanism of

interaction, effectors directly come in contact with the receptors (Fig. 22.8a).
Direct interaction is seen between P. infestans effector Avrblb1 and Rpi-blb1 in
potato plants.
2. Indirect perception of pathogen effectors by R proteins. An alternative model
“guard hypothesis” postulates that R proteins recognize effectors indirectly via
their conformational changes in the guarded host targets (Van der Biezen and
Jones 1998). It was proposed that effectors target host proteins instead of R
proteins directly and that conformational changes of those host proteins are the
trigger that leads to R protein activation. Thus, these types of R proteins guard the
target of effectors and induce defence responses when those targets are disturbed
due to the interaction with effectors. This kind of interaction is evident between
Pseudomonas syringae effector protein (AvrPphB) and RPS5 (R protein) of
Arabidopsis thaliana (Fig. 22.8b). In this case, protein kinase PBS1 acts as a
guardee. Similarly, RIN4, which acts as a guardee protein, has the ability to
interact with several effector proteins, viz. AvrB, AvrRpm1, Rpm1, AvrRpt2 and
Rps2 (Fig. 22.8c).
Effectors can influence the various crucial cellular processes and manipulate as
well as direct them towards the growth and colonization of the pathogens and
infection in the host cells; the following are the few examples of key cellular
processes that are being affected by the effectors of pathogens. Effectors can
facilitate an effective penetration and early invasion of host tissues. For example,
Ustilago maydis secretes an effector called Pep1 from its hyphae that is required
for an effective invasion of the host tissue; Pep1 inhibits plant peroxidases to
suppress the early maize defence responses (Doehlemann et al. 2009).
486
Fig. 22.7 Plant defence responses associated with ETI

C. S. Karibasappa et al.
Fig. 22.8 Modes of pathogen effectors and host receptors interaction: (a) compatible reaction
resulting in resistance development and incompatible interaction results in susceptibility; (b) direct
interaction between effectors and receptors; (c) indirect interaction between effectors and receptor
via guard protein
3. Bacterial effectors for stomatal manipulation. The tobacco wildfire pathogen

P. syringae pv. tabaci secretes the effector HopX1, a cysteine protease (CP) that
can degrade multiple JAZ transcriptional repressors, leading to the activation of
JA-regulated genes (JA signalling) and inducing stomatal reopening on the leaf
exterior (Melotto et al. 2006).
4. Effectors for enhancing tissue colonization. Cytosolic effectors of M. oryzae,
such as PWL2 and BAS1, can translocate from cell to cell, possibly through
plasmodesmata, to enhance subsequent colonization (Khang et al. 2010).
5. Effectors interfere with plant hormones metabolism. P. sojae and Verticillium
dahliae secrete the virulence-promoting effectors Pslsc1 and Vdlscl, respectively;
these are isochorismatases, enzymes that can hydrolyse the SA precursor
isochorismate to disrupt SA metabolism (Liu et al. 2014).
6. Manipulating host gene expression is carried out by TALEs (transcriptional
activator-like effectors), which act as plant transcription factors present in
multiple Xanthomonas and Ralstonia bacterial pathogens, for example,
Xanthomonas oryzae pv. oryzae TALE PthXo1 binds to the promoter region of
a sucrose transporter gene called OsSWEET11 to induce its expression and to

promote bacterial pathogenicity (Yang et al. 2006).
7. Effectors targeting RNA silencing machinery as plant viruses have evolved
suppressors of RNA interference machinery, which helps them to multiply well
inside the host system, for example, P1/HC-Pro protein (helper component
proteinase) from Tobacco etch potyvirus, 2b from Cucumber mosaic virus and
P19 from Tomato bushy stunt virus (Csorba et al. 2015).
22.10 Effect on Various Cellular Processes of the Host by

the Effectors
Effectors can influence various crucial cellular processes and manipulate as well as
direct them towards the growth and colonization of the pathogens and infection in
the host cells. Following are the few examples of key cellular processes that are
being affected by the effectors of pathogens:
1. Effectors can facilitate effective penetration and early invasion of host

tissues. For example, Ustilago maydis secretes effector called Pep1 from its
hyphae that is required for effective invasion of host tissue; Pep1 inhibits plant
peroxidases to suppress early maize defence responses (Doehlemann et al. 2009).
2. Bacterial effectors for stomatal manipulation. The Tobacco wildfire pathogen
P. syringae pv. tabaci secretes the effector HopX1, a cysteine protease (CP) that
can degrade multiple JAZ transcriptional repressors, leading to activation of
JA-regulated genes (JA signalling) and to induce stomatal reopening on the leaf
exterior (Melotto et al. 2006).
3. Effectors for enhancing tissue colonization. Cytosolic effectors of M. oryzae
such as PWL2 and BAS1 can translocate from cell to cell possibly through
plasmodesmata to enhance subsequent colonization (Khang et al. 2010).
4. Effectors interfere with plant hormones metabolism. P. sojae and Verticillium
dahliae secrete the virulence-promoting effectors Pslsc1 and Vdlscl, respectively;
these are isochorismatases, enzymes that can hydrolyse the SA precursor
isochorismate to disrupt SA metabolism (Liu et al. 2014).
5. Manipulating host gene expression is carried out by TALEs (transcriptional
activator-like effectors), which act as plant transcription factors present in
multiple Xanthomonas and Ralstonia bacterial pathogens; for example,
Xanthomonas oryzae pv. oryzae TALE PthXo1 binds to the promoter region of
a sucrose transporter gene called OsSWEET11 to induce its expression and to
promote bacterial pathogenicity (Yang et al. 2006).
6. Effectors targeting RNA silencing machinery, as plant viruses have evolved
suppressors of RNA interference machinery which helps them to multiply well
inside the host system. For example, P1/HC-Pro protein (helper component
proteinase) from Tobacco etch potyvirus, 2b from Cucumber mosaic virus and
P19 from Tomato bushy stunt virus (Csorba et al. 2015)
22.11 Different Ways to Utilize Effectors as Well as Receptors

for Enhancing Plant Immunity
1. Effectors for screening R genes: As the great amount of research work has been
taking place in the field of molecular plant pathology over the last 30 years, it has
enabled scientists to effectively integrate ETI into crop improvement programs. It
will first be essential to identify multiple NLRs, which can recognize conserved
effectors of pathogens in order to aptly harness ETI for the development of
disease resistance. If effector repertoires attained through genomics studies are
opted, they are helpful in breeding programmes for disease resistance. Effectors
can be employed to identify R genes/NLRs in plant systems, so such extensive
screening approach that uses effectors to identify specific unknown R genes is
known as ‘effectoromics’ (Vleeshouwers et al. 2008). This method mainly
depends on the transient expression of candidate effector gene in plant leaves;
subsequently, the appearance of cell death responses due to hypersensitive
reaction (HR) indicates the recognition of the effector by a suitable matching
plant immune receptor in the host. This approach has the ability to quicken the
identification of a large number of immune receptors when the matching specific
effectors are present in the plant host system, since it has the ability to replace the
slow process of stable transformants’ development (Vleeshouwers et al. 2008).
This approach was initiated for P. infestans and potato; in the last few years, a
catalogue of Avr (Effector) and R (receptors) genes has become available.
Specific HR responses to AVRblb1 were quickly detected in S. stoloniferum
that is directly crossable with cultivated potato S. tuberosum and was found to
carry Rpi-sto1, a functional homologue of Rpi-blb1; presently, the Rpi-sto1 gene
is efficiently utilized for the classic introgression into cultivated potato breeding
material (Hein et al. 2009).
2. Stacking multiple NLRs to confer resistance for durable resistance: Multiple
NLRs recognizing the core effectors can be stacked into one genotype, since the
pathogen would rarely be able to mutate or lose multiple core effectors simulta-
neously. For example, three Rpi genes have been stacked transgenically into
potato simultaneously, and it resulted into the development of resistance against
P. infestans (Zhu et al. 2012).
3. Engineering new NLR-mediated resistance specificities: To engineer novel
resistance specificities, much effort has targeted at the receptor level and defined
mutations in the NLRs’ nucleotide-binding site. NLR receptors have developed
for enhanced effector recognition: Tomato NLR I2, which weakly responds to
Avr3a effector of P. infestans. A point mutation in coiled-coil domain of I2 was
carried out, which resulted in enhanced perception of Avr3a (Giannakopoulou
et al. 2015). A great promise for synthetic NLR engineering for designing of NLR
receptors to recognize diverse pathogen effectors has seen when mutation in the
Rx CNL expanded the recognition of Potato virus X (PVX) strains. In
Arabidopsis, RPS5 NLR guards the host kinase PBS1. AvrPphB effector of
P. syringae is a protease which can cleave PBS1 at a defined region. The resulting
conformational change due to cleavage will be detected by RPS5 (Fig. 22.8c).
Since effector proteases are common in both bacterial and viral pathogens,
recently, Kim et al. (2016) engineered host proteins guarded by NLRs to generate
new resistance specificities, by substituting the cleavage site of AvrPphB within
PBS1 with those from other bacterial or viral proteases, which enabled RPS5
recognition of these proteases upon infection. This approach could also be
employed to engineer resistance against a wide variety of other pathogens using
well-characterized NLRs.
4. Combining NLR-mediated resistance (ETI) with pattern recognition
receptors (PRRs)-mediated resistance (PTI): The transfer of Arabidopsis
EFR, which recognizes elongation factor Tu (EF-Tu) to Nicotiana benthamiana
and tomato confers responsiveness to EF-Tu, resulted in resistance against
bacterial pathogens from different genera (Lacombe et al. 2010). This research
suggests that PRRs could be used to engineer broad-spectrum disease resistance
to diverse pathogens, potentially enabling more durable resistance in the field.
Additional layers of disease resistance can also be combined with stacks of PRRs
and NLRs.
5. Genome editing of susceptibility loci: TALEs, also called as transcription
activator-like effectors, are mostly found in Xanthomonas spp. These are deliv-
ered into host cells during infection which later bind to loci of susceptible genes
in the host cells and induce expression of susceptibility genes, by acting as
transcription factors, thereby facilitating bacterial pathogen growth and virulence
(Streubel et al. 2013), to provide resistance against susceptibility genes, viz. plant
SWEET sugar transporters, which are TALE targets. Genome editing mediated
by CRISPR/Cas9 approach and engineering of synthetic TALE nucleases have
been carried out (Fig. 22.9). Jiang et al. (2013) developed resistance to
Xanthomonas citri pv. citri through CRISPR/Cas9-targeted genome editing of
citrus susceptibility gene CsLOB1 and its promoter.
6. Promoter traps and executor genes: In resistant host genotypes, TAL effectors
are recognized by plant cells through trap promoters, which are coupled to an
executor-type resistance gene (E-gene). Trap promoters possess recognition sites
for specific TAL effectors, so that, upon infection, the delivery of that TAL
effector by pathogen induces the expression of the executor gene. Expression
of the executor genes triggers hypersensitive (HR) reaction, and this limits the
further growth of the pathogen. The first such E-type resistance gene shown to be
triggered by a TAL effector was the Xa27 gene of rice; it has a binding site for the
X. oryzae pv. oryzae TAL effector AvrXa27 in its promoter region (Fig. 22.10a).
This principle can be further utilized by combining different EBEs for multiple
TAL effectors from individual and different pathogen strains and species into one
promoter to achieve broad-spectrum resistance (Fig. 22.10b). Hummel et al.
(2012) demonstrated that combining six EBEs that correspond to three TAL
effectors from Xoo and three from X. oryzae pv. oryzicola into the Xa27 promoter
resulted in a gene inducible by all six of the TAL effectors and plant lines resistant
to both the bacterial pathogens.
7. TAL effector-based antiviral approaches: TAL effectors are typically known
to bind to double-stranded DNA. There are only very few double-stranded DNA
Fig. 22.9 Engineering resistance to Xanthomonas by utilizing TAL effector knowledge. (a)
Customized TAL effector pairs fused to FokI nuclease domains are used to mutate a TAL
effector-binding element (EBE) upstream of a key host susceptibility gene (S gene). (b, c, d)
Mechanisms of plant resistance against transcription activator-like (TAL) effectors
plant viruses; Geminiviruses possess single-stranded DNA but go through a

double-stranded DNA rolling circle replicative stage. Examples include Maize
streak virus, African cassava mosaic virus and Tomato yellow leaf curl virus
Fig. 22.10 EBEs for TAL effectors of pathogen strains are construed and inserted into promoters
driving executor gene (E gene)
(TYLCV). Rolling circle replication could be obstructed in transgenic plants

expressing one or more TAL DNA-binding domains designed against the origin
of replication sequence (ori). In addition, pathogen genomes can also be
destroyed through TAL nucleases, which are designed to cleave sequences within
the viral genomes. Transgenic plants engineered with TAL nucleases targeting
the specific DNA of other pathogens, like fungi and nematodes, may also be
feasible, provided such molecules (TAL nucleases) can be delivered to the
pathogen, for instance, through transfer across haustorial membranes to fungi
or parasitic plants or by feeding in nematodes.
8. Host-induced gene silencing (HIGS): It is based on exploiting an RNA interfer-
ence mechanism to target a selected pathogen gene, i.e. effector gene, via the host
plant to nullify the effector production capacity. Plants can be transformed with
hairpin RNA constructs that target selected pathogen effector genes to suppress
their expression and production of effectors. For example, HIGS in barley
expressing dsRNA targeting the effector gene Avra10 from the fungus Blumeria
graminis led to reduced disease incidence (Nowara et al. 2010) (Table 22.1).
Table 22.1 Receptors present in different plant species and their corresponding effector (Avr)
proteins present in different pathogen species
Pathogen
NLR Type Host species Avr species References
Pi2 NB-LRR Rice Unknown Magnaporthe Zhou et al.
oryzae (2006)
Pi37 NB-LRR Rice AvrPi37 Magnaporthe Lin et al.
oryzae (2007)
RPI- CNL Solanum AvrBLB1 Phytophthora van der Vossen
BLB1 bulbocastanum infestans et al. (2003)
R1 CNL Solanum Avr1 Phytophthora Vleeshouwers
demissum infestans et al. (2011)
R8 CNL Solanum Avr8 Phytophthora Vossen et al.
demissum infestans (2016)
Rpi- NB-LRR Solanum AvrBLB1 Phytophthora Wang et al.
sto1 stoloniferum infestans (2008)
N TNL Tobacco Helicase Tobacco Padgett and
direct domain mosaic virus Beachy (1993)
RPG1b CNL Soybean AvrB Pseudomonas Kessens et al.
glycinea (2014)
Prf CNL Tomato AvrPto/ Pseudomonas Rathjen et al.
AvrPtoB syringae (1999)
Lr10/ CNL/CC- Wheat AvrLr10 Puccinia Loutre et al.
RGA2 NB- triticina (2009)
NBLRR
sr22 CNL Wheat PGTAUSPE- Puccinia Upadhyaya
10-1 graminis et al. (2014)
RPS1k CNL Soybean Avr1k/ Phytophthora Dou et al.
Avr1b sojae (2008)
Pm3b CNL Wheat AvrPm3b Blumeria Yahiaoui et al.
graminis (2004)
Sw5b CNL Tomato NSm Tomato Yahiaoui et al.
spotted wilt (2004)
Virus
Bs4 TNL Tomato AvrBs4 Xanthomonas Schornack et al.
campestris (2004)
I2 CNL Tomato Avr2-SIX5 Fusarium Ma et al. (2015)
pair oxysporum
RPG1r CNL Soybean AvrRPM1 Pseudomonas Ashfield et al.
glycinea (2003)
R3A CNL Solanum Avr3a Phytophthora Engelhardt
demissum infestans et al. (2012)
Pi9 NB-LRR Rice AvrPi9 Magnaporthe Wu et al.
oryzae (2015)
22.12 Limitations
Even though we identify different NLRs in wild germplasm, it is difficult to transfer

into commercial cultivars through conventional plant breeding approaches; to
achieve this, we need to rely on transgenic approaches, but transgenic-based crops
are not widely accepted in most of the countries in the world. Sometimes combining
multiple NLRs together may negatively regulate each other, for example, Rye Pm8
and wheat Pm3 resistance genes form heteromeric receptor complex.
22.13 Conclusion
Since the discovery of H. H. Flor’s gene-for-gene concept, there has been a signifi-
cant progress in understanding the genetic and molecular basis of ETI. Accelerating
climate change is predicted to generate new strains of plant pathogens carrying novel
effectors, demanding rapid responses by plant breeders. Rational design of plant
immune systems will be one tool among many that enables agricultural systems to
keep pace with pathogens. Thus, it will be necessary to utilize concept of effectors
and receptors to improve plant immunity in combination with different types of
resistance acting at different stages in pathogen infection.
22.14 Future Prospects
For better understanding of how individual NLR domains interact with one another,
we need to understand how NLRs activate downstream (signalling) events, How
disease resistance and cell death are triggered remains, remarkably, a black box. To
gain deeper insights, the modes of action of individual effectors and their
interactions with effector targets and matching immune receptors should be studied.
References
Ashfield T, Bocian A, Held D, Henk AD, Marek LF, Danesh D, Penuela S, Meksem K, Lightfoot
DA, Young ND, Shoemaker RC, Innes RW (2003) Genetic and physical localization of the
soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly
linked families of NBS-LRR genes. Mol Plant-Microbe Interact MPMI 16:817–826
Bhattacharjee S, Garner CM, Gassmann W (2013) New clues in the nucleus: transcriptional
reprogramming in effector-triggered immunity. Front Plant Sci 4:364
Bianchi ME (2007) DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc
Biol 81:1–5
Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular
patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60:379–406
Caplan J, Padmanabhan M, Dinesh-Kumar SP (2008) Plant NB-LRR immune receptors: from
recognition to transcriptional reprogramming. Cell Host Microbe 3:126–135
Csorba T, Kontra L, Burgyan J (2015) Viral silencing suppressors: tools forged to fine-tune host-
pathogen coexistence. Virology 479–480:85–103
de Jonge R et al (2010) Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in
plants. Science 329:953–955
Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant-pathogen
interactions. Nat Rev Genet 11:539–548
Doehlemann G, Hemetsberger C (2013) Apoplastic immunity and its suppression by filamentous
plant pathogens. New Phytol 198:1001–1016
Doehlemann G, van der Linde K, Assmann D, Schwammbach D, Hof A et al (2009) Pep1, a
secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells.
PLoS Pathog 5:e1000290
Dou D, Kale SD, Wang X, Jiang RHY, Bruce NA, Arredondo FD, Zhang X, Tyler BM (2008)
RXLR-mediated entry of Phytophthora sojae effector avr1b into soybean cells does not require
pathogen-encoded machinery. Plant Cell Online. 20:1930–1947
Engelhardt S, Boevink PC, Armstrong MR, Ramos MB, Hein I, Birch PRJ (2012) Relocalization of
late blight resistance protein R3a to endosomal compartments is associated with effector
recognition and required for the immune response. Plant Cell Online 24:5142–5158
Giannakopoulou A, Steele JFC, Segretin ME, Bozkurt TO, Ji Z, Robatzek S, Banfield MJ, Pais M,
Kamoun S (2015) Tomato I2 immune receptor can be engineered to confer partial resistance to
the oomycete Phytophthora infestans in addition to the fungus Fusarium oxysporum. Mol Plant-
Microbe Interact 28:1316–1329
Hein I, Birch PRJ, Danan S et al (2009) Progress in mapping and cloning qualitative and quantita-
tive resistance against Phytophthora infestans in potato and its wild relatives. Potato Res
52:215–227
Hemetsberger C, Herrberger C, Zechmann B, Hillmer M, Doehlemann G (2012) The Ustilago
maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity. PLoS
Pathog 8:e1002684
Hou S, Liu Z, Shen H, Wu D (2019) Damage-associated molecular pattern-triggered immunity in
plants. Front Plant Sci 10:646
Hummel A, Doyle E, Bogdanove A (2012) Addition of transcription activator-like effector binding
sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against
additional strains and against bacterial leaf streak. New Phytol 195:883–893
Ionis S, de Witt PJGM (2009) Fungal effector proteins. Annu Rev Phytopathol 47:233–263
Jiang W et al (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat
Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329
Kamoun S (2006) A catalogue of the effector secretome of plant pathogenic oomycetes. Annu Rev
Kessens R, Ashfield T, Kim SH, Innes RW (2014) Determining the GmRIN4 requirements of the
soybean disease resistance proteins rpg1b and rpg1r using a Nicotiana glutinosa based
agroinfiltration system. PLoS One 9
Khang CH, Berruyer R, Giraldo MC, Kankanala P, Park SY et al (2010) Translocation of
Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement.
Plant Cell 22:1388–1403
Kim SH, Qi D, Ashfield T, Helm M, Innes RW (2016) Using decoys to expand the recognition
specificity of a plant disease resistance protein. Science 351:684–687
Lacombe S, Rougon-Cardoso A, Sherwood E, Peeters N, Dahlbeck D, van Esse HP, Smoker M,
Rallapalli G, Thomma BP, Staskawicz B, Jones JD, Zipfel C (2010) Nat Biotechnol 28:365–369
Leisner SM, Schoelz JE (2018) Joining the Crowd: Integrating Plant Virus Proteins into the Larger
World of Pathogen Effectors. Annu Rev Phytopathol 56:5.1–5.22
Lin F, Chen S, Que Z, Wang L, Liu X, Pan Q (2007) The blast resistance gene pi37 encodes a
nucleotide binding site–leucine-rich repeat protein and is a member of a resistance gene cluster
on rice chromosome 1. Genetics 177:1871–1880
Liu Y, Schiff M, Czymmek K, Talloczy Z, Levine B, Dinesh Kumar SP (2005) Autophagy
regulates programmed cell death during the plant innate immune response. Cell 121:567–577
Liu T, Song T, Zhang X, Yuan H, Su L et al (2014) Unconventionally secreted effectors of two

filamentous pathogens target plant salicylate biosynthesis. Nat Commun 5:4686
Loutre C, Wicker T, Travella S, Galli P, Scofield S, Fahima T, Feuillet C, Keller B (2009) Two
different CC-NBS-LRR genes are required for Lr10-mediated leaf rust resistance in tetraploid
and hexaploid wheat. Plant J 60:1043–1054
Ma L, Houterman PM, Gawehns F, Cao L, Sillo F, Richter H, Clavijo-Ortiz MJ, Schmidt SM,
Boeren S, Vervoort J, Cornelissen BJC, Rep M, Takken FLW (2015) The AVR2-SIX5 gene
pair is required to activate I-2-mediated immunity in tomato. New Phytol 208:507–518
Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate
immunity against bacterial invasion. Cell 126:969–980
Nicaise V, Roux M, Zipfel C (2009) Recent advances in PAMP triggered immunity against
bacteria: pattern recognition receptors watch over and raise the alarm. Plant Physiol
150:1638–1647
Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Hensel G, Kumlehn J, Schweizer
P (2010) HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen
Blumeria graminis. Plant Cell 22(9):3130–3141
Padgett HS, Beachy RN (1993) Analysis of a Tobacco mosaic virus strain capable of overcoming N
gene-mediated resistance. Plant Cell Online. 5:577–586
Rathjen JP, Chang JH, Staskawicz BJ, Michelmore RW (1999) Constitutively active Pto induces a
Prf-dependent hypersensitive response in the absence of avrPto. EMBO J 18:3232–3240
Rivas S (2012) Nuclear dynamics during plant innate immunity. Plant Physiol 158:87–94
Rivas S, Deslandes L (2013) Nuclear components and dynamics during plant innate immunity.
Front Plant Sci 4:481
Rooney HC et al (2005) Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2-
dependent disease resistance. Science 308:1783–1786
Schornack S, Ballvora A, Gurlebeck D, Peart J, Ganal M, Baker B, Bonas U, Lahaye T (2004) The
tomato resistance protein Bs4 is a predicted non-nuclear TIR-NBLRR protein that mediates
defense responses to severely truncated derivatives of AvrBs4 and overexpressed AvrBs3. Plant
J 37:46–60
Streubel J, Pesce C, Hutin M, Koebnik R, Boch J, Szurek B (2013) Five phylogenetically close rice
SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae.
New Phytol 200:808–819
Upadhyaya NM, Mago R, Staskawicz BJ, Ayliffe MA, Ellis JG, Dodds PN (2014) A bacterial type
III secretion assay for delivery of fungal effector proteins into wheat. Mol Plant-Microbe
Interact 27:255–264
van den Burg HA et al (2004) Binding of the AVR4 elicitor of Cladosporium fulvum to chitotriose
units is facilitated by positive allosteric protein-protein interactions: the chitin-binding site of
AVR4 represents a novel binding site on the folding scaffold shared between the invertebrate
and the plant chitin-binding domain. J Biol Chem 279:16786–16796
Van der Biezen EA, Jones JDG (1998) Plant disease resistance proteins and the gene-for-gene
concept. Trends Plant Sci 23:454–456
Van der Vossen E, Sikkema A, te B, Hekkert L, Gros J, Stevens P, Muskens M, Wouters D,
Pereira A, Stiekema W, Allefs S (2003) An ancient R gene from the wild potato species Solanum
bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated
potato and tomato. Plant J. Cell Mol. Biol 36:867–882
Vleeshouwers VGAA, Rietman H, Krenek P, Champouret N, Young C, Oh SK, Wang M,
Bouwmeester K, Vosman B, Visser R (2008) Effector genomics accelerates discovery and
functional profiling of potato disease resistance and Phytophthora Infestans Avirulence genes.
PLoS One 3:e2875
Vleeshouwers VGAA, Raffaele S, Vossen JH, Champouret N, Oliva R, Segretin ME, Rietman H,
Cano LM, Lokossou A, Kessel G, Pel MA, Kamoun S (2011) Understanding and exploiting late
blight resistance in the age of effectors. Annu Rev Phytopathol 49:507–531
Vossen JH, van Arkel G, Bergervoet M, Jo KR, Jacobsen E, Visser RGF (2016) The Solanum
demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed
worldwide in late blight resistant varieties. Theor Appl Genet 129:1785–1796
Wang M, Allefs S, van den Berg RG, Vleeshouwers VGAA, van der Vossen EAG, Vosman B
(2008) Allele mining in Solanum: conserved homologues of Rpi-blb1are identified in Solanum
stoloniferum. TAG Theor Appl Genet Theor Angew Genet 116:933–943
Wu J, Kou Y, Bao J, Li Y, Tang M, Zhu X, Ponaya A, Xiao G, Li J, Li C, Song MY, Cumagun CJR,
Deng Q, Lu G, Jeon JS, Naqvi NI, Zhou B (2015) Comparative genomics identifies the
Magnaporthe oryzae avirulence effector AvrPi9 that triggers Pi9-mediated blast resistance in
rice. New Phytol 206:1463–1475
Yahiaoui N, Srichumpa P, Dudler R, Keller B (2004) Genome analysis at different ploidy levels
allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat. Plant J
37:528–538
Yang B, Sugio A, White FF (2006) Os8N3 is a host disease-susceptibility gene for bacterial blight
of rice. Proc Natl Acad Sci U S A 103:10503–10508
Yu X, Feng B, He P, Shan L (2017) From Chaos to harmony: responses and Signaling upon
microbial pattern recognition. Annu Rev Phytopathol 55:5.1–5.29
Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang GL (2006) The eight amino-acid
differences within three leucine-Rich repeats between pi2 and piz-t resistance proteins deter-
mine the resistance specificity to Magnaporthe grisea. Mol Plant-Microbe Interact
19:1216–1228
Zhu S, Li Y, Vossen JH, Visser RG, Jacobsen E (2012) Functional stacking of three resistance
genes against Phytophthora infestans in potato. Transgenic Res 21:89–99
Transgenic Technology for Disease
Resistance in Crop Plants 23
T. Makeshkumar, K. Divya, and S. Asha
Keywords
Transgenic for disease resistance · Pathogen-derived resistance · RNAi · Genome

editing tools
23.1 Introduction
The ever increasing demand for food production proportionate to exponential

growth of global population evoked the need for applying innovative techniques
for developing disease-resistant crop varieties, as the pest and pathogen attack causes
considerable yield loss which in turn putting the agriculture sector and crop produc-
tion in crisis. This is highly significant when the resistance conferred by conven-
tional measures, like artificial hybridization, mutation breeding, marker-assisted
selection, etc. appears to be inadequate in many cases, especially due to the evolution
of new/more virulent strains/ pathovars/ isolates of pathogens and their unexpected
host range expansion. Development of recombinant DNA technology, transgenic
expression, and RNA silencing strategies lead to a new era of transgenically
engineered resistance in several crop species, many of which succeeded in field
trials and got commercialized. Elucidation of genetic and molecular mechanisms
underlying host-pathogen interactions, resistance, susceptibility, and different levels
of plant immune responses (PTI, ETI, HR, etc.) revealed the key genes in the host as
well as pathogens that can be manipulated by transgenic approaches/ techniques for
conferring effective resistance. Reprogramming of phytohormonal regulatory
pathways determining defense response and remodeling of molecular receptors/
T. Makeshkumar (*) · K. Divya · S. Asha

ICAR- Central Tuber Crops Research Institute, Thiruvananthapuram, Kerala, India
e-mail: makeshkumar.t@icar.gov.in

https://doi.org/10.1007/978-981-15-6275-4_23
500 T. Makeshkumar et al.
transcription factors involved in resistance or disease development can be brought

about by transgenic methods to enhance the resistance in host plants. Targeting of
conserved sequences or molecular components could provide broad-spectrum resis-
tance in certain cases. Mining of R genes, transgenic expression of foreign R gene,
etc. is another useful strategy. Potential of genome editing based on engineered
nucleases like ZFNs, TALENs, and CRISPR/ Cas9 to precisely mutate the genomic
sequence of interest can be exploited for specific targeting of the host/ pathogenic
genes associated with the biotic stress response. Application of all these approaches
for the management of plant pathogens were discussed in this chapter.
During the earlier times, resistant varieties were created by artificial hybridization
with wild-type resistant varieties, which was purely depended on phenotypic selec-
tion. Diminishing genetic variability and lack of resistant germplasm limited the
possibilities of resistant gene introgression via conventional breeding. Moreover, it
is time-consuming and associated linkage drag may result in introgression of
undesirable traits too. Mutation breeding was an alternative to this, where chemical
mutagens are used for inducing mutation in target genes to create desirable pheno-
type. Resistance to powdery mildew has been successfully engineered in this way by
mutating Mlo locus. As mutagenesis is random, the chances of off-target mutations
is high. QTL mapping and marker-associated selection and breeding are other ways
to create disease-resistant varieties but require large mapping population and are
time-consuming and labour-intensive as in the case of hybridization. Even though
these conventional strategies enabled crop protection to a great extent, over the
centuries, rapid genetic variation and adaptive evolution of pathogens engendered
for novel, robust and sustainable methods for controlling pathogens and pest
management.
The development of genetic engineering (GE) technology for designing tailor-
made plants by transforming plant with the desired gene from other organism was a
major breakthrough in crop improvement programs. Initially, transgenic
experiments were confined to model plants merely for demonstration and proof-of-
concept purpose; later, species wide dispersal of technology was made possible by
the development of optimum transformation and regeneration protocols, and this
widened the applicability of GE for many economically important crops. Since
disease resistance is an important yield determinant, transgenic technologies have
given considerable focus on developing/reinforcing resistance in economically
important crop species. Crop improvement program for disease resistance gained a
significant progress by deploying various GE techniques. Rapid introgression of
promising candidate genes, even into phylogenetically unrelated species, precise
alteration or modification of host factors involved in the perception of pathogenic
effectors and deployment of defence signals, metabolic pathways and biomolecules
involved in direct defence responses as well as targeted by pathogen effectors have
been successfully implemented using transgenic strategies. Similarly, multitude of
pathogenic genes and effectors have also been manipulated transgenically, in order
to reduce virulence and pathogenicity. Furthermore, transgenic strategies permit
precise spatio-temporal regulation of trait of interest. The emergence of new breed-
ing techniques (NBT), including targeted mutagenesis and precision breeding,
23 Transgenic Technology for Disease Resistance in Crop Plants 501
further revolutionized crop improvement strategies. Genome editing tools, like

ZFNs, TALENs and CRISPR/Cas9, based on site-directed nucleases enabled precise
editing of targeted genomic loci to confer/modify resistance traits in many of the
crop plant species. Altogether transgenic strategies facilitated the fine-tuning of the
plant immune system for ensuring sustainable production. In this chapter, we will
describe about the strategies adopted for creating resistance in transgenic plants
against virus, bacteria and fungi and at then the upcoming strategies based on the
information obtain from plant–pathogen interactions.
23.2 Transgenic Technology for Virus Resistance
Viruses are notorious plant pathogens causing devastating damage to crop yield.
These obligate parasites invade, replicate and proliferate in host cell and result in
infection, which reduces fitness and productivity of crop plants and decreases market
and aesthetic values of the products. Attributes like great evolvability, large popula-
tion sizes, error-prone replication and efficient host-dependence render the control of
plant viruses extremely difficult. Development of sustained resistance against path-
ogenic virus, broad-spectrum as well as durable, throughout the productive stage
while infection pressure persists is always a challenge in crop improvement. Farmers
rely on combining traditional cultural management practices, such as field sanitation,
crop rotation, planting of trap plants, spraying for vector, rouging and manual
removal of infected plants upon detection of disease symptoms, use of certified
virus-free seeds or planting materials (Kamala and Makeshkumar 2015; Deepthi and
Makeshkumar 2016), as there is no specific direct control strategy; even chemical
pesticides are not available. All these measures are laborious and time-consuming.
Chemical control of vectors, in addition, causes health and environmental hazards.
The most efficient practical solution available is the use of resistant varieties. But
elite cultivars might not always be endowed with resistance traits, which further
make the problem complicated and uneconomical. Even though resistance genes can
be introgressed from wild varieties, either by conventional breeding or marker-
assisted selection, linkage drag and other inherent shortcomings of these techniques
render it inadequate for successful crop improvement. Resistance genes in unrelated
species or sometimes related species cannot be introgressed by hybridization due to
barriers, like sexual incompatibility, male sterility, etc. Similarly, attaining broad-
spectrum resistance requires stacking of resistance genes from different sources,
which is again a laborious and less efficient strategy. Moreover, virus resistance
conferred by R genes is less durable due to the suppression of R–Avr interaction by
creating variant Avr protein, which might be unrecognizable by the host R protein,
by quickly evolving viral genome, rendering the host susceptible for compatible
host-pathogen interaction. However, transgenic approaches have succeeded to a
great extend in engineering efficient virus resistance in crop species.
Recessive resistance, conferred by mutating host factors required for infection
cycle completion and thereby making the host non-permissive to virus, is more
efficient and durable strategy. Resistance thus conferred is passive as there is no
active involvement of plant immune system. Resistance mechanism, to be adopted,

and respective targets vary with stage of infection (Johnson 1981).
Transgenic antiviral approaches used so far were based on the expression of
various viral proteins; RNAs; nonviral genes like nucleases, antiviral inhibitors and
plantibodies; plant defence response elicitors; host-derived resistance genes (domi-
nant resistance genes and recessive resistance genes); and various factors involved in
host defence responses. Viral proteins usually used for engineering resistance are
capsid protein, replicase proteins and movement proteins. Several RNA molecules,
viz. sense RNAs, antisense RNAs, satellite RNAs, defective interfering RNAs,
hairpin RNAs and artificial microRNAs, noncoding RNAs, antisense RNAs,
ribozymes, double-stranded RNAs (dsRNAs) and inverted repeat RNAs (irRNAs),
have been employed for conferring virus resistance by post-transcriptional or tran-
scriptional gene silencing. Transgenic virus resistance strategies are deployed prin-
cipally by three mechanisms: pathogen-derived resistance, pathogen-targeted
resistance and RNA interference.
23.2.1 Pathogen-Derived Resistance
Pathogen-derived resistance (PDR) is protein-mediated resistance conferred by viral

protein expressed in host cells (Sanford and Johnston 1985). PDR is accomplished
by different a mode of action, which varies with strains and stages of infection cycle,
like whether it is in movement/transport/replication phase. It can be inhibition of
replication and viral particle accumulation in the early stages of infection while
limiting the spread via apoplastic/symplastic/phloem stream during the movement
stage (Galvez et al. 2014). The first type of PDR is based on the silencing of
pathogenic gene by expression of a part of that gene in host.
23.2.2 Coat Protein–Mediated Resistance (CPMR)
Since coat protein (CP) has implications in almost all stages of infection, like
uncoating, systemic movement, long-distance transport, replication, symptom devel-
opment, etc., it can be the ideal candidate for engineering PDR, even in non-host
plants (Galvez et al. 2014). CPMR is manifested by expression of coat protein gene
in the host cell and the subsequent interaction between transgenic CP and viral CP
(Koo et al. 2004). The very first time application of CPMR was showed in tobacco
(Nicotiana tabacum) plants, expressing the capsid protein-encoding sequences of
Tobacco mosaic virus (TMV), and this resulted in partial resistance to TMV (Abel
et al. 1986). Later it was expanded to tomato, using the same CP construct, with
resistance to Tomato mosaic virus (ToMV) (Nelson et al. 1988). Similarly, capsid
protein gene of a potyvirus was expressed in a non-host plant, tobacco, conferring
resistance to other potyviruses. CPMR has been used to confer resistance to at least
35 viruses, representing more than 15 different taxonomic groups (Table 23.1).
Several virus-resistant transgenic crop plants were developed by using a suitable
Table 23.1 Transgenic plant species showing coat protein-mediated resistance

Host plants Virus References
Citrus Citrus mosaic virus (CiMV) Iwanami et al. (2004)
Citrus psorosis virus (CPsV) Reyes et al. (2011)
Citrus tristeza virus (CTV) Domínguez et al. (2002)
Febres et al. (2008)
Loeza-Kuk et al. (2011)
Cucumber Zucchini yellow mosaic virus (ZYMV) Wako et al. (2001)
Squash Squash mosaic virus (SqMV) Pang et al. (2000)
Watermelon Cucumber green mottle mosaic virus Park et al. (2005)
(CGMMV)
Cucumber mosaic virus (CMV) and Lin et al. (2012)
Watermelon mosaic virus (WMV)
WMV Wang et al. (2003)
Zucchini yellow mosaic virus (ZYMV)
ZYMV
ZYMV and Papaya ringspot virus -W Yu et al. (2011)
(PRSV-W) PRSV-W
Brinjal CMV Pratap et al. (2011)
Lettuce Lettuce big-vein associated virus Kawazu et al. (2006)
(LBVaV)
Melon CMV Xu et al. (2005)
ZYMV Wu et al. (2009)
ZYMV and PRSV-W Wu et al. (2010)
Soybean Bean pod mottle virus (BPMV) Reddyet al. (2001)
Soybean dwarf virus (SDV) Tougou et al. (2007)
Wang et al. (2001)
Soybean mosaic virus (SMV) Furutani et al. (2006)
Rice Rice stripe virus (RSV) Park et al. (2012)
Rice tungro bacilliform virus (RTBV) Ganesan et al. (2009)
Rice tungro spherical virus (RTSV) Verma et al. (2012)
Rice yellow mottle virus (RYMV) Kouassi et al. (2006)
Maize Maize dwarf mosaic virus (MDMV) Liu et al. (2005)
Sugarcane mosaic virus (SCMV) Liu et al. (2009)
Wheat Wheat streak mosaic virus (WSMV) Sivamani et al. (2002)
Li et al. (2005)
Orchid Cymbidium mosaic virus (CymMV) Chang et al. (2005)
CymMV Liao et al. (2004)
Papaya Papaya ringspot virus (PRSV) Lines et al. (2002)
Ferreira et al. (2002)
Fermin et al. (2004)
Souza et al. (2005)
Kertbundit et al. (2007)
PRSV and Papaya leaf-distortion mosaic Kung et al. (2009)
virus (PLDMV) Kung et al. (2010)
(continued)

Host plants Virus References
Groundnut Peanut stripe virus (PStV) Higgins, et al. (2004)
Tobacco streak virus (TSV) Mehta et al. (2013)
Capsicum CMV Lee et al. (2009)
Pack et al. (2012)
CMV and Pepper mild mottle virus Shin et al. (2002)
(PMMoV)
CMV and Tomato mosaic virus (ToMV) Shin et al. (2002)
Sugarcane Sugarcane mosaic virus (SCMV) Guo et al. (2008)
Sugarcane yellow leaf virus (SCYLV) Zhu et al. (2011)
Sweet potato Sweet potato feathery mottle virus Okada et al. (2001)
(SPFMV) Okada and Saito (2008)
Okada and Yoshinaga
(2010)
Potato Potato virus Y (PVY) Rachman et al. (2001)
Plum Plum pox virus (PPV) Scorza et al. (1997); Ilardi
and Tavazza (2015)
Sweet pepper Cucumber mosaic virus Zhu et al. (1996)
Nicotiana tabacum Cucumber mosaic virus (CMV) subgroup Dubey et al. (2015)
cv. Petit Havana IA
Tomato CMV Pratap et al. (2012)
Physalis mottle virus (PhMV) Vidya et al. (2000)
Tomato leaf curl virus (ToLCV) Raj et al. (2005)
Tomato leaf curl Taiwan virus Sengoda et al. (2012)
(ToLCTWV)
Tomato spotted wilt virus (TSWV) Gubba et al. (2002)
coat protein gene (Mundembe et al. 2009; Nomura et al. 2004). CPMR has been
successfully established in host plants, including potato, tomato, tobacco and
papaya, exhibiting resistance to Potato virus Y (PVY) and Potato leaf roll virus
(PLRV), Cucumber mosaic virus (CMV) and Papaya ring spot virus
(PRSV) (Makeshkumar et al. 2002). The efficiency of CPMR varies for each virus
with different stages of infection cycle (Bendahmane et al. 2007). The underlying
molecular mechanism of resistance manifestation is either through recoating of
invading viral particles or by blocking of receptors in transgenic plants (Saharan
et al. 2016).
23.2.3 Replicase- or Rep-Associated Protein-Mediated Resistance
Replicase is another potential candidate to engineer resistance against viral genome.

Expression of intact or truncated or mutant virus-encoded replicase in host can
confer resistance to that virus. It was reported for the first time when a 54 kDa
truncated protein of TMV replicase protein was expressed in N. benthamiana, where
it conferred high level of resistance against TMV infection (Golemboski et al. 1990).
Table 23.2 List of various transgenic plants developed with replicase-mediated resistance
Plant species Virus
Potato Potato leafroll virus (PLRV)
Potato virus Y (PVY-N) tobacco rattle virus (TRV)
Rice Maize dwarf mosaic virus (MDMV)
Rice tungro spherical virus (RTSV)
Rice yellow mottle virus (RYMV)
Tomato CMV
Watermelon CMV, Zucchini yellow mosaic virus (ZYMV), and WMV
Wheat Wheat streak mosaic virus (WSMV)
Wheat yellow mosaic virus (WYMV)
Papaya Papaya ringspot virus (PRSV)
Citrus Citrus tristeza virus (CTV)
Cucumber Cucumber fruit mottle mosaic virus (CFMoMV)
Barley Barley yellow dwarf virus (BYDV-PAV)
This strategy has been extended to many food crops, including rice, bean, potato etc.,
and mostly resulted in narrow-spectrum resistance towards a particular race of the
pathogen (Saharan et al. 2016) (Table 23.2). Rep-associated protein, which interacts
with host DNA polymerase during replication of ssDNA virus, can also be
manipulated in a similar manner. Resistance to two ssDNA viruses, Tomato yellow
leaf curl virus-Israel (TYLCV-Is [Mild]) and Bean golden mosaic virus were
conferred by transgenic expression of a truncated replication-associated protein
gene and rep gene, respectively, in tomato and P. vulgaris (Brunetti et al. 1997;
Faria et al. 2006). As in the case of CPMR, the active entity conferring resistance can
either be protein or RNA or both.
23.2.4 Movement Protein–Mediated Resistance
Movement proteins (MP) facilitate the intracellular or cellular movement of viral

particles through plasmodesmata, by modifying the gating channels of
plasmodesmata. Resistance conferred by dysfunctional or mutated MP is mostly
broad-spectrum in nature when compared to CP or replicase-mediated resistance
(Prins et al. 2008). The first MP-mediated resistance was shown in transgenic
tobacco plants expressing a 30 kDA mutant defective MP (dMP), which competed
with the wild-type virus-encoded MP for the binding sites in the plasmodesmata and
conferred resistance against eponymous virus infection. dMP conferred broad-
spectrum resistance by preventing the systemic spread of distantly related and
unrelated viruses (Lapidot et al. 1993; Cooper et al. 1995). Similar broad-spectrum
resistance was observed in transgenic potato expressing wild-type Potato leafroll
virus (PLRV) movement protein against PLRV, PVY and PVX (Tacke et al. 1996),
while narrow-spectrum resistance was shown in transgenic N. benthamiana
expressing wild-type movement proteins of Cowpea mosaic virus (CPMV) and
transgenic tobacco expressing PVX movement protein (Sijen et al. 1995).
23.2.5 Other Viral Protein-Mediated Resistance
Transgenic expression of viral proteins other than those discussed previously, such
as replication-associated protein, NIa protease, P1 protein and HC- Pro, has been
tried out in order to achieve resistance against viruses (Cillo and Palukaitis 2014).
Transgenic expression of partial or complete Rep gene was found to confer resis-
tance against Tomato golden mosaic virus (TGMV), Tomato yellow leaf curl virus
(TYLCV) (Yang et al. 2004; Antignus et al. 2004; Lucioli et al. 2003), African
cassava mosaic virus (ACMV) (Chellappan et al. 2004), Bean golden mosaic virus
(BGMV) (Faria et al. 2006), Maize streak virus (Shepherd et al. 2007), Cotton leaf
curl virus (CLCuV) (Hashmi et al. 2011) and Tomato leaf curl Taiwan virus
(ToLCTWV) (Lin et al. 2012) in transgenic plants.
Transgenic tobacco plants expressing the NIa protein of Tobacco vein mottling
virus (TVMV) exhibited resistance against TVMV, whereas it failed to confer
resistance to two other potyviruses, Tobacco etch virus (TEV) and PVY (Maiti
et al. 1999). Transgenic tobacco lines expressing paired NIa protease coding
sequences for two viruses, like TEV–PVY, TEV–TVMV and TVMV–PVY, were
assessed for virus resistance and mostly resulted in the recovery-type resistance
(Fellers et al. 1998). However, expression of multiple genes (NIa/NIb/capsid pro-
tein) from a single potyvirus using a single construct failed to confer any enhanced
resistance in most cases (Maiti et al. 1999).
Transgenic expression of complete or partial sequence encoding P1 protein
conferred resistance, of varying degree, against viruses like PVY, PPV, TVMV
and PVA. A recovery-type resistance was obtained in most cases rather than
complete resistance (Germundsson and Valkonen 2006). Expression P1 coding
sequence of PVY-O and PPV, respectively, showed either complete resistance or a
recovery-type resistance against PVY-O infection in potato and PPV infection in
N. benthamiana (Maki-Valkama et al. 2001; Tavert-Roudet et al. 1998).
Expression of viral HC-Pro protein resulted in recovery phenotypes instead of
conferring resistance to PVA and PPV in transgenic N. benthamiana and SMV in
transgenic soybean (Savenkov and Valkonen 2002; Barajas et al. 2004; Lim et al.
2007). Expression level of transgene considerably influenced the resistance mediated
by HC-Pro in most of the experiments, wherein low expression levels are mostly
favoured for resistant or recovery phenotype rather than high expression levels.
Deletion of central domain of HC-Pro protein has found to confer recover disease
symptoms of Cowpea aphid-borne mosaic virus (CABMV) in transgenic
N. benthamiana compared to intact protein (Mlotshwa et al. 2002).
Other viral genes, such as VPg-protease coding region of Tomato ringspot virus
(ToRSV), capsid protein domain of BNYVV (Andika et al. 2005) and p23 silencing
suppressor protein of Citrus tristeza virus (CTV) (Fagoaga et al. 2006), were also
used as potential transgene candidates for providing resistance against ToRSV
infection in N. benthamiana, olymyxa betae infection in transgenic
N. benthamiana plants and CTV infection in transgenic Mexican lime plants,
respectively (Table 23.3).
Table 23.3 List of transgenic crop plants with hairpin-mediated resistance against viruses
Transgenic
host plant Disease/virus Hairpin construct used References
Cassava ACMV Rep gene of ACMV Vanderschuren et al. 2012
Cassava brown Capsid protein of CBSV Ogwok et al. (2012)
streak virus
Cassava brown Rep gene of CBSV Chauhan et al. (2015), Wagaba
streak Uganda et al. (2017), Vanderschuren
virus et al. (2012), Yadav et al. (2011)
SLCMV AV1 and AV2 Ntui et al. (2015)
Citrus Citrus psorosis RNA3 of CPsV Reyes et al. (2009)
virus (CPsV) p24 gene on RNA1 Reyes et al. (2011)
CTV p23 gene and 3’NTR Lopez et al. (2010)
Capsid protein gene Muniz et al. (2012)
RNA silencing Soler et al. (2012)
suppressor (CTV)
Cucurbits PRSV Capsid protein coding Krubphachaya et al. (2007)
sequences of PRSV
CGMMV Capsid protein gene of Kamachi et al. (2007)
CGMMV
Melon necrotic Cm-eIF4E translation Rodríguez-Hernández et al.
spot virus initiation factors (eIF) (2012)
(NSV)
ZYMV HC-pro encoding Leibman et al. (2011)
sequences of ZYMV
Common BGMV C1 gene of BGMV Bonfim et al. (2007)
bean
Soybean Soybean mosaic Coat protein, VSR Kim et al. (2016), Gao et al.
virus Hc-pro, P3 cistron (2015), Yang et al. (2018)
Soybean dwarf Coat protein Tougou et al. (2006)
virus (SbDV)
Cowpea Cowpea aphid- Coat protein Cruz et al. (2014)
borne mosaic
virus
White WCIMV Replicase gene of Ludlow et al. (2009)
clover WClMV
Maize SCMV NIb coding sequences of Zhang et al. (2010)
SCMV
Maize dwarf P1 coding sequence or Zhang et al. (2011)
mosaic virus capsid protein coding
(MDMV) sequence of MDMV
Potato PVY-N, certain 3’part of the PVY-N Missiou et al. (2004)
isolates of capsid protein coding
PVY-NTN and sequences of PVY-N
PVY-O
PVX, PVY-O, Fused PVX capsid Bai et al. (2009)
PVY-N, PVY-C protein and PVY Nib
encoding region
(continued)

Transgenic
PVX, PVY and Fused sequences from Arif et al. (2012)
PLRV PVX, PVY and PLRV
PVY-O and PVY capsid protein McCue et al. (2012)
PVY-NTN
PVA, PVY, Fused hpRNA sequences Chung et al. (2013)
PLRV of three viruses (PVA,
PVY and PLRV)
Sweet SPFMV and Capsid protein of Sweet Nyaboga et al. (2008)
potato SPCSV potato feathery mottle
virus (SPFMV)
Tomato TYLCV C1 (rep) gene Fuentes et al. (2006)
TYLCV TYLCV capsid protein Zrachya et al. (2007)
TYLCV Rep gene of TYLCV Tamarzizt et al. (2009)
(Sardinia (Sardinia variant)
variant),
TYLCV
PSTVd hpRNAs derived from Schwind et al. (2009)
PSTVd
CMV Replicase Ntui et al. (2015)
Wheat WSMV NIa coding sequence Fahim et al. (2010)
(WSMV)
BYDV (PAV) Polymerase gene of Yassaie et al. (2011)
BYDV (PAV)
Maize Maize dwarf Coat protein Zhang et al. (2011)
mosaic virus
(MDMV)
Barley Barley yellow Polymerase gene Wang et al. (2000)
dwarf virus
(BYDV)
Banana Banana bunchy Master replication Elayabalan et al. (2013)
top virus initiation protein of
bunchy top virus
Rice RDV Pns4 or Pns12 genes of Shimizu et al. (2009)
RDV
RSV pC1 (replicase), pC3 Shimizu et al. (2011)
(nucleocapsid) and pC4
(movement protein) of
RSV
RSV Nucleocapsid protein Ma et al. (2011)
gene or disease-specific Zhou et al. (2012)
protein gene of RSV Park et al. (2012)
Rice black Non-structural gene Shimizu et al. (2012)
streaked dwarf (P9–1) of Fijivirus
virus (RBSDV) (RBSDV)
(continued)

Transgenic
Rice gall dwarf Non-structural gene Shimizu et al. (2012)
virus (Pns9) of Rice gall dwarf
virus
Rice ragged Nucleocapsid protein or Shimizu et al. (2013)
stunt virus movement protein gene
of Rice ragged stunt
virus
Tobacco CMV Coat protein Chen et al. (2004)
PPV VSR P1 and HC- pro Di Nicola-Negri et al. (2005)
CGMMV Coat protein Kamachi et al. (2007)
23.2.6 Viral RNA-Mediated Resistance
Although virus resistance has been successfully manifested in several crop species
by transgenic expression of intact or dysfunctional or mutated structural proteins of
virus, resistance in many plants has been found to be mediated by corresponding
mRNAs rather than the encoded protein moieties (Saharan et al. 2016). The active
role of RNA in conferring resistance was first revealed when an untranslatable coat
protein gene in transgenic tobacco plants provided resistance against Tobacco etch
virus (TEV) (Lindbo and Dougherty 1992a, b). Many cases of transgenic expression
of untranslatable viral proteins further substantiated the direct participation of RNA
in developing resistance. Later, it has been shown that the virus resistance can be
RNA-mediated, and viral protein expression is not necessary for the same
(Dougherty et al. 1994). Viral RNA-mediated resistance is carried out by transcrip-
tional gene silencing (PTGS), RNA interference or RNAi or RNA silencing, where a
transgenic viral RNA introduced into the host plant drives sequence-specific homol-
ogy-dependent degradation of viral genomic RNA or viral mRNAs by employing
the small interfering RNA (siRNA) pathway of host cell machinery (Voinnet 2008;
Waterhouse et al. 2001; Lindbo and Dougherty 2005). This homology-dependent
gene silencing mechanism is conserved among higher eukaryotes and operates for
gene expression regulation and in host defence against transposable elements and
viruses (Hannon 2002; Mawassi and Gera 2012).
23.2.7 RNA Interference: Mechanism
RNA interference is widely accepted as a potential gene silencing strategy that can
be easily manipulated to obtain desirable trait in organism of interest. RNA silencing
process requires a double-stranded RNA precursor to trigger the silencing process.
This dsRNA precursor is derived from viral RNA intermediates or viral RNA
secondary structures. After recognition of this dsRNA by the RNase III-like enzyme,
namely, DICER, it is cleaved into 21–25 nucleotide duplexes, called as small
interfering RNAs (siRNAs). The siRNAs thus formed are incorporated and
converted to ssRNAs by Argonaut (Ago) containing multisubunit ribonuclease
named as RNA-induced silencing complex (RISC). Subsequently RISC targets the
specific mRNAs that share sequence similarity with siRNA through degradation of
transcript (Waterhouse et al. 1998; Hamilton and Baulcombe 1999; Voinnet 2008).
RNA-mediated resistance engineering approaches include expression of
non-coding regions of viral genome, viral CP mRNA, satellite (sat) RNA, defective
interfering (DI) RNA and viral sequences in sense or antisense orientation or in
double-stranded forms in host plants (Cillo and Palukaitis 2014).
Non-coding ssRNA Viral non-coding RNAs like 50 and 30 NTRs, intergenomic

regions and non-coding RNAs, either in sense or antisense orientation, have been
used for engineering resistance in various crop species. Transgenic expression of 50
and 30 NTRs and intergenomic regions could successfully confer resistance in crop
plants. Sometimes simultaneous expression of non-coding NTR regions with
mRNAs also confer resistance. For instance, transgenic tobacco expressing
30 -NTR of Andean potato mottle virus (APMoV), which was expressed with a
smaller portion of capsid protein-coding region, showed excellent resistance in
several lines (Vaskin et al. 2001). Transgenic oilseed rape plant expressing
30 -NTR of Turnip yellow mosaic virus (Zaccomer et al. 1993) and intergenic region
of PLRV expressing transgenic potato (Dong et al. 1999) showed resistance to
respective pathogens.
Non-translatable Sense RNAs Non-translatable sense RNAs of coat protein cre-

ated by frameshift mutation have been widely used to confer resistance to a large
number of crops, including tobacco, rice, papaya, peanut, grape, sugar cane, etc.
Expression of frameshifted, capsid protein coding sequences of PVY-NTN in
transgenic potato was shown to be resistant to PVYNTN infection (Rachman et al.
2001). There are some other viral proteins which can be suitable candidates to be
manipulated in similar manner to engineer resistance. Potential of non-translatable
coding sequences of movement protein, nucleoprotein, Vpg, NSm, etc. to mediate
viral gene silencing has been proved through different experiments. Expression of
non-translatable form of the capsid protein gene has been conferred resistance to
Papaya leaf-distortion mosaic virus (PLDMV) (Kung et al. 2010), Zucchini yellow
mosaic virus (ZYMV) and PRSV-W (Wu et al. 2010; Yu et al. 2011) and Sugarcane
yellow leaf virus (Zhu et al. 2011). Transgenic tobacco expressing a non-translatable
form of the NSM coding region of TSWV and a non-translatable form of the TEV
6-kDa/VPg encoding region exhibited resistance towards TSWV and TEV, respec-
tively (Prins et al. 1996; 1997; Swaney et al. 1995). Moreover, hybrid constructs,
designed by a fusion of non-translatable coding sequence of coat protein of different
viruses, have found to be efficient for developing broad-spectrum resistance.
Antisense RNAs Transformation of host plant with antisense RNAs of certain viral
genes is a powerful strategy to silence invading viral genes. Transgenic plants
expressing sense RNAs for viral replicase, transcription activator protein (TrAP),
replication enhancer protein (Ren), AV1, etc. had been developed. Antisense
RNA-mediated silencing mostly resulted in attenuated or delayed symptoms and
complete resistance in some cases. Trials to engineer resistance to Tomato leaf curl
virus (Praveen et al. 2005), African cassava mosaic virus (Zhang et al. 2005) and
Mung bean yellow mosaic India virus (Singh et al. 2013) have been performed.
Satellite RNA Some viruses exhibit a supernumerary RNA component that does
not show any apparent homology to the viral RNA genome but depends on its helper
virus for replication, encapsidation and transmission and is defined as a satellite
RNA (Simon et al. 2004). These sequences have found to have a highly variable
range of effects on various components like viral replication, pathogenesis and
symptom expression in certain host-pathogen interactions. Sat variants with
attenuating effects are regarded as potential biocontrol agents in transgenic plants.
Transgenic expression of an attenuating CMV-associated satRNA suppressed viral
replication and symptom development and thereby conferred tolerance to CMV in
tobacco and other solanaceous plants including tomato (Baulcombe et al. 1986;
Harrison et al. 1987; Kim et al. 1997; Kim et al. 1995)). Transgenic N. benthamiana
and A. thaliana plants expressing Bamboo mosaic virus (BaMV) satRNA showed
high resistance to helper virus (Lin et al. 2013).
Defective Interfering DNAs and RNAs Defective DNAs and RNAs are produced
during the replication of certain viral species, which can reduce the full-length
genome accumulation and results in the denomination of DI nucleic acids, associated
with the modulation of symptom expression, and thus serves as a source for
transgenic resistance strategies. ssDNA geminiviruses, like ACMV, and ssRNA
Tombusvirus, like Cymbidium ringspot virus (CymRSV), are known to form DIs.
Interfered viral replication and milder symptoms were observed in transgenic
N. benthamiana expressing naturally occurring subgenomic ACMV DNA B upon
ACMV infection (Stanley et al. 1990). Tolerance to other viral species, including
Beet curly top virus (BCTV) (Frischmuth and Stanley 1998; Stenger 1994), Cucum-
ber necrosis virus (CNV), Carnation Italian ringspot virus and CymRSV (Kollàr
et al. 1993; Rubio et al. 1999), has been obtained by employing DI DNA and
DI RNA.
Silencing by Ds Inverted Repeat Sequence/hpRNA Combined expression of

sense and antisense transcripts in the same transgenic plants is advantageous for
obtaining more transgenic lines exhibiting resistance. It is more efficient than the
expression of either strand alone (Waterhouse et al. 1998; Smith et al. 2000;
Waterhouse and Helliwell 2003). This was made possible by designing a single
transcript of both polarities, with sense and antisense sequences separated by introns
or other spacer sequences, to generate dsRNAs with loops, known as hpRNAs,
intron hpRNAs (ihpRNAs) or irRNAs, which stabilize the inverted repeat DNA
sequences in Escherichia coli (Smith et al. 2000; Wesley et al. 2001). dsRNA thus
produced is then acted upon by siRNA pathway, resulting in virus resistance (Castel
and Martienssen 2013; Csorba et al. 2009; Ding 2010; Eames et al. 2008; Pumplin
and Voinnet 2013). Broad-spectrum resistance can be achieved by creating a

chimeric, fused hpRNAs expressing sequences of several viruses from the same
vector (Cillo and Palukaitis 2014). The application of hpRNAs as a transgenic
resistance strategy was first demonstrated in transgenic tobacco and barley
expressing a hpRNA specifically targeting NIa protease coding sequences of PVY
and polymerase coding sequences of BYDV, respectively (Smith et al. 2000, Abbott
et al. 2000). Development of viral resistance in crop plants was tremendously
advantaged from the establishment of simple and efficient technique for stable
integration of self-complementary hairpin construct, designed specifically for the
cognate RNA target. dsRNA and siRNA are generated in host cell and silence the
pathogen gene expression by cleaving target RNA. The major crop plants, like rice,
maize, citrus, cassava, legumes, etc., have been conferred durable and efficient
resistance using this method (Cillo and Palukaitis 2014). Although RNAi confers
efficient and durable resistance, plant viruses have evolved smart measures, like
suppressors of RNA-induced gene silencing, to escape RNAi which is a challenge
before the advancements of transgenic resistance (Table 23.3).
Silencing by Artificial MicroRNAs MicroRNAs are small (20–250 nt),

non-coding RNAs present in eukaryotic cells and facilitate gene regulation at post-
transcriptional or translational level (Bartel 2004). The precursor miRNA transcripts
are processed into mature miRNA and directed to target mRNA by the same
mechanism involving DICER, Ago and RISC, as that of siRNAs (Jones-Rhoades
et al. 2006). Customized miRNA precursors producing target-specific siRNAs,
which are having no similarity with mature endogenous miRNAs, known as artificial
microRNAs (amiRNAs), can be transgenically expressed to target invading viral
sequences. amiRNA-mediated virus resistance was first reported in transgenic
Arabidopsis showing resistance against Turnip yellow mosaic virus (TYMV) and
Turnip mosaic virus (TuMV) by stable expression of amiRNAs targeting the RNA
sequences encoding silencing suppressors P69 and HC-Pro of the virus (Niu et al.
2006). amiRNAs targeting RNA silencing suppressors of virus have found to be
more efficient in conferring resistance either complete resistance or delayed infec-
tion/susceptibility, when compared to those targeting CP or other structural proteins
(van Vu et al. 2013). Silencing suppressors Hc-Pro and TGB1/p25 of PVY and PVX,
respectively, were targeted efficiently by amiRNAs (Ai et al. 2011). amiRNAs have
been demonstrated to confer resistance against a vast range of viruses, including
positive-sense ssRNA genome such as CMV, PVY and PVX; negative-sense ssRNA
viruses like WSMoV (genus Tospovirus); and ssDNA viruses of the genus
Begomovirus like ToLCV New Delhi variant (van Vu et al. 2013) and Cotton leaf
curl Burewala virus (Ali et al. 2013). Efficiency of amiRNA-induced viral gene
silencing is determined by several factors, like expression levels of pre-miRNA
backbone and sequence complementarity of amiRNA with target and structural
features of target RNA and accessibility of amiRNA to target RNA (Ali et al.
2013; Cillo and Palukaitis 2014; Simón-Mateo and García 2011; Duan et al.
2008). Viruses may quickly evolve new strains with mutations in amiRNA target
site and escape silencing, which can be circumvented by simultaneous targeting of
multiple regions of highly conserved RNA motifs of viral genome with multiple
amiRNAs. A polycistronic amiRNA designed from a modified rice miRNA395,
targeting different conserved regions of the WSMV, conferred resistance to WSMV
in wheat (Fahim et al. 2012).
Silencing by Co-suppression Resistance to virus can be achieved by activating

co-suppression by introducing surplus amount of viral transcripts in the host, where
overabundance of sense strand induces downregulation or suppression of transgene
and invaded viral gene. Such overabundance of sense strand leads to the removal of
all homologous transcripts, beyond a critical threshold, by respective cellular
machineries (Stam et al. 1997). Overexpressed transcripts, when recognized by
plant RNA-dependent RNA polymerase, act as primers for dsRNA synthesis,
which then subjected to cleavage and degradation by DICER and RISC complex
(Dougherty and Parks 1995).
23.2.8 Transgenic Technology to Engineer Pathogen-Targeted Virus

Resistance
PDR and R gene often do not confer complete resistance to invading viruses, and
mostly resistance is not stable over several generations. Pathogen-targeted resistance
is manifested by introducing silencing constructs into host plants, where it targets
viral genome. Synthetic constructs designed to target viral sequences are neither of
plant origin nor pathogen-derived. Such non-viral-mediated resistance is manifested
by transgenic expression of synthetic nucleases like zinc finger nucleases, transcrip-
tion activator like effector nucleases, CRISPR/Cas9, antiviral inhibitor proteins like
ribosome-inhibiting proteins, peptide aptamers and plantibodies (Bastet et al. 2018).
Nucleases Resistance against Fijivirus RBSDV and TMV was conferred by trans-
genic expression of an E. coli dsRNase gene (Cao et al. 2013), bovine pancreatic
RNase (Trifonova et al. 2007) and an inducible extracellular RNase from Zinnia
elegans (Trifonova et al. 2012). E. coli dsRNase (RNase III) conferred high-level
resistance to a tomato isolate of TSWV also (Langenberg et al. 1997). Feasibility of
transgenic expression of antiviral pathways or invading nucleic acid targeting
pathways from heterologous systems in host plant for targeting infectious viruses
has been demonstrated using OAS system 2,5A-oligoadenylate synthetase (OAS)/
RNase L system, which is also called as 2,5A oligoadenylate pathway, an animal
antiviral pathway induced by interferons in mammalian cells. The OAS catalyses the
polymerization of ATP producing 2-5-linked oligoadenylates, pppA (2_p5_A) nor
2,5A, when it recognizes a dsRNA, which can be replicative intermediates of single-
stranded RNA viruses or viral dsRNA genomes. The subsequent activation of the
latent ribonuclease, RNAse L, by the 2,5A oligonucleotides produced by OAS,
carries out the degradation of both viral and cellular RNAs, thereby hampering the
viral replication and infection (Floyd-Smith et al. 1981). The efficacy of this RNA
targeting mechanism to confer broad-spectrum resistance has been demonstrated in
tobacco plants (Mitra et al. 1996). Resistance to several DNA and RNA viruses has
been engineered employing ZFNs, TALENs and CRISPR/Cas9. Artificial TALE
proteins and FokI nuclease expressing Nicotiana benthamiana plants showed resis-
tance to different Begomoviruses. Transgenic expression of yeast-derived dsRNase,
Pac1, confer broad-spectrum resistance to phytopathogenic viruses. Pac1 has been
successfully used to confer resistance to CMV, Tomato mosaic virus (ToMV) and
PVY in tobacco (Watanabe et al. 1995) and PSTVd in potato (Sano et al. 1997).
Ribosome-Inactivating Proteins Ribosome-inhibiting proteins or ribosome-

inactivating proteins found in certain plant species possess anti-viral activities.
Transgenic expression of RIP from the pokeweed (Phytolacca americana) conferred
broad-spectrum resistance to virus infection in several plant species, including
tobacco, potato and N. benthamiana and Brassica napus against PVX, PVY and
TuMV (Lodge et al. 1993; Zhang et al. 1999), respectively. Transgenic expression of
RIP from Dianthus caryophyllus, Trichosanthes kirilowii and Iris hollandica also
was observed to provide varying degrees of resistance to ACMV, TMV and CMV
and TEV and TMV, respectively. However, any of the RIPs evaluated so far has not
found to have an efficient broad-spectrum anti-viral activity.
Peptide Aptamers There are some short-peptide molecules, which can confer
low-level broad-spectrum resistance and are advantageous in that the need for
producing high level of transgene to induce PTGS can be avoided. Such short-
peptide-mediated resistance sometimes provides better resistance than RNAi- and
protein-mediated PDR as in the case of transgenic N. benthamiana with an
introduced target-specific peptide aptamer showing broad-spectrum resistance to
Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV) and Chry-
santhemum stem necrosis virus (CSNV) (Rudolph et al. 2003). Similarly, a
conformationally constrained peptide aptamer was found to interfere the replication
of Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus (CaLCuV)
(Lopez-Ochoa et al. 2006). Further investigation of such peptides and understanding
of their mode of action enable their application for engineering broad-spectrum
resistance.
Plantibodies Antibody-mediated resistance to a plant virus was first demonstrated

in transgenic N. benthamiana plants expressing an scFv against Artichoke mottled
crinkle virus (Tavladoraki et al. 1993). Later, transgenic expression of scFv in
different crop plants, like Chinese cabbage, citrus, gladiolus, potato and tomato,
conferred them with virus resistance. Initial experiments resulted in delayed or
reduced disease symptoms only, and complete resistance was not observed.
Improvements in plantibody targeting and stabilization and development of non-
structural viral proteins targeting plantibodies enhanced the level of resistance
provided by them (Gargouri-Bouzid et al. 2006; Nickel et al. 2008, Boonrod et al.
2004; Gil et al. 2011). Transgenic N. benthamiana expressing scFvs targeted against
the TBSV replicase showed broad-spectrum resistance to TBSV, two members of
family Tombusviridae (CNV and TCV) and Dianthovirus Red clover necrotic
mosaic virus (Boonrod et al. 2004).
23.3 Engineering Resistance to Vectors
Many pathogens, particularly viruses, spread by different insect vectors, mostly

hemipterans, aphids, dipterans, etc. Efficient vector control strategies significantly
reduce the infection and crop damage. In certain cases, a single vector may harbour
multiple pathogens, which are infecting same or different species. Transgenic
strategies for vector control can be implemented by taking cues from vector-
pathogen–host interactions and underlying genetic, molecular and biochemical
processes. Persistent, semi-persistent and non-persistent vectors differ highly in
their feeding nature, subcellular localization of virus in them and the status of
virus in vector, whether replicating or non-replicative, circulating (Jones 2014;
Brault et al. 2010). Viruses themselves influences host interaction with vectors and
environment and modulate host immune response accordingly to regulate the feed-
ing pattern and preference in order to manage the acquisition as well as injection of
pathogens from viruliferous and non-viruliferous plants (Ingwell et al. 2012;
Stafford et al. 2011). All these are accomplished by regulating host defence signal-
ling hubs, manipulation of plant biochemistry for enhanced vector performance and
virus-mediated suppression of plant immunity. Suppression of JA- and SA-mediated
immune response to reduce insect repellents signals, i.e. different volatile
compounds is a typical example for how pests reprogramme plant immunity for
enhancing infection (Westwood et al. 2013). Interestingly, such negative regulation
of defence-related signalling can be manipulated transgenically to reverse the effects
so that resistance to both pests and pathogens can be achieved.
Understanding of involvement of semiochemicals and subsequent and signal
generation and transduction can give an insight into potential semiochemical targets
that can be modified transgenically to control vectors. Transgenic plants constitu-
tively emitting the semiochemical (E)-b-farnesene, an aphid alarm pheromone and
predator attractant, showed repulsion to aphid species and attraction to aphid-
parasitizing wasps under controlled conditions (Bruce et al. 2015). However, the
metabolically engineered plants showed no differences in aphid infestation or
presence of natural enemies in field. Stimulated expression or mixed expression of
different semiochemicals can be an alternative strategy to effectively control vectors.
Introgression of R genes conferring vector resistance, like the melon Vat (‘virus
aphid transmission’) gene and the tomato Mi-1.2 gene, is useful for controlling
vectors. Vat confers effective resistance to multiple viruses but only when transmit-
ted by specific aphid species (Goggin et al. 2006), whereas Mi-1.2 provide resistance
to the nematode Meloidogyne incognita and many phloem-feeding insects, including
B. tabaci (Guo et al. 2016; Peng et al. 2016). As the vectors that are identified by
NB-LRR protein domains of plants are limited, modifying these receptors in order to
expand the range of defendable vectors using transgenic methods can be a promising
strategy (Harris et al. 2013; Kim et al. 2016). The vector genes/transcripts encoding
effectors can be targeted by transgenic expression of cognate RNA molecules in

host, which is taken up by feeding invertebrates and silences the respective target.
Fusion protein consisting of luteoviral coat protein–spider toxin was expressed in
host, where viral coat protein moiety facilitates the uptake of toxin by vector
(Whitfield and Rotenberg 2015; Bonning et al. 2014). Disruption of transmission
of three groups of vectors – whiteflies, aphids and mealy bugs – was obtained by
phloem-specific expression of a spider-derived toxin and a lectin (Javaid et al. 2016).
Engineering plant as vectors of viruses, which are pathogenic on aphids and
leafhoppers, including Densoviruses (DNA) and Dicistroviruses (positive-sense
RNA), is a potential strategy, where host plants act as biocontrol agents. It was
tested using cricket paralysing virus, and it disrupted normal aphid responses to
olfactory cues from other aphids, causing them to scatter and predisposing them to
attack by predators and parasitoids or promote transition to winged morphs, enhanc-
ing virus dissemination (Kerr et al. 2015; Ban et al. 2008; Ryabov et al. 2009).
23.4 Transgenic Approach for Resistance Against Viroids
Viroids are small (~250 to 400 nt) non-protein-coding, circular, single-stranded

RNAs, which autonomously replicate through an RNA–RNA rolling-circle mecha-
nism that is catalysed by host enzymes (RNA polymerases, RNases and RNA
ligases) in the nucleus (Pospiviroidae), or in plastids, primarily chloroplasts
(Avsunviroidae). These small infectious nucleic acids can infect economically
important higher plants potato, tomato, citrus, hop and temperate fruits, like peach,
apple and pear. Systemic infection of viroids involves entry into specific subcellular
organelles, replication, exit of the organelles and cell-to-cell trafficking, access into
and long-distance trafficking within the vasculature and exit and invasion of non-
vascular tissue to restart the cycle. Interaction of the viroid RNA with cellular
proteins involved in its replication/trafficking or from defensive responses is trig-
gered by the host results in infection and disease development (Navarro et al. 2012;
Flores et al. 2005). As conventional control measures sound insufficient to offer
complete protection against these pathogens, transgenic approaches, like targeting
the viroid RNA for degradation or manipulating the host defensive response in order
to disrupt host–viroid interactions, may efficiently alleviate the disease symptoms. A
peculiar resistance mechanism, namely, cross-protection, wherein a previous infec-
tion from mild strain induces development of resistance against further infection
from a severely virulent strain, operates against viroids. The underlying molecular
mechanism of cross-protection can be sequence specific, which may be related to
RNA silencing (Niblett et al. 1978; Khoury et al. 1988; Flores et al. 2005).
As viroid replicates via dsRNA intermediates, yeast-derived dsRNA-specific
RNase pac1 has been engineered to target this dsRNA of PSTVd in potato, which
resulted in reduced infection and symptoms. Such RNAi approach can confer
additional resistance to some RNA viruses as well. These pac1 expressing transgenic
potato lines showed resistance to Tomato spotted wilt virus also (Flores et al. 2017).
Expression of a catalytic single-chain variable antibody (3D8 scFv) with intrinsic
RNase (and DNase) activities, in chrysanthemum plants, resulted in resistance to

CSVd infection. This strategy is advantageous in that antibody accumulates to very
low levels and antibody expression does not induce any phenotypic alteration, even
though it acts in sequence-independent manner, and protects plants not only against
CSVd but also confers resistance to DNA and RNA viruses (Lee et al. 2013; Tran
et al. 2016). Different trials for antisense RNA-mediated silencing of viroid genomic
RNA couldn’t establish a stable resistance strategy based on RNAi. Even so, CSVd
resistance was conferred by Agrobacterium-mediated transformation of a commer-
cial chrysanthemum cultivar with four different constructs carrying sense or anti-
sense CSVd-specific RNAs of 75–82 nt (Matoušek et al. 1994; Jo et al. 2015).
Although initial attempts to engineer hammerhead ribozymes to target the plus and
minus strands of CEVd did not confer viroid resistance in vivo, hammerhead
ribozyme with shorter recognition sequences (9–11 bases) engineered to target
PSTVd minus RNA suppressed viroid accumulation in transgenic potato lines
(Yang et al. 1997). Tertiary stabilizing motifs (TSMs) of hammerhead ribozymes
is suspected to play a critical role in their potential to cleave specific RNA in trans,
and this was substantiated when hammerhead ribozymes, with their tertiary
stabilizing motifs (TSMs) preserved, engineered to target PSTVd minus RNA
successfully interfered with systemic PSTVd infection in in transgenic
N. benthamiana. Target accessibility of the substrate and the subcellular
co-localization of ribozyme and substrate are major hurdles to extract the complete
potential of these small RNAs for crop protection (Flores et al. 2017). RNA
interference strategy for targeting viral genome can be applied to viroids also.
Transgenic tomato and N. benthamiana expressing hpRNA construct with near
full-length (340-nt) and specific truncated sequences of PSTVD, respectively,
showed resistance to this viroid. As RISC is active in cytoplasm, it may not interfere
with viroid replication and accumulation host in nucleus but can be targeted while
they move to cytoplasm for invasion into neighbouring cells (Schwind et al. 2009).
Overexpression of AGO also has found to attenuate viroid accumulation (Minoia
et al. 2014). Artificial small RNAs, artificial miRNAs (amiR-NAs) and synthetic
trans-acting siRNAs (syn-tasiRNAs) are another efficient transgenic strategy for
controlling viroids, when stably expressed in transgenic plants (Carbonell et al.
2014). Concurrent application of more than one strategy might have synergistic
effects, resulting in enhanced resistance to viroids.
23.5 Transgenic Technology for Resistance Against Fungi
Fungal pathogens cause several diseases in plants resulting in catastrophic effects on

crop yield (10% of global crop loss). Conventional control strategies include the
application of chemical fungicides and biocontrol agents, phytosanitation, crop
rotation, destruction of intermediate hosts, etc., which are inadequate to provide
complete protection from infection. Transgenic strategies can be adopted to confer/
enhance the resistance to fungal pathogens so that yield loss and disease manage-
ment expenses can be considerably reduced. Several plants naturally produce
antifungal compounds, and genes encoding these compounds or regulating their

biosynthesis can be manipulated to confer fungal resistance. Prime plant-derived
antifungal compounds are chitinase and glucanases, enzymes that degrade major
components of fungal cell wall, chitin and glucan, thereby imparting resistance
(Silva et al. 2018, 2019). Stacking of these two genes can yield enhanced resistance
against fungal pathogens. Transgenic carrot expressing chitinase and β1,3-
glucanase, together with AP24 gene, exhibited broad-spectrum resistance against
fungal pathogens (Ram and Mohandas 2003). Transgenic grape and cotton plants
constitutively expressing chitinase from Trichoderma species conferred resistance to
several fungal diseases (Rubio et al. 2015; Emani et al. 2003). Plant-derived
inhibitors of microbial cell wall degrading enzymes are other potential candidates
for engineering resistance. Transgenic expression of inhibitors of cell wall degrading
enzymes has conferred resistance against many dreaded fungal pathogens in crop
plants. Reduced susceptibility to necrotrophic fungus Botrytis cinerea has been
observed in PGIP overexpressing transgenic tomato, grapevine, tobacco and
Arabidopsis thaliana (Ferrari et al. 2012; Liu et al. 2017; Manfredini et al. 2005).
PGIP degrades microbial polygalacturonases and thereby delays plant cell pectin
hydrolysis, which in turn restricts fungal infection (Ferrari et al. 2013). Xylanase
inhibitor protein controls the xylanases, which degrades the main component of cell
wall, xylan, as in the case of effective inhibition and counteraction of the
F. graminearum necrotic xylanase activity by constitutive expression of Triticum
aestivum xylanase inhibitor III (TAXI-III) in GM wheat (Tundo et al. 2016).
Expression of pectin methyl esterase (PME) inhibitors in Arabidopsis could prevent
damage to the plant cell wall during Botrytis cinerea infection (Lionetti et al. 2017).
Transgenic strategies to control insect pests, including cry genes from Bacillus
thuringiensis, provide dual protection by reducing the chances of fungal infection
and mycotoxin (fumonisin, Fusarium spp.; aflatoxin ergotoxine, Claviceps spp.;
aflatoxins, Aspergillus spp.) contamination by reducing the rate of insect wounding.
This would reduce management costs and yield loss considerably. Overexpression
of PnAMP-h2 gene from Pharbitis nil and barley chitinase (chi-2) genes were found
to provide enhanced resistance to fungal pathogens. Antimicrobial peptides derived
from non-plant, non-phytopathogenic microbes can also be used for imparting
disease resistance as in the case of enhanced resistance to R. solani and soil-borne
pathogen A. alternata observed in transgenic cotton and tobacco plants expressing
endochitinase gene from a mycoparasitic fungus Trichoderma virens. Phytoalexin
capsidiol provided resistance to the potato blight pathogen, Phytophthora infestans
(P. infestans) in pepper (Capsicum spp.) (Lee et al. 2017) (Table 23.4).
Defensins are cysteine-rich antimicrobial peptide of plant origin, which is a
potential candidate to protect crop plants with phytopathogenic fungal infection by
their transgenic expression in plants. Constitutive expression of an alfalfa seed
defensin MsDef1 and NmDef02 defensin in potato provided strong resistance to
Verticillium dahliae and Phytophthora infestans, respectively (Gao et al. 2000).
Constitutive expression of defensins mostly causes undesirable side effects, includ-
ing reduced growth and yield performance (Chen and Chen 2002). Regulation of
defensin expression by using (native or heterologous) pathogen-induced promoter
Table 23.4 List of transgenic plants developed for resistance to fungal pathogens
Plant species Antifungal genes transferred Resistance against References
Rice Chitinase (chi11); glucanase Rhizoctonia solani Sridevi et al.
(gluc) (2008)
stress-inducible b-glucanase Magnaporthe grisea Nishizawa et al.
(Gns1) (2003)
Ribosome-inactivating protein Rhizoctonia solani Kim et al.
(MOD1) (2003)
Chitinase (RCH10)
Chitinases (RCH10 and Magnaporthe grisea Zhu et al.
RAC22); glucanase (2007)
(b-Glu); ribosome-inactivating
protein
(B-RIP)
ER-CecA; Ap-CecA (cecropin Magnaporthe grisea Coca et al.
A) (2006)
Dahlia merckii defensin Magnaporthe oryzae and Jha et al. (2009)
DM-AMP1) Rhizoctonia solani
Potato Chitinase (ChiC) Alternaria solani Khan et al.
(2008)
Chitinase (CHIT); glucanase Rhizoctonia solani Moravcikova
(GLUC) et al. (2007)
Moravcikova
et al. (2004)
Chitinase (BjCHI1); glucanase Rhizoctonia solani Chye et al.
(HbGLU) (2005)
Cationic peptide (msrA3) Phytophthora infestans Osusky et al.
Phytophthora (2004)
erythroseptica
Erwinia carotovora
RPI-BLB2 Phytophthora infestans van der Vossen
et al. (2005)
Tomato Glucanase (GLU); antifungal Ralstonia solanacearum Chen et al.
protein (alfAFP); (2006)
glucanase (GLU-AFP)
Carrot Chitinase383; glucanase638; Botrytis cinerea and Wally et al.
cationic peroxidise (POC1) Sclerotinia sclerotiorum (2009)
Taro Chitinase (ricchi11) Sclerotium rolfsii He et al. (2008)
Cucumber Chitinase (RCC2) Botryti cinerea Kishimoto et al.
(2002)
Nicotiana Polygalacturonase-inhibiting Botrytis cinerea, Joubert et al.
benthamiana protein from grapevine Bipolaris sorokiniana (2007)
Tobacco Polygalacturonase-inhibiting Phytophthora capsici Wang et al.
protein (PGIPs) (pepper) (2013)
(continued)

Plant species Antifungal genes transferred Resistance against References
Alfalfa RCT1 from (Medicago Colletotrichum trifolii Yang et al.
truncatula) (2008)
Potato RPI-BLB2 (Solanum Phytophthora infestans Van der Vossen
bulbocastanum) et al. (2005)
Wheat Fusarium Tri101 F. graminearum Okubara et al.
(2002)
Peanut Chitinase Cercospora arachidicola, Prasad et al.
Aspergillus flavus (2013)
like barley GER4c promoter or tissue-specific promoter, which mainly depends on

the infection biology and tissues affected, would be advantageous to avoid the
negative fitness effects associated with their constitutive expression (Himmelbach
et al. 2010; Rushton et al. 2002). Expression of antifungal defensins under root-
specific promoter confers resistance to sudden death syndrome in soybean, because
it is caused by a root-colonizing pathogen Fusarium virguliforme. Similarly,
lifestyle-specific expression of defensins by fine-tuning their subcellular localiza-
tion, which in turn depends upon whether pathogen is biotrophic or hemibiotrophic,
is also effective to confer durable resistance without compromising yield. Extracel-
lularly targeted defensins enable protection from biotrophic fungi, whereas extra-
and intracellularly targeted defensins should be coexpressed in order to confer
resistance to hemibiotroph. However, the effect of transgene encoded antimicrobial
compound on animal/mammalian system should be assessed before employing this
strategy for transforming edible crops. A detailed understanding of the underlying
molecular mechanisms of their mode of action would permit the development of
much sophisticated strategies for the regulated expression of antifungal defensins to
confer broad-spectrum durable resistance against many notorious fungal pathogens
(Kaur et al. 2011). Synergistic effects by coexpression of different plant defensins
with different mode of action or plant defensin with antifungal pathogenesis-related
protein (PR) or HIGS with plant defensins enhance resistance to a wide range of
fungal infection (Chen et al. 2009; Ntui et al. 2011).
Silencing of pathogen genes by RNAi-based host-induced gene silencing (HIGS)
is another efficient transgenic strategy to develop resistance. Resistance to Blumeria
graminis and F. verticillioides was achieved by HIGs of their endogenous gene and
fungal transgene, respectively (Nowara et al. 2010; Tinoco et al. 2010).
Plant resistance genes have extensively used for engineering resistance against
phytopathogenic fungi. Introgression of R genes from related or unrelated species is
facilitated by transgenic technologies. Overexpression of NPR1, a key regulator of
SAR, conferred resistance to blight and blast disease causing fungal and bacterial
pathogens, respectively (Chern et al. 2005; Yuan et al. 2007). Transgenic rice plants
overexpressing AtNPR1 gene and translational suppressor uORFs from the TBF1
gene displayed resistance towards bacterial and fungal pathogens and reduced
negative fitness costs associated with constitutive expression of NPR (Xu et al.
2017). In another case, introgression of the gene involved in non-host resistance,

Phytophthora soaje susceptible 1 (AtPSS1), from Arabidopsis into soybean by
transformation enhanced resistance to the soybean host fungal pathogen Fusarium
virguliforme, by a suspected autophagy mechanism without yield penalty (Wang
et al. 2018). Transgenic expression of R gene RPI-BLB2 from Solanum
bulbocastanum in cultivated potato showed resistance to Phytophthora infestans
(van der Vossen et al. 2005). Resistance conferred by single R gene is mostly
not/less durable as pathogen is smart enough to circumvent the resistance. Stacking
of multiple R genes, either targeting single or different pathogens, in single plant is
an efficient alternative to impart durable and/or broad-spectrum resistance against
destructive fungal pathogens. Robust transgenic strategies for gene pyramiding are
required for executing this. Host genes manipulated by fungus for apressoria forma-
tion and haustoria development can be a potential target to be modified in order to
confer pre-haustorial resistance. Arabidopsis PENETRATION genes involved in
haustoria establishment is a suitable candidate in that regard (Fonseca and Mysore
2019). Integrated transgenic approach combining resistance genes, defensins and
antifungal proteins is expected to confer effective durable resistance.
23.6 Transgenic Technology for Bacterial Resistance in Crop

Plants
Bacteria are causing many diseases in economically important plants, where it

severely impairs the growth, development and yield potential of plants. Plants
defend bacterial pathogens with common components of immunity, viz. R genes,
ETI-associated HR, antimicrobial compounds, inhabitants of bacterial enzymes, etc.
Transgenic approaches for engineering resistance to bacteria in plants can be
executed in the following ways: introduction of host R genes and bacterial
avirulence genes, incorporation of pathogen-derived genes for resistance to bacterial
phytotoxins and expression of antibacterial proteins from plants, insects or
bacteriophages as bactericidal or bacteriolytic agents (Panopoulos et al. 1996).
Transfer of maize R gene RESISTANCE TO XANTHOMONAS ORYZAE
1 (RXO1) to rice conferred enhanced resistance to the rice host pathogen
Xanthomonas oryzae pv. oryzicola causing bacterial streak disease. Similarly, single
R gene obtained from pepper was introgressed to tomato in order to control highly
destructive bacterial leaf spot (Zhao et al. 2004, 2005; Tai et al. 1999). Unravelling
novel candidate receptors or enzymes or structural proteins from bacteria that can be
targeted by transgenic methods will have promising results in controlling bacterial
diseases (Table 23.5).
Antimicrobial peptides of plant origin as well as those from heterologous
non-plant systems are efficient candidates for engineering disease resistance. Plant-
derived antimicrobial peptides contain cysteine-rich short amino acid sequence and
target the outer membrane structures. Overexpression of pepper antimicrobial
peptide-encoding gene CaAMP1 in Arabidopsis conferred broad-spectrum resis-
tance to bacterial, fungal and oomycete pathogens (Lee et al. 2008).
Table 23.5 List of transgenic plants developed for resistance to bacterial pathogens
Plant species Transgene Target species Reference
Nicotiana Magainin analogue, Myp30, P. syringae Li et al. (2001)
tabacum var. MSI-99 pv. tabaci De Gray et al.
Petit Havana (2001)
Tobacco Cecropin B, mutant (SB37, MB39) P. syringae pv. Huang et al. (1997)
Potato and synthetic (Shiva-1, D4E1) tabaci Arce et al. (1999)
Apple, Erwinia Norelli et al.
Poplar carotovora subsp. (1998)
atroseptica
Mentag et al.
E. amylovora
(2003)
Agrobacterium
tumefaciens and
Xanthomonas
populi
Tobacco LTPs (lipid transfer protein) Pseudomonas Sarowar et al.
syringae (2009)
pv. tabaci.
Arabidopsis Plant PRRs (RLPs) Pseudomonas Saur et al. (2016)
N. benthamiana (NbCSPR) syringae
Tomato R proteins (NB-LRR) pepper Bs2 Xanthomonas Horvath et al.
perforans (2012)
Tomato Pto X. campestris Tang et al. (1999)
pv. vesicatoria
Bs2 X. campestris Tai et al. (1999)
pv. vesicatoria
Tomato Plant antimicrobial defence Clavibacter Balaji and Smart
(tomato proteins (plant encoded defensins michiganensis (2012)
snakin-2) (PR-12), thionins (PR- 13), lipid
transfer proteins (PR-14), snakins,
cyclotides, knottins and hevein-
like proteins
Banana Hrap and Pflp (sweet pepper) X. campestris Tripathi et al.
pv. musacearum (2010, 2014),
Namukwaya et al.
(2012)
Rice Rxo1 X. oryzae Zhao et al. (2005)
pv. oryzicola
Npr1 X. oryzae Chern et al. (2005)
pv. oryzae
NH1 X. oryzae Yuan et al. (2007)
pv. oryzae
A significant reduction of fire blight symptoms in pear and partial resistance to

bacterial blight in tomato was achieved by expression of lactoferrin, a mammalian
glycoprotein (Malnoy et al. 2003). Attacin is another heterologous protein that has
been used for conferring resistance against Erwinia amylovora, causing fire blight in
transgenic apple (Aldwinckle et al. 2003). Transgenic expression of another protein,

pectate lyase 3 (PL3), in potato resulted in increased resistance to Erwinia soft rot
(Wegener 2002).
Cecropins are antimicrobial amphipathic peptides of 31–39 aa long and interact
with bacterial membranes or induce pore formation in membrane. Transgenic tomato
expressing high level of cecropin displayed enhanced resistance against bacterial
speck disease. An increased resistance to bacterial blight in rice was engineered by
targeting cecropin towards intercellular species (Alan et al. 2004; Jan et al. 2010).
Increased expression and accumulation of ROS, hydrolytic enzymes, antimicro-
bial peptides and proteins in transgenic host plant can enhance resistance, including
HR, to bacterial pathogens and avoid the energy and resource loss in inducing
immune responses, which in turn enable plant to channelize the saved energy and
nutrients/metabolites into growth and development process so that yield is not
compromised. Resistance to several bacterial and fungal pathogens has been
achieved by transgenic expression of genes encoding cell wall degrading hydrolytic
enzymes, defence-related proteins with protease inhibitor activity, etc. (Shin et al.
2008; Senthilkumar et al. 2010).
23.7 Genome Editing Techniques for Engineering Disease

Resistance
Genome editing technology, which emerged in the 1990s, enabled targeted muta-
genesis of genomic loci of interest by exploiting cell’s intrinsic DNA repair
pathways. Site-directed nuclease fused with sequence-specific DNA-binding protein
domains or RNAs creates double-stranded breaks in target genomic site. DSBs thus
formed are subsequently repaired either by error-prone DNA repair pathway
non-homologous end joining (NHEJ) or high-fidelity homology-directed repair
pathway (HDR). NHEJ usually results in point mutations at the site of DSB, and
HDR enables replacement or introduction of a DNA fragment, while a repair
template is provided. Mutations thus induced are stably inherited over generations.
Genome editing techniques are useful for functional genomic studies as well as for
creating or modifying desired phenotype or trait in organism of interest.
Meganucleases, zinc finger nucleases (ZFN), TALENs and CRISPR/Cas are the
commonly used genome editing tools. ZFNs and TALENS are fusion proteins in
which specific DNA-binding proteins domains are fused with endonuclease domain
of FokI nuclease, so that protein guides FokI to respective DNA targets. CRISPR/
Cas9, in contrast, is an RNA-guided engineered nuclease, where a single guide RNA
with 50 target-specific 20 nt spacer directs Cas nuclease to target DNA. CRISPR/Cas
is a part of bacterial adaptive immune system, providing resistance to invading
viruses or nucleic acids. There are different classes of CRISPR/Cas systems, of
which dsDNA targeting class 2, type II system seen in Streptococcus pyogenes,
popular as CRISPR/Cas9 is the most common CRISPR system exploited for genome
editing. Several variants of CRISPR systems and Cas protein have been discovered,
each of which varies in their target recognition and cleavage properties.
Genome editing techniques have been successfully employed to develop resis-

tance against bacterial, fungal and viral pathogens. ZFNs conferred broad-spectrum
resistance to various Begomoviruses, including Tomato yellow leaf curl China virus
(TYLCCNV) and Tobacco curly shoot virus (TbCSV) by targeting a single site in
viral genome (Chen et al. 2014). Modified ZFN called as artificial zinc finger protein
(AZP), which lacks the cleavage domain compared to ZFN, has been used to confer
resistance to Beet severe curly top virus (BSCTV, family Geminiviridae) and Rice
tungro bacilliform virus (RTBV) in Arabidopsis by targeting the intergenic region
(IR) of BSCTV and blocking viral promoter sequences of RTBV (Ordiz et al. 2010).
TALEs have been engineered to confer resistance to TbCSV and TYLCCNV in
tobacco (Cheng et al. 2015). Complexity of protein engineering and off-target effects
limited the use of these modular protein toolboxes. CRISPR/Cas9, whose genome
editing potential was revealed recently only, quickly superseded and emerged as a
promising tool for crop improvement with high efficiency, precision, robustness and
simple designing strategy.
CRISPR/Cas9 has been widely used for engineering disease resistance in many
crop plants by targeting pathogenic as well as host factors. Pathogenic genes that are
inevitable for survival and infection cycle are appropriate candidate targets for
CRISPR/Cas9-mediated knockout. Adaptive potentials enable pathogens to over-
come the dominant R gene-mediated resistance. Thus, susceptibility genes are
considered as potential candidates to be targeted in order to develop durable resis-
tance. Disruption of susceptibility genes or S proteins by CRISPR/Cas9-mediated
targeted mutagenesis has been successfully demonstrated. The first report of the
application of CRISPR/Cas9 for virus resistance came when double-stranded repli-
cative from geminiviral DNA was targeted to confer resistance. Later, this strategy
was used to provide resistance to several DNA viruses. Apart from the demonstra-
tion for proof of concept of the CRISPR/Cas9 in model plants, highly efficient
resistance against different viral, bacterial and fungal pathogens has been obtained in
various crops, including major food crops like wheat and rice (Table 23.6, 23.7 and
23.8). For instance, mutagenesis of ERF transcription factor (OsERF922) gene using
CRISPR/Cas9 imparted resistance in rice against fungal pathogen Magnaporthe
oryzae (Wang et al. 2016). Similarly, CRISPR/Cas9-induced mutation in
homeoalleles of mildew resistance locus (MLO), encoding a transmembrane protein,
and enhanced disease resistance 1 (EDR1), encoding a Raf-like mitogen-activated
protein, conferred resistance to powdery mildew in hexaploid wheat (Wang et al.
2014). CRISPR/Cas9-mediated loss-of-function mutation in eukaryotic translation
initiation factor eIF4e, which is essential for infection of Potyviridae family viruses,
conferred resistance against potyvirus (Zucchini yellow mosaic virus and Papaya
ringspot mosaic virus-W) and Ipomovirus in Arabidopsis and cucumber (Cucumber
vein yellowing virus) (Chandrasekaran et al. 2016). CRISPR/Cas9-mediated targeted
mutagenesis of promoter sequence of susceptibility gene CsLOB1 conferred resis-
tance to bacterial pathogen Xanthomonas citri subsp. citri in citrus (Peng et al.
2017). This tool has also been shown to be effective to control dsDNA viruses, as in
the case of resistance to Cauliflower mosaic virus (CaMV) in Arabidopsis (Liu et al.
2018).
23
Table 23.6 Crop plants harnessing CRISPR/Cas-mediated resistance to viruses

GM/
Target transgene-
Virus/viruses Plant (viral/host) Function Strategy free References
BSCTV N. benthamiana IR, CP and RCA mechanism Agrobacterium-mediated GM Ji et al. (2015)
and A. thaliana rep transformation of leaves with
Cas9/gRNA expression
plasmid vectors
BeYDV N. benthamiana LIR and RCA mechanism Agrobacterium-mediated GM Baltes et al.
rep/RepA transformation of leaves with (2015)
Cas9/gRNA expression
plasmid vectors
TYLCV, BCTV and N. benthamiana IR, CP, and RCA mechanism Agrobacterium-mediated GM Ali et al. (2015)
MeMV rep transformation of leaves with
a TRV vector in Cas9
overexpressing plants
CLCuKoV, TYLCV N. benthamiana IR, CP, and RCA mechanism Agrobacterium-mediated GM Ali et al. (2016)
2.3, TYLCSV-Logan, rep transformation of leaves with
BCTV-Worland, a TRV vector in Cas9
MeMV overexpressing plants
TuMV A. thaliana Host factor Host factor for RNA virus Agrobacterium-mediated Transgene- Pyott et al.
eIF(iso)4E translation transformation with Cas9/ free (2016)
Transgenic Technology for Disease Resistance in Crop Plants
gRNA recombinant plasmid

binary vectors (floral
dipping)
CVYV, ZYMV and Cucumis Host factor Host factor for RNA virus Agrobacterium-mediated Transgene- Chandrasekaran
PRSMV sativus eIF4E translation transformation of cut free et al. (2016)
cotyledons (without embryo)
with Cas9/gRNA binary
vectors
(continued)
525
526
GM/
Target transgene-
Virus/viruses Plant (viral/host) Function Strategy free References
CMV, TMV Nicotiana ORF1, 2, 3, Replication mechanism Agrobacterium-mediated GM Zhang et al.
benthamiana CP and transformation of leaves with (2018)
and 30 UTR FnCas9/gRNA expression
Arabidopsis binary vectors floral dipping
thaliana for Arabidopsis
RTSV Oryza sativa eIF4G Host factor for RNA virus Agrobacterium-mediated GM Macovei et al.
L. japonica translation transformation of immature (2018)
embryos with Cas9/gRNA
expression plasmid vectors
TYLCV Solanum CP, rep RCA mechanism Agrobacterium-mediated GM Tashkandi et al.
lycopersicum transformation of cotyledons (2018)
with Cas9/gRNA expression
vectors
TuMV Nicotiana GFP1, Replication mechanism Agrobacterium-mediated GM Aman et al.
benthamiana GFP2, transformation of leaves with (2018)
HC-Pro, CP a TRV vector in Cas13a
Endogenous Banana Musa ORF 1, 2 Transcription or/and Agrobacterium mediated GM Tripathi et al.
streak virus (eBSV) balbisiana and translation/post- transformation of cell (2019)
3 (aspartic translational modification suspension culture with
protease of vital viral proteins CRISPR/Cas9 construct
gene)
T. Makeshkumar et al.
23
Table 23.7 Crop plants harnessing CRISPR/Cas-mediated resistance to bacterial pathogens

Disease and causative Target
Plant species bacteria gene Gene function Strategy Reference
Oryza sativa Bacterial blight SWEET13 Sucrose transporter gene Agrobacterium-mediated Li et al. (2012),
(Xanthomonas oryzae transformation of embryogenic callus Chen et al.
pv. oryzae) with Cas9/gRNA expression plasmid (2012), Zhou et al.
vectors and TALEN (2015)
Citrus Citrus canker LOB1 Susceptibility (S) gene Agrobacterium-mediated Jia et al. (2016)
paradisi (Xanthomonas citri promoting pathogen growth transformation of epicotyl with Cas9/
subspecies citri) and pustule formation gRNA expression plasmid vectors
Citrus Citrus canker LOB1 Susceptibility (S) gene Agrobacterium-mediated Peng et al. (2017)
sinensis (Xanthomonas citri promoting pathogen growth transformation of epicotyl with Cas9/
Osbeck subspecies citri) and pustule formation gRNA expression plasmid vectors
Malus Fire blight (Erwinia DIPM-1 Susceptibility factor PEG-mediated protoplast Malnoy et al. (2016)
domestica amylovora) DIPM- involved in fire blight transformation with CRISPR
2 DIPM-4 disease ribonucleoproteins
Solanum Pseudomonas syringae and DMR6 Susceptibility (S) gene Agrobacterium-mediated Thomazella et al.
Transgenic Technology for Disease Resistance in Crop Plants
lycopersicum Xanthomonas spp. involved in downy mildew transformation of cotyledons with (2016)
disease Cas9/gRNA expression vectors
Tomato bacterial speck JAZ2 Regulation of stomatal Agrobacterium-mediated Ortigosa et al.
disease (Pseudomonas opening by coronatine transformation of cotyledons with (2018)
syringae pv. tomato (Pto)) Cas9/gRNA expression vectors
527
528
Table 23.8 Crop plants harnessing CRISPR/Cas-mediated fungal resistance

Disease and target Target
Plant species species gene Gene function Strategy Reference
Vitis vinifera Powdery mildew MLO-7 Susceptibility (S) gene PEG-mediated protoplast transformation with Malnoy et al.
(Erysiphe necator) involved in powdery CRISPR ribonucleoproteins (2016)
mildew disease
Oryza sativa Rice blast disease ERF922 Transcription factor Agrobacterium-mediated transformation of Wang et al. (2016)
L. japonica (Magnaporthe oryzae) implicated in multiple stress embryogenic calli with Cas9/gRNA
responses expression binary vectors
Oryza sativa Rice blast disease SEC3A Subunit of the exocyst Protoplast transformation with Cas9/gRNA Ma et al. (2018)
L. japonica (Magnaporthe oryzae) complex expression binary vectors
Solanum Powdery mildew MLO1, Major responsible for Agrobacterium-mediated transformation of Nekrasov et al.
lycopersicum (Oidium PMR4 powdery mildew cotyledons with Cas9/gRNA expression (2017), Koseoglou
neolycopersici) vulnerability plasmid vectors (2017)
Triticum Powdery mildew MLO- Susceptibility (S) gene Particle bombardment of immature wheat Wang et al. (2014)
aestivum (Blumeria graminis A1 involved in powdery embryos with Cas9/gRNA expression plasmid
f. sp. tritici) mildew disease vectors
Theobroma Black pod disease NPR3 Regulator of the immune Agrobacterium-mediated transient Fister et al. (2018)
cacao (Phytophthora system transformation of stage C leaves with Cas9/
tropicalis) gRNA expression binary vectors
T. Makeshkumar et al.
Loss-of-function mutations created by CRISPR/Cas9 is useful for identifying

resistance and susceptibility-determining genes and corresponding regulatory
elements like promoters or enhancers. As the NHEJ-induced mutations are stably
inherited and the CRISPR/Cas expression cassettes can be eliminated in successive
generations, the resulting plant remains transgene-free and thus overcomes the
regulatory issues associated with Agrobacterium transformed conventional trans-
genic plants. Delivery of CRISPR/Cas9 constructs as ribonucleoprotein (RNP)
complex composed of sgRNA and Cas9 protein into host plant also yields
non-transgenic edited plant with disease resistance. Non-transgenic tomato, resistant
to powdery mildew, has been created in this manner (Nekrasov et al. 2017). Multiple
DNA sequences can be targeted simultaneously by designing gRNA cassettes using
golden gate cloning/tRNA: gRNA/gRNA: ribozyme assembly strategy. The ease of
multiplexing is highly beneficial for conferring host with sound resistance against
single pathogen as well as with broad-spectrum resistance. Multiple regions of
pathogen genome can be targeted to alleviate/reduce the possibilities for the evolu-
tion of cleavage or recognition resistant variants by overcoming mutations at single
site. Although incorporation of resistance gene from foreign species can also be
performed by inducing HDR repair pathway of DSB created, efficiency of such
events is very less and still needs to be optimized for obtaining high rate of HDR.
Cisgenic applications of genome editing can be useful for enhancing disease resis-
tance in species, which are difficult to hybridize by introducing a desirable gene
fragment from its natural gene pool using CRISPR/Cas9-induced HDR-based path-
way. Genetic engineering approaches manipulating endogenous genetic variations
related to specific traits are preferred to exogenous genomic targeting recombinant
DNA technology.
Apart from dsDNA targeting Cas9, there are certain CRISPR systems with RNA
targeting properties, which can be engineered to confer resistance to RNA viruses or
silencing of viral transcripts in host. Class II, type VI Cas effector proteins, Cas13a
(C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d and Cas9 from Francisella novicida
(FnCas9), RNA targeting SpCas9 (RCas9), are the RNA-guided RNA targeting Cas
effector variants identified in different microbial genomes so far (Abudayyeh et al.
2017). Expression of Fncas9 and sgRNA targeting conserved 30 UTR of Cucumber
mosaic virus (CMV) and Tobacco mosaic virus (TMV) developed stable immunity
in N. benthamiana against CMV and/or TMV (Zhang et al. 2018). CRISPR-Cas13a
was used to confer resistance to Turnip mosaic virus (TuMV) in N. benthamiana by
targeting Hc-Pro and GFR regions of genome (Aman et al. 2018).
A class II, type V CRISPR/Cpf1 system that efficiently target DNA was discov-
ered from Prevotella and Francisella species and named as Cas12a (Zetsche et al.
2015; Makarova et al. 2015). Cpf1 from Acidaminococcus sp. BV3L6 Cpf1
(AsCpf1) and Lachnospiraceae bacterium Cpf1 (LbCpf1) are other two promising
nucleases that can be used for genome editing purposes (Zetsche et al. 2015).
Gene expression regulation and epigenetic modification is also facilitated by
mutant Cas9, which lacks cleavage activity. Transcriptional activators or repressors
can be recruited to a particular gene of interest by fusing them with dCas9, which
then is directed by specific sgRNA to the target site. Similar strategy can be adopted
for editing epigenetic marks using appropriate epigenetic modifiers (Dominguez

et al. 2016). This enable fine-tuning of defence regulatory pathways and metabolic
pathways involved in immune response. Transcriptional activation of positive
regulators of immunity and biomolecules like pathogenesis-related proteins or
hydrolytic enzymes, which are directly involved in defence manifestation, repres-
sion of negative regulators of immunity, etc., can be achieved using CRISPR/dCas9
system. Resistance traits that are unexpressed due to epigenetic suppression can be
activated by reversing the methylation and similarly epigenetically controlled sus-
ceptibility also modified. Tissue and developmental stage or stress status specific
activity of CRISPR components and target editing events can be achieved by
employing tissue-specific/inducible promoters for the expression of Cas effector
and guide RNA. This is particularly beneficial for reducing unwanted or off-target
editing.
Genome-wide mining of S genes may unravel novel candidate S genes that can
confer resistance, durable as well as broad-spectrum, without having any fitness
costs. Several trace elements, like Fe, Ca, Cu, K, Mn and Zn, are required by
pathogens to meet their nutritional requirements, whereas the same are required in
host plants for nutrition as well as to activate/drive certain critical defence responses
like Cu-dependent binding of SA binding to its NPR1 receptor (Yuan et al. 2010).
Coordinated activities of plant immune signalling pathways to inhibit/suppress
bacterial iron acquisition mechanisms substantiated the role of Fe in establishing
infection (Nobori et al. 2018). Pathogens compete with host to acquire these mineral
nutrients, which is counteracted by plant, by limiting their availability to invading
pathogens by a mechanism called as nutritional immunity. Pathogenic mineral
transporters strive to acquire Fe, Mn, Zn, K and Cu, which are essential for their
virulence/survival, from the host, leaving host deprived of these trace elements and
thereby inhibiting immune responses that require these minerals (Ren et al. 2016;
Hood and Skaar 2012). Thus, such trace element transporters or their acquisition
mechanisms can be potential nutritional immunity-related S gene targets for genome
editing (Zaidi et al. 2018).
Complete knockout of S genes mostly has negative fitness effects, like reduced
growth, yield and fertility; early senescence; and reduced tolerance to abiotic stress,
as most of them being primary in function. Even though fitness cost is not lethal, it
can cause phenotypic abnormalities. Alternative strategies, like introduction of S
gene variant, creation of intermediate alleles by promoter targeting, transient knock-
out of S genes by using pathogen-inducible promoter, etc., using CRISPR/Cas9
would efficiently confer resistance, avoiding any cost of fitness. Identification of S
gene allelic variants, which can confer resistance, leads to create resistant S gene
variant by specifically editing the respective SNP using CRISPR/Cas9 base editors
(Rodríguez-Leal et al. 2017; Yan et al. 2018).
Even though the characteristics, like specificity, efficiency, simplicity, flexibility,
etc., of this genome editing tool are highlighted, several shortcomings and
complications, including off-target effects, low rate of successful transformation
events and recalcitrance for regeneration and establishment of edited plants, are
associated with CRISPR/Cas. Each stage of experiment, from the selection of target
sequence to stable integration of mutations, needs to be optimized for each and every
species for utilizing maximum potential of this genome editing tool for disease
resistance.
23.8 Strategies Based on the Mechanism of Plant–Pathogen

Interaction
Plants are constantly subjected to stress from various biotic agents/pathogens, like
virus, bacteria, fungi, oomycetes, nematodes, insects and parasitic plants, that are
causing serious crop loss. Plant–pathogen interaction studies have revealed various
components involved in immunity and their mechanism of action during defence
response. Interaction is specific characteristic of each group of pathogens. A vast
array of well-programmed defensive mechanisms driven by multiple biomolecules
which are involved in each stage, viz. invasion of pathogen into host, recognition
and initiation of immune response, active deployment of defensive strategies and
establishment of resistance, together constitute plant immunity. Pathogen-derived
signals during infection process is perceived by specific receptors, mostly located in
the cell membrane, which then trigger a cascade of defence activities/pathways
making use of a large spectrum of biomolecules and molecular mechanisms (Silva
et al. 2018).
Plant innate immunity has organized as a three-layered system with pathogen-
triggered immunity (PTI), effector-triggered susceptibility (ETS) and effector-
triggered immunity (ETI). The first active line of plant immunity is PTI, which is
triggered when pathogen-associated molecular patterns (PAMP), together with this
damage-associated molecular patterns (DAMP) released during pathogen invasion
and damage, are recognized by transmembrane pattern recognition receptors (PRRs)
(Boutrot and Zipfel 2017; Uma et al. 2011). PTI is suppressed by effector-triggered
susceptibility (ETS) induced by pathogen-derived susceptibility proteins/effector
and results in infection (Jones and Dangl 2006; Chisholm et al. 2006). This activates
the second line of defence, effector-triggered immunity induced by recognition of
specific effectors/cognate factors, pathogen avirulence (Avr) proteins by another
group of receptors encoded by resistance genes (R). A co-evolutionary gene-to-gene
molecular arm race occurs between pathogen effectors and host R genes, while
pathogen evolves a new effector to restore the compatible interaction to facilitate
infection, parallel to which host evolves a new R protein, to strengthen immunity.
PTI is conserved over a range of organisms, whereas ETI specific to Avr protein is
produced by each organism. ETI mostly/usually activates localized cell death path-
way, otherwise known as hypersensitive response (HR), to restrain infection by
transmitting defence signals to neighbouring non-infected cells via plasmodesmata
and to other systemic organs via phloem, resulting in distal resistance responses,
namely, local acquired resistance (LAR) and systemic acquired resistance (SAR),
respectively (Coll et al. 2011; Dangl and Jones 2001). Like HR, ETI responses also
involve production of salicylic acid (SA), reactive oxygen species (ROS), necrosis
and also structural changes, such as lignification and callose deposition. Various
biomolecules, like pathogenesis-related proteins (PR), antimicrobial peptides

(AMP), ribosome-inhibiting proteins (RIPs), defensive secondary metabolites, etc.,
are produced by plants as a part of defence response (Kachroo and Robin 2013;
Dempsey and Klessig 2012). Increasing the cytosolic calcium levels in response to
exogeneous signals, like H2O2/PAMP/DAMPs, etc., produced during infection is
sensed by cellular and membrane calcium receptors, which in turn transduce signals
to elicit defence responses, like HR, ROs production, transcriptional regulation of
stress responsive genes, etc. (Seybold et al. 2014; Silva et al. 2018).
Incompatible interaction between non-host species and pathogen results in immu-
nity against non-host pathogen in non-host plant. Preinvasive NHR is mostly passive
mainly based on different physical and chemical barriers to interfere proliferation
and accumulation of pathogen. Sometimes, NHR involves active defence response
mediated by incompatible R–Avr gene-to-gene interaction result in ETI, and this
shows that common mechanisms converge at some points in NHR and HR (Flor
1971; Glazebrook 2005; Gill et al. 2015). Prolonged interaction between host and
pathogen occurs, wherein pathogens are under constant dual selection pressures, for
increased resistance from host and on pathogen for increased performance of
pathogen, which results in co-evolution of pathogen and host with virulence
specificities on specific hosts (Allen et al. 2004). Such co-evolved pathogens become
unable to infect phylogenetically unrelated hosts, resulting in non-host resistance,
which is highly beneficial to control pathogens. NHR is a quantitative trait with
multiple genes and pathways, whereas HR has specific R gene (Senthil-Kumar and
Mysore 2013). Based on plant response, NHR is considered to be of two types. Type
1 NHR involves no visible symptom as pathogen fails to penetrate tissues. It is
manifested by different physical, chemical and metabolical barriers that block
pathogen penetration without activating ETI and PTI. Type II NHR involves some
degree of pathogen penetration or entry in plant tissue, not as strong as a susceptible
host pathogen infection but in a dose sufficient to trigger HR (cell death), and
involves ETI. During post-invasive NHR, some pathogens penetrate host by
overcoming PTI responses and involve activation of defence responses, like HR,
ROS, cell death or the formation of cell wall appositions in infected cells (Collins
et al. 2003; Rojas et al. 2012).
23.9 Enhancing Immunity: Transgenic Approaches

to Manipulate Plant Innate Immunity
23.9.1 Upregulation of Defence Pathways
Upregulation of molecules involved in defence regulation, signalling and other allied

cellular processes can boost hosts’ general immune responses, including ROs
production, callose deposition, PR proteins and activation of SAR, etc. Resistance
to several microbial (bacterial and fungal) pathogens has been achieved by
employing this strategy without introducing new metabolic pathway or new gene
but exploiting plant’s own immune system. Resistance to Rhizoctonia solani and
Magnaporthe oryzae (rice blast causative fungi) has been achieved by expressing a
native rice gene under the control of a constitutive promotor from maize (Bundó and
Coca 2016; Chen et al. 2016; Vincelli 2016). Genome editing techniques, like
CRISPR/cas9-mediated transcriptional regulation, can be a potential strategy to
serve this purpose.
23.9.2 Production of Antimicrobial Compounds
Microbial pathogens secrete different cell wall degrading enzymes (CWDE) like
cellulases, polygalacturonases, xylanases, xyloglucan endoglucanase, chitinases and
protease inhibitors to damage cell wall for making their way into host cells. In order
to combat CWDEs, plants initiate cell wall strengthening/damage preventing
mechanisms, like production of polygalacturonase-inhibiting proteins (PGIPs),
xylanase-inhibiting proteins (XIPs) and xyloglucan-specific endoglucanase-
inhibiting proteins (XEGIPs) (Schüttelkopf et al. 2010; Xu et al. 2011). As these
mechanisms are mostly evolutionarily conserved, their manipulation could confer
durable and broad-spectrum resistance in many crop species. Reduced susceptibility
to Phytophthora capsici by expressing a pepper PGIP in GM tobacco; resistance to
Verticillium and Fusarium wilts by expressing protein GhPGIP1 in Arabidopsis and
cotton; resistance to Phytophthora sojae in soybean by constitutive expression of
elevated levels of a Glycine max XEGIP (GmGIP1), an inhibitor of a Phytophthora
sojae, xyloglucan-specific endoglucanase (PsXEG1); etc. have proved the efficacy
of enhancing plant’s own immune mechanisms by transgenic strategies to confer
resistance (Silva et al. 2013; Liu et al. 2017; Wang et al. 2013; Ma et al. 2015).
23.9.3 Enhancing Plant Recognition of Infection
PTI results from perception of self-derived DAMPs and non-self PAMPs signals by
PRR comprising receptor like kinases and receptor like proteases, whose active
domains include leucine-rich repeats (LRR), lysine M domain (LysM), epidermal
growth factor (EGF) like domain and lectin motif (Wu and Zhou 2013; Silva et al.
2018). Transfer and expression of PRRs in heterologous species by transgenic
methods broaden the range of pathogens that can be recognized by the host while
plant’s own defence system or metabolic pathways are left unmodified. Expansion of
pathogen recognition window is of prime importance in field conditions, where
multiple infections prevail. Even though certain microbes are highly adapted with
species-/race-/strain-specific PAMPs and have specific residues/motifs to block
perception by PRRs, most of the PAMPs are conserved across microbial species,
and thus individual PRR genes can confer broad-spectrum resistance (Zipfel and
Felix 2005). Manipulation of PRRs and other receptors or downstream components
involved, from a non-host species or unrelated family, is useful for boosting plant
immunity as it will be difficult for pathogen to overcome the resulting new
PRR/PAMP recognition system and the downstream signalling pathways.
Resistance to Xanthomonas citri in transgenic Hamlin sweet orange expressing

NbFLS2 and resistance to Pseudomonas syringae in Arabidopsis expressing Nicoti-
ana benthamiana receptor-like protein are required for Csp22 responsiveness
(NbCSPR); NbCSPR exemplifies the effectiveness of transgenic expression of
PRRs, even in heterologous system (Hao et al. 2016). Similarly, the transgenic
expression of AtEFR in tomato (against P. syringae pv. syringae, Ralstonia
solanacearum and Xanthomonas perforans), tomato PRR Ve1 in Arabidopsis and
the Arabidopsis PRR AtEFR-Tu in wheat and potato against bacterial wilt
(BW) causing soil-borne pathogen Ralstonia solanacearum is also expected to
confer durable and effective resistance to microbial pathogens (Fradin et al. 2011;
Lacombe et al. 2010; Schoonbeek et al. 2015). As most of the PAMPs are conserved
and metabolically vital molecules of pathogen, it may not evolve quickly along with
transferred PRRs in order to widen the host range.
23.9.4 Targeting Susceptible Gene/Protein
PTI is modulated or suppressed by the binding of pathogen effector proteins to host

S proteins and thereby triggering ETS, which facilitate pathogen’s invasion and
establishment. Plant S genes are responsible for susceptibility to pathogen, ETS
establishment and supporting compatibility. Molecular mechanisms associated with
S genes are exercised in three levels: basic compatibility or early establishment in
preinvasive stage when recognition and invasion occurs, sustained compatibility
during which proliferation and spread of pathogen occurs and, finally, negative
regulation of immune signals via respective signalling during post-penetration
stage (van Schie and Takken 2014; Pavan et al. 2010). S proteins can either be
positive regulators of immunity by promoting infection and disease development or
negative regulators of immunity, and S gene products either facilitate pathogen’s
growth in host system or negatively regulate the plant immunity (Silva et al. 2018).
Disarming/dysfunctioning, such susceptibility genes, by knockout or loss-of-func-
tion mutation is an efficient strategy to develop immunity, and the resulting resis-
tance is mostly recessive and may be associated with pleiotropic effects and negative
fitness costs, which may be due to constitutive activation of defence response.
Durable and broad-spectrum resistance to powdery mildew in different plant species
has been conferred in the same manner, by mutating mildew resistance locus O
(MLO). Effector targets in host are activated by pathogenic effectors, which then
negatively regulate plant immunity. S gene-mediated resistance can be pathogen
specific, if the impaired pathway is inevitable for pathogen in any of the
pre-penetration/penetration/post-penetration events, and it can be broad-spectrum,
if the target S gene has implications in constitutive defence response. Another point
to be noted while S proteins are encoded by multiple S genes is that all of them need
to be targeted simultaneously; otherwise, the feasibility of same also should be
checked. S gene LOF confers plants with broad-spectrum durable resistance than
dominant R gene-mediated resistance as this is race/pathovar/strain non-specific. In
order to overcome a modified or silenced susceptibility gene conferring host-
pathogen compatibility, pathogen should acquire a new function to replace the host
factor it was exploiting, which is a tough task.
Eukaryotic translation initiation factors eIF4e and eIF4g usually interact with the
cap structure of transcripts. Upon Potyviridae family virus attack, this interaction is
disrupted and initiates interaction with Vpg proteins from virus and facilitates
translation of viral proteins in host cell, thereby eIF4e and eIF4g act as negative
regulators of plant immunity (Zhang et al. 2006; Callot and Gallois 2014). Similar
factor rwm1 has been identified in watermelon under Cucumber mosaic virus
infection (Ouibrahim et al. 2014). Loss-of-function mutation of such factors confers
recessive resistance towards respective pathogens and is advantageous as far as it
does not cause any serious pleiotropic effect. Sugar efflux from the host cell is
activated by TAL activator from Xanthomonas oryzae, by binding to promoter
region of SWEET14 gene in order to nourish bacterial pathogen (Yuan and Wang
2013; Boch et al. 2009; Chen et al. 2012; Li et al. 2012). Since this may have an
adverse impact on plant health and agronomic performance due to altered sugar flux,
modification of binding sites or specific residues of SWEET14 gene render is
non-accessible or useless for pathogen. This can be accomplished by CRISPR/
Cas9 genome editing tool.
23.9.5 Mining of R Genes
Overexpression or transfer of resistance genes from other related/unrelated species

can confer durable broad-spectrum resistance. This is particularly advantageous in
some crop species, where natural R gene is lacking or endogenous R genes fail to
confer effective broad-spectrum resistance to most dreaded pathogens. In such cases,
resistance genes can be introgressed from distantly related or even from unrelated
species by genetic engineering, which in any way is impossible with conventional
breeding. Conventional breeding itself is a hurdle in the case of polyploid crops, like
potato, grape, banana, apple, etc. In such cases, the useful Cis genes also can be
introgressed by genetic engineering, by avoiding major drawbacks of breeding
techniques, viz, time consumption and linkage drag (Jones et al. 2014). Resistance
to potato late blight has been achieved by this strategy (Jo et al. 2014). Though
resistance genes efficiently confer resistance, their durability in heterologous hosts,
over the course of time in field conditions, is limited. Since R gene action is strain-/
race-specific, it confers species/subspecies level durable, broad-spectrum resistance,
particularly in phylogenetically related hosts by heterologous expression of R genes
(Eg: Bs2 gene). GM tomato plants expressing Bs2, an R gene from pepper that
recognizes AvrBs2 present in some Xanthomonas campestris pathovars, were
conferred with resistance against Xanthomonas perforans (Horvath et al. 2012).
The efficacy of R genes in heterologous systems in conferring disease resistance has
been demonstrated by similar transgenic plants. Bs2 gene functions as a solid source
of resistance to different Xanthomonas species and X. campestris pathovars (e.g. pv.
vesicatoria (Xcv)) in multiple solanaceous species. Co-transformation of R genes
with cognate Avr gene can be followed by stacking of heterologous R genes, once
the R gene becomes able to overcome phytopathogen. Such interspecific R gene

transfer confers host with broad-spectrum resistance in unrelated host as well
(Gururani et al. 2012; Horvath et al. 2012). Overexpression of certain post-invasion-
induced non-host resistance genes (PINGs) and bright trichomes (BRT1) genes that
encode UDP glycosyltransferase 84A2, both of which have been found to be
involved in NHR, conferred enhanced resistance against P. pachyrhizi in soybean
(Langenbach et al. 2016). It has been found that expression of translational repressor
domain from the TBF1 gene (uORFs), along with the defence-related gene, can
circumvent the negative fitness costs associated with expression of defence-related
genes in plants. For example, transgenic rice plants overexpressing the AtNPR1
gene and translational suppressor uORFs from the TBF1 gene displayed the
expected resistance phenotype towards bacterial and fungal pathogens and reduced
negative fitness costs associated with constitutive expression of NPR (Xu et al.
2017).
The principal functional domains of R genes are Toll/interleukin-1 receptor
(TIR), nucleotide-binding site (NBS), leucine-rich repeat (LRR), LRR transmem-
brane domain (TrD), coiled coil (CC), nuclear localization signal (NLS), mitogen-
activated protein kinase (MAPK) and MAPK kinase (MAPKK) domains. R proteins
are classified into eight groups based on the arrangement of functional elements, and
the major subset among them is LRR-NBS protein (LRR interact to Avr proteins
with their highly variable, specificity-determining C- terminus. Mostly, R–Avr
interactions occur via intermediate molecules regarded as effector targets. Mutation
of host receptors mostly by altering or modifying functional domains by transgenic
methods would enhance resistance and can be used in agriculture even though it is
quite difficult to achieve broad-spectrum resistance by this strategy due to high R–
Avr specificities. Mutation of functional domains will alter binding specificities of R
proteins, which in turn strengthen the defence response either by blocking perception
of specific pathogen by native R gene or by broadening the range of pathogens that
can be recognized by R protein. This strategy handles pathogen recognition
mechanisms only, while the rest of the defence response is part of the innate
immunity only. Solanum tuberosum R3a gene (from the CNL group) confers
resistance to the P. infestans Avr3aKI isolate but not to the Avr3aEM isolate.
N. benthamiana expressing R3a* mutant variants showed significantly improved
recognition of AVR3aEM (Chapman et al. 2014). Such mutated plant R genes
enhance possibilities of recognizing pathogens/races that usually escape original R
gene perception and thus confer durable resistance in field.
23.9.6 Modifying Pathogen Effector Targets in Host and Targeting

Pathogen Effectors
Pathogenic virulence factors involved in virulence usually bind to certain host

targets during their activity. Genetic modification of such host molecules targeted
by pathogen can efficiently inhibit infection process. Modification/deletion of bind-
ing site residues in host target without compromising its other biological functions
can be an alternative strategy to obstruct infection. Pathogen-derived proteins either

sustain pathogen growth in susceptible host or induce HR in resistant host, and some
of them have proved to be effective in enhancing plant defence response while
expressed in host systems and thus can serve as functional genes for creating
resistant GM plants (Silva et al. 2018). Expression of such defence promoting
effectors from pathogens may confer resistance in host plants. N. benthamiana
expressing P. sojae CRN effector PsCRN115 was conferred with upregulated
defence responses, such as ABC transporters, cytochrome P450 and PRRs, and
increased the level of plant resistance to P. capsici and P. parasitica oomycetes
(Zhang et al. 2015).
23.9.7 Silencing Pathogen Genes
RNA interference is a potential strategy to control microbial pathogens of all

classes – viral, bacterial, fungal (bio-necrotrophic and oomycetes) and nematode –
wherein expression of essential genes of pathogen required for proliferation and
establishment of infection is silenced post-transcriptionally by siRNAs complemen-
tary to pathogen-derived mRNAs. A prerequisite for RNAi is a double-stranded
DNA precursor derived from a replicating viral genome or viral RNA secondary
structure, which will be cleaved by DICER-like proteins (ribonuclease III – DCL),
and the resulting ssRNA is loaded into silencing complex (RISC complex). siRNA-
guided activated complex further recognizes and degrades the target mRNA by
Argonaut (Ago) protein in cytosol (Seo et al. 2013; Weiberg et al. 2014). Conserved
domains of vital genes related to cellular processes, morphogenesis or pathogenesis
can be potential target sites for host-induced gene silencing, which may confer
broad-spectrum durable resistance. Similarly, host genes, which are inevitable for
the infection process, whose silencing does not have any/have minimal negative
fitness costs also, can be targeted. Transgenic plants expressing target-specific
siRNAs successfully confer resistance by sequence-specific degradation of
target mRNA.
23.9.8 Transgenic Expression of Detoxifying Pathogenic Toxins
Several pathogens produce toxins, which adversely affect various metabolic

pathways/processes of host system. The expression of detoxifying enzymes, either
native or transgenic, specific to each pathogen under native or heterologous pro-
moter would considerably reduce/alleviate the detrimental effects of such pathogenic
toxins. For instance, detoxification of phytotoxin oxalic acid from Cryphonectria
parasitica causing chestnut blight disease was achieved in American chestnut
transformed with wheat gene coding for the production of the degradative enzyme,
oxalate oxidase, and the resultant GM chestnut plant showed considerable reduction
in chestnut blight disease development (Zhang et al. 2013). Similarly, transgenic
wheat expressing a toxin-degrading enzyme from barley showed resistance to

Fusarium head blight (Li et al. 2015).
23.9.9 Expression of Antimicrobial Compounds
Introduction of genes encoding antimicrobial compounds into host plant can confer
efficient resistance to specific pathogens. Defensins, an antimicrobial peptide of
plant origin, chitin-degrading enzyme from other microbial species, is a well-
established example for this strategy. Resulting transgenic crops must display
pathovar-/strain-/race-specific resistance to a respective pathogen in the field,
while exhibiting normal growth and development pattern without compromising
the yield and their responses to other biotic as well as abiotic stress stimuli. Spatio-
temporally regulated expression of these genes using tissue-/development-specific
promoters or pathogen-inducible promoter would be advantageous to minimize the
negative fitness effects, if any. The effect of transgene encoded antimicrobial
compound on animal/mammalian system should be assessed before employing
this strategy for transforming edible crops. The manipulation of bacterial cry genes
is an efficient strategy for controlling insect pests and thereby prevents vector-borne
diseases (Mayee et al. 2003). A detailed understanding of the underlying molecular
mechanisms of their mode of action would permit the development of much sophis-
ticated strategies for the regulated expression of appropriate antimicrobial
compounds to confer broad-spectrum durable resistance to crop plants.
23.10 Cisgenesis: Alternative to Transgenics
Cisgenesis is emerging as an efficient alternative strategy of crop improvement,

which combines the beneficial aspects of classical breeding and modern biotechnol-
ogy. In cisgenesis, desirable genes are transferred among crossable species/species
capable of sexual hybridization (Schouten et al. 2006). In this method, the cisgene to
be transferred is same as that in breeding procedure, and the resulting plant also is the
same as a classically bred plant. Thus, cisgenesis eliminates biosafety concerns
regarding transgenic plants, thereby overcoming the regulatory issues for field
cultivation and marketing. As cisgenesis facilitates precise gene transfer, negative
effects of linkage drag that associated with conventional breeding can be avoided.
Additionally, cisgenesis is not time-consuming as the conventional breeding. Thus,
cisgenesis can be adopted for specifically stacking resistance genes from crossable
gene pools.
23.11 Conclusion
Genetic engineering technology has made tremendous contributions in developing

disease-resistant crop plants. Transgenic technology has evolved multitude of
options for precise alteration of crop genome, even at nucleotide level over a very
short time span, and crop improvement programmes for disease resistance have
taken advantage of these methods in order to circumvent the prevailing as well as
expanding challenges/vulnerabilities posed by pathogenic organisms. From the
incorporation of resistance genes or pathogen targeting genes and manipulation of
plant’s biochemistry for harming pathogens to editing of single bases to disrupt the
entire plant–pathogen interaction and thereby disease development, genetic engi-
neering techniques have opened up every possible way for crops to combat
pathogens. Sequence databases and functional annotation information are expanding
on a daily basis with the establishment of high-throughput sequencing and
genotyping platforms. This further unravels novel molecular and genomic targets
that can be manipulated for conferring robust – broad-spectrum – durable resistance
to all crop plant species.
Despite the availability of transgenic technology for developing crops with
desirable resistance traits, field-level cultivation and commercialization of produce
are still a major challenge in the case of these plants due to stringent regulations.
Although some transgenic crops have been commercialized, many are not going for
field trials. Stability of resistant phenotypes without any negative fitness effects
under field conditions needs to be ensured. Protocols for transformation and regen-
eration of farmer-preferred cultivars/local cultivars of crop of interest need to be
optimized. Stigma associated with transgenic plants is another major issue that
restricts the GE crops in laboratories or experimental glasshouses. Even in the case
of engineering resistance, GE strategies, which modify or alter the endogenous
immune components rather than those adding/introducing novel/foreign gene or
sequence or metabolic pathways, are preferred, when such issues are concerned.
Ecological and health concerns regarding possibilities of horizontal gene flow,
especially the bacterial marker genes used in the transformation process and the
loss of endogenous agrobiodiversity, are the prominent underlying causes of such
regulations. The evaluation of transgenic crop products to ensure biosafety should be
followed before releasing. For transgene-free editing options opened up by third-
generation genome editing tool, CRISPR/Cas is a promising strategy to overcome
regulatory barriers associated with transgenic crops. Recently, the definitions of
genetically modified plants are getting revised, enabling field-level cultivation of
many transgenic disease-resistant crops. However, the innovations that render trans-
genic technology free of any risks need to be emerged for better public acceptance of
genetically modified crops. But lastly, the conventional breeding techniques, which
created the large pool of disease-resistant cultivars we had all this time, cannot be
replaced completely and are powerful to sustain crop production, despite its
shortcomings. Genetic engineering can supplement and renovate at points where
breeding techniques are struggling to prevail over and that will lead us to the prime
goal, sustainable crop/food production.
References
Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986) Delay of
disease development in transgenic plants that express the tobacco mosaic virus coat protein
gene. Science 232:738–743
Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Lander ES (2017)
RNA targeting with CRISPR–Cas13. Nature 550:280
Ai T, Zhang L, Gao Z, Zhu CX, Guo X (2011) Highly efficient virus resistance mediated by
artificial microRNAs that target the suppressor of PVX and PVY in plants. Plant Biol
13:304–316
Ali I, Amin I, Briddon RW, Mansoor S (2013) Artificial microRNA-mediated resistance against the
monopartite begomovirus cotton leaf curl Burewala virus. Virol J 10(1):231
Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM (2015) CRISPR/Cas9-mediated
viral interference in plants. Genome Biol 16:238
Ali Z, Ali S, Tashkandi M, Zaidi SSEA, Mahfouz MM (2016) CRISPR/Cas9-mediated immunity to
geminiviruses: differential interference and evasion. Sci Rep 6:1–13
Alan AR, Blowers A, Earle ED (2004) Expression of a magainin-type antimicrobial peptide gene
(MSI-99) in tomato enhances resistance to bacterial speck disease. Plant Cell Rep 22(6):388–
396
Allen RL, Bittner-Eddy PD, Grenville-Briggs LJ, Meitz JC, Rehmany AP, Rose LE, Beynon JL
(2004) Host-parasite coevolutionary conflict between Arabidopsis and downy mildew. Science
306:1957–1960
Aldwinckle HS, Borejsza-Wysocka EE, Malnoy M, Brown SK, Norelli JL, Beer SV, Jin QL (2003)
Development of fire blight resistant apple cultivars by genetic engineering. Acta Hortic
622:105–111
Antignus Y, Vunsh R, Lachman O, Pearlsman M, Maslenin L, Hananya U, Rosner A (2004)
Truncated Rep gene originated from Tomato yellow leaf curl virus-Israel [Mild] confers
strainspecific resistance in transgenic tomato. Ann Appl Biol 144:39–44
Andika IB, Kondo H, Tamada T (2005) Evidence that RNA silencing-mediated resistance to Beet
necrotic yellow vein virus is less effective in roots than in leaves. Mol Plant Microbe Interact
18:194–204
Arce P, Moreno M, Gutierrez M, Gebauer M, Dell’Orto P, Torres H, Acuna I, Oliger P, Venegas A,
Jordana X, Kalazich J, Holuigue L (1999) Enhanced resistance to bacterial infection by Erwinia
carotovora subsp. atroseptica in transgenic potato plants expressing the attacin or the cecropin
SB-37 genes. Am J Potato Res 76:169–177
Arif M, Azhar U, Arshad M, Zafar Y, Mansoor S, Asad S (2012) Engineering broad-spectrum
resistance against RNA viruses in potato. Transgenic Res 21:303–311
Aman R, Ali Z, Butt H, Mahas A, Aljedaani F, Khan MZ, Mahfouz M (2018) RNA virus
interference via CRISPR/Cas13a system in plants. Genome Biol 19:1
Bai Y, Guo Z, Wang X, Bai D, Zhang W (2009) Generation of double-virus-resistant marker-free
transgenic potato plants. Prog Nat Sci 19:543–548
Balaji V, Smart CD (2012) Over-expression of snakin-2 and extensin-like protein genes restricts
pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp.
michiganensis in transgenic tomato (Solanum lycopersicum). Transgenic Res 21:23–37
Baltes NJ, Hummel AW, Konecna E, Cegan R, Bruns AN, Bisaro DM, Voytas DF (2015)
Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system.
Nat Plants 1:1–4
Baulcombe DC, Saunders GR, Bevan MW, Mayo MA, Harrison BD (1986) Expression of
biologically active viral satellite RNA from the nuclear genome of transformed plants. Nature
321:446–449
Ban L, Ahmed E, Ninkovic V, Delp G, Glinwood R (2008) Infection with an insect virus affects
olfactory behaviour and interactions with host plant and natural enemies in an aphid. Entomol
Exp Appl 127:108–117
Barajas D, Tenllado F, González-Jara P, Martínez-García B, Atencio FA, Díaz-Ruíz JR (2004)

Resistance to plum pox virus (PPV) in Nicotiana benthamiana plants transformed with the PPV
HC-Pro silencing suppressor gene. Plant Pathol 88:239–248
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Bastet A, Lederer B, Giovinazzo N, Arnoux X, Retana S, Reinbold C, Lemaire O (2018) Trans-
species synthetic gene design allows resistance pyramiding and broad-spectrum engineering of
virus resistance in plants. Plant Biotechnol J 16:1569–1581
Bendahmane M, Chen I, Asurmendi S, Bazzini AA, Szecsi J, Beachy RN (2007) Coat protein-
mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of
interference by coat protein. Virology 366:107–116
Boonrod K, Galetzka D, Nagy PD, Conrad U, Krczal G (2004) Single-chain antibodies against a
plant viral RNA-dependent RNA polymerase confer virus resistance. Nat Biotechnol 22:856–
862
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U
(2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science
326:1509–1512
Bonning BC, Pal N, Liu S, Wang Z, Sivakumar S, Dixon PM, Miller WA (2014) Toxin delivery by
the coat protein of an aphid-vectored plant virus provides plant resistance to aphids. Nat
Biotechnol 32:102
Boutrot F, Zipfel C (2017) Function, discovery, and exploitation of plant pattern recognition
receptors for broad-spectrum disease resistance. Annu Rev Phytopathol 55:257–286
Bonfim K, Faria JC, Nogueira EO, Mendes ÉA, Aragão FJ (2007) RNAi-mediated resistance to
bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol
Plant Microbe Interact 20:717–726
Brault V, Uzest M, Monsion B, Jacquot E, Blanc S (2010) Aphids as transport devices for plant
viruses. C R Biol 333:524–538
Bruce TJ, Aradottir GI, Smart LE, Martin JL, Caulfield JC, Doherty A, Jones HD (2015) The first
crop plant genetically engineered to release an insect pheromone for defence. Sci Rep 5:11183
Brunetti A, Tavazza M, Noris E, Tavazza R, Caciagli P, Ancora G, Accotto GP (1997) High
expression of truncated viral Rep protein confers resistance to tomato yellow leaf curl virus in
transgenic tomato plants. Mol Plant-Microbe Interact 10:571–579
Bundó M, Coca M (2016) Enhancing blast disease resistance by overexpression of the calcium-
dependent protein kinase Os CPK 4 in rice. Plant Biotechnol J 14:1357–1367
Carbonell A, Takeda A, Fahlgren N, Johnson SC, Cuperus JT, Carrington JC (2014) New genera-
tion of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient
gene silencing in Arabidopsis. J Plant Physiol 165:15–29
Cao X, Lu Y, Di D, Zhang Z, Liu H, Tian L (2013) Enhanced virus resistance in transgenic maize
expressing a dsRNA-specific endoribonuclease gene from E. coli. PLoS One 8:e60829
Castel SE, Martienssen RA (2013) RNA interference in the nucleus: roles for small RNAs in
transcription, epigenetics and beyond. Nat Rev Genet 14:100–112
Callot C, Gallois JL (2014) Pyramiding resistances based on translation initiation factors in
Arabidopsis is impaired by male gametophyte lethality. Plant Signal Behav 9:e27940
Chang C, Chen YC, Hsu YH, Wu JT, Hu CC, Chang WC, Lin NS (2005) Transgenic resistance to
Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene. Transgenic
Res 14:41–46
Chapman S, Stevens LJ, Boevink PC, Engelhardt S, Alexander CJ, Harrower B, Champouret N,
McGeachy K, Van Weymers PS, Chen X, Birch PR (2014) Detection of the viru lent form of
AVR3a from Phytophthora infestans following artificial evolution of potato resistance gene
R3a. PLoS One 9:e110158
Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Gal-On A (2016)
Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 tech-
nology. Mol Plant Pathol 17:1140–1153
Chauhan RD, Beyene G, Kalyaeva M, Fauquet CM, Taylor N (2015) Improvements in

agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) for large-scale
production of transgenic plants. Plant Cell Tissue Organ Cult 121:591–603
Chen C, Chen Z (2002) Potentiation of developmentally regulated plant defense response by
AtWRKY18, a pathogen-induced Arabidopsis transcription factor. Plant Physiol 129:706–716
Chen YK, Lohuis D, Goldbach R, Prins M (2004) High frequency induction of RNA-mediated
resistance against cucumber mosaic virus using inverted repeat constructs. Mol Breed 14:215–
226
Chen SC, Liu AR, Zou ZR (2006) Overexpression of glucanase gene and defensin gene in
transgenic tomato enhances resistance to Ralstonia solanacearum. Russ J Plant Physiol
53:671–677
Chen SC, Liu AR, Wang FH, Ahammed GJ (2009) Combined overexpression of chitinase and
defensin genesin transgenic tomato enhances resistance to Botrytis cinerea. Afr J Biotechnol
8:5182–5188
Chen LQ, Qu XQ, Hou BH, Sosso D, Osorio S, Fernie AR, Frommer WB (2012) Sucrose efflux
mediated by SWEET proteins as a key step for phloem transport. Science 335:207–211
Chen W, Qian Y, Wu X, Sun Y, Wu X, Cheng X (2014) Inhibiting replication of begomoviruses
using artificial zinc finger nucleases that target viral-conserved nucleotide motif. Virus Genes
48:494–501
Cheng X, Li F, Cai J, Chen W, Zhao N, Sun Y, Guo Y, Yang X, Wu X (2015) Artificial TALE as a
convenient protein platform for engineering broad-spectrum resistance to begomoviruses.
Viruses 7:4772–4782
Chern M, Fitzgerald HA, Canlas PE, Navarre DA, Ronald PC (2005) Overexpression of a rice
NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light.
Mol Plant Microbe Interact 18:511–520
Chung BN, Yoon J, Palukaitis P (2013) Engineered resistance in potato against potato leaf roll
virus, potato virus A and potato virus Y. Virus Genes 47:86–92
Chen XJ, Chen Y, Zhang LN, Xu B, Zhang JH, Chen ZX, Xu JY (2016) Overexpression of
OsPGIP1 enhances rice resistance to sheath blight. Plant Dis 100:388–395
Chellappan P, Masona MV, Vanitharani R, Taylor NJ, Fauquet CM (2004) Broad spectrum
resistance to ssDNA viruses associated with transgene-induced gene silencing in cassava.
Plant Mol Biol 56:601–611
Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the
evolution of the plant immune response. Cell 124:803–814
Chye ML, Zhao KJ, He ZM, Ramalingam S, Fung KL (2005) An agglutinating chitinase with two
chitin-binding domains confers fungal protection in transgenic potato. Planta 220:717–730
Cillo F, Palukaitis P (2014) Transgenic resistance. Adv Virus Res 90:35–146
Cruz ARR, Aragão FJL (2014) RNAi-based enhanced resistance to Cowpea severe mosaic virus
and Cowpea aphid-borne mosaic virus in transgenic cowpea. Plant Pathol 63:831–837
Coca M, Penas G, Gómez J, Campo S, Bortolotti C, Messeguer J, San Segundo B (2006) Enhanced
resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A
gene in transgenic rice. Planta 223:392–406
Coll NS, Epple P, Dangl JL (2011) Programmed cell death in the plant immune system. Cell Death
Differ 18:1247
Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL, Schulze-Lefert P (2003)
SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425:973
Cooper B, Lapidot M, Heick JA, Dodds JA, Beachy RN (1995) A defective movement protein of
TMV in transgenic plants confers resistance to multipleviruses whereas the functional analog
increases susceptibility. Virology 206:307–313
Csorba T, Pantaleo V, Burgyán J (2009) RNA silencing: an antiviral mechanism. Adv Virus Res
75:35–230
Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature
411:826
De Gray G, Rajasekaran K, Smith F, Sanford J, Daniell H (2001) Expression of an antimicrobial

peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol
127:852–862
Dempsey DMA, Klessig DF (2012) SOS–too many signals for systemic acquired resistance. Trends
Deepthi DC, Makeshkumar T (2016) Elimination of cassava mosaic disease through meristem
culture and field evaluation for yield loss assessment in cassava genotypes. J Root Crops 42
(1):45–52
Di Nicola-Negri E, Brunetti A, Tavazza M, Ilardi V (2005) Hairpin RNA-mediated silencing of
plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus.
Transgenic Res 14:989–994
Ding SW (2010) RNA-based antiviral immunity. Nat Rev Immunol 10:632–644
Dong J, Li T, Hasi A, Zhang H (1999) Resistance of transgenic potato expressing intergenic
sequence of potato leafroll virus. Virol Sin 14:66–72
Domínguez A, de Mendoza AH, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2002)
Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime (Citrus
aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene. Mol Plant Breed 10:1–
10
Dominguez AA, Lim WA, Qi LS (2016) Beyond editing: repurposing CRISPR–Cas9 for precision
genome regulation and interrogation. Nat Rev Mol Cell Biol 17:5
Dougherty WG, Lindbo JA, Smith HA, Parks TD, Swaney S, Proebsting WM (1994) RNA-
mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly
involved in RNA degradation. Mol Plant Microbe Interact 7:544–552
Dougherty WG, Parks TD (1995) Transgene and gene suppression: telling us something new. Curr
Opin Cell Biol 7:399–405
Duan CG, Wang CH, Fang RX, Guo HS (2008) Artificial microRNAs highly accessible to targets
confer efficient virus resistance in plants. J Virol 82:11084–11095
Dubey VK, Chandrasekhar K, Srivastava A, Aminuddin Singh VP, Dhar K, Arora PK (2015)
Expression of coat protein gene of cucumber mosaic virus (CMV-subgroup IA) Gladiolus
isolate in Nicotiana tabacum. J Plant Interact 10:296–304
Eames A, Wang MB, Smith NA, Waterhouse PM (2008) RNA silencing in plants: yesterday, today
and tomorrow. Plant Physiol 147:456–468
Elayabalan S, Kalaiponmani K, Sreeramanan S, Selvarajan R, Radha P, Ramlatha M, Kumar KK,
Balasubramanian P (2013) Development of Agrobacterium-mediated transformation of highly
valued hill bananacultivar Virupakshi (AAB) for resistance to BBTV disease. World J Microb
Biotech 29:589–596
Emani C, Garcia JM, Lopata-Finch E, Pozo MJ, Uribe P, Kim DJ, Rathore KS (2003) Enhanced
fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma
virens. Plant Biotechnol J 1:321–336
Fagoaga C, López C, de Mendoza AH, Moreno P, Navarro L, Flores R, Pena L (2006) Post-
transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers
resistance to the virus in transgenic Mexican lime. Plant Mol Biol 60:153–165
Fahim M, Ayala-Navarrete L, Millar AA, Larkin PJ (2010) Hairpin RNA derived from viral NIa
gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants. Plant
Biotechnol J 8(7):821–834
Fahim M, Millar AA, Wood CC, Larkin PJ (2012) Resistance to Wheat streak mosaic virus
generated by expression of an artificial polycistronic microRNA in wheat. Plant Biotechnol J
10:150–163
Faria JC, Albino MM, Dias BB, Cançado LJ, da Cunha NB, Silva LDM, Aragao FJ (2006) Partial
resistance to Bean golden mosaic virus in a transgenic common bean (Phaseolus vulgaris L.)
line expressing a mutated rep gene. Plant Sci 171:565–571
Febres VJ, Lee RF, Moore GA (2008) Transgenic resistance to Citrus tristeza virus in grapefruit.
Plant Cell Rep 27:93–104
Fellers JP, Collins GB, Hunt AG (1998) The NIa-proteinase of different plant potyviruses provides
specific resistance to viral infection. Crop Sci 38:1309–1319
Fermin G, Inglessis V, Garboza C, Rangel S, Dagert M, Gonsalves D (2004) Technology transfer
for engineered resistance against PRSV in Venezuelan transgenic papayas. Plant Dis 88:516–
522
Ferreira SA, Pitz KY, Manshardt R, Zee F, Fitch M, Gonsalves D (2002) Virus coat protein
transgenic papaya provides practical control of papaya ringspot virus in Hawaii. Plant Dis
86:101–105
Ferrari S, Sella L, Janni M, De Lorenzo G, Favaron F, D’ovidio R (2012) Transgenic expression of
polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the
flower pathogen Fusarium graminearum. Plant Biol 14:31–38
Ferrari S, Savatin DV, Sicilia F, Gramegna G, Cervone F, De Lorenzo G (2013)
Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth
and development. Front Plant Sci 4:49
Fister AS, Landherr L, Maximova SN, Guiltinan MJ (2018) Transient expression of CRISPR/Cas9
machinery targeting TcNPR3 enhances defense response in Theobroma cacao. Front Plant Sci
9:268
Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev Phytopathol 9:275–296
Flores R, Hernández C, Alba AEMD, Daròs JA, Serio FD (2005) Viroids and viroid-host
interactions. Annu Rev Phytopathol 43:117–139
Flores R, Navarro B, Kovalskaya N, Hammond RW, Di Serio F (2017) Engineering resistance
against viroids. Curr Opin Virol 26:1–7
Floyd-Smith G, Slattery E, Lengyel P (1981) Interferon action: RNA cleavage pattern of a (20 -50 )
oligoadenylate-dependent endonuclease. Science 212:1030–1032
Fonseca JP, Mysore KS (2019) Genes involved in nonhost disease resistance as a key to engineer
durable resistance in crops. Plant Sci 279:108–116
Fradin EF, Abd-El-Haliem A, Masini L, van den Berg GC, Joosten MH, Thomma BP (2011)
Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis. Plant
Physiol 156:2255–2265
Frischmuth T, Stanley J (1998) Recombination between viral DNA and the transgenic coat protein
gene of African cassava mosaic geminivirus. J Gen Virol 79:1265–1271
Furutani N, Hidaka S, Kosaka Y, Shizukawa Y, Kanematsu S (2006) Coat protein gene-mediated
resistance to soybean mosaic virus in transgenic soybean. Breed Sci 56:119–124
Fuentes A, Ramos PL, Fiallo E, Callard D, Sánchez Y, Peral R (2006) Intron-hairpin RNA derived
from replication association protein C1 gene confers immunity to tomato yellow leaf curl virus
infection in transgenic tomato plants. Transgenic Res 15:291–304
Ganesan U, Suri SS, Rajasubramaniam S, Rajam MV, Dasgupta I (2009) Transgenic expression of
coat protein gene of Rice tungro bacilliform virus in rice reduces the accumulation of viral DNA
in inoculated plants. Virus Genes 39:113–119
Gargouri-Bouzid R, Jaoua L, Rouis S, Saïdi MN, Bouaziz D, Ellouz R (2006) PVY-resistant
transgenic potato plants expressing an anti-NIa protein scFv antibody. Mol Biotechnol
33:133–140
Germundsson A, Valkonen JP (2006) P1-and VPg-transgenic plants show similar resistance to
Potato virus A and may compromise long distance movement of the virus in plant sections
expressing RNA silencing-based resistance. Virus Res 116:208–213
Galvez LC, Banerjee J, Pinar H, Mitra A (2014) Engineered plant virus resistance. Plant Sci
228:11–25
Gao AG, Hakimi SM, Mittanck CA, Wu WBM, Stark DM, Shah DM, Liang J, Rommens CM
(2000) Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat
Gao L, Ding X, Li K, Liao W, Zhong Y, Ren R, Liu Z, Adhimoolam K, Zhi H (2015) Characteri-
zation of soybean mosaic virus resistance derived from inverted repeat-SMV-HC-Pro genes in
multiple soybean cultivars. Theor Appl Genet 128:1489–1505
Gil M, Esteban O, García JA, Pena L, Cambra M (2011) Resistance to plum pox virus in plants
expressing cytosolic and nuclear single-chain antibodies against the viral RNA NIb replicase.
Gill US, Lee S, Mysore KS (2015) Host versus nonhost resistance: distinct wars with similar
arsenals. Phytopathology 105:580–587
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic
pathogens. Annu Rev Phytopathol 43:205–227
Goggin FL, Jia L, Shah G, Hebert S, Williamson VM, Ullman DE (2006) Heterologous expression
of the Mi-1.2 gene from tomato confers resistance against nematodes but not aphids in eggplant.
Mol Plant-Microbe Interact 19:383–388
Golemboski DB, Lomonossoff GP, Zaitlin M (1990) Plants transformed with a tobacco mosaic
virus nonstructural gene sequence are resistant to the virus. Proc Natl Acad Sci 87:6311–6315
Guo Y, Ruan MH, Yao W, Chen L, Chen RK, Zhang MQ (2008) Difference of coat protein
mediated resistance to sugarcane mosaic virus between Badila and Funong 91–4621. J Fujian
Agric For Univ (Natural Science Edition) 37:7–12
Guo HJ, Huang LC, Sun YC, Guo H, Ge F (2016) The contrasting effects of elevated CO2 on
TYLCV infection of tomato genotypes with and without the resistance gene, Mi-1.2. Front Plant
Sci 7:1680
Gubba A, Gonsalves C, Stevens MR, Tricoli DM, Gonsalves D (2002) Combining transgenic and
natural resistance to obtain broad resistance to tospovirus infection in tomato (Lycopersicon
esculentum Mill). Mol Breed 9:13–23
Gururani MA, Venkatesh J, Upadhyaya CP, Nookaraju A, Pandey SK, Park SW (2012) Plant
disease resistance genes: current status and future directions. Physiol Mol Plant Pathol 78:51–65
Hannon GJ (2002) RNA interference. Nature 418:244–251
Hashmi JA, Zafar Y, Arshad M, Mansoor S, Asad S (2011) Engineering cotton (Gossypium
hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences.
Virus Genes 42:286–296
Hao G, Pitino M, Duan Y, Stover E (2016) Reduced susceptibility to Xanthomonas citri in
transgenic citrus expressing the FLS2 receptor from Nicotiana benthamiana. Mol Plant-
Harris CJ, Slootweg EJ, Goverse A, Baulcombe DC (2013) Stepwise artificial evolution of a plant
disease resistance gene. Proc Natl Acad Sci 110:21189–21194
Harrison BD, Mayo MA, Baulcombe DC (1987) Virus resistance in transgenic plants that express
cucumber mosaic virus satellite RNA. Nature 328:799–802
Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene
silencing in plants. Science 286:950–952
He X, Miyasaka SC, Fitch MM, Moore PH, Zhu YJ (2008) Agrobacterium tumefaciens-mediated
transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improved
tolerance to a fungal pathogen Sclerotium rolfsii. Plant Cell Rep 27:903–909
Himmelbach A, Liu L, Zierold U, Altschmied L, Maucher H, Beier F, Muller D, Hensel G, Heise A,
Schutzendubel A, Kumlehn J, Schweizer P (2010) Promoters of the barley germin-like GER4
gene cluster enable strong transgene expression in response to pathogen attack. Plant Cell 22
(3):937–952
Hood MI, Skaar EP (2012) Nutritional immunity: transition metals at the pathogen–host interface.
Nat Rev Microbiol 10:525
Horvath DM, Stall RE, Jones JB, Pauly MH, Vallad GE, Dahlbeck D, Scott JW (2012) Transgenic
resistance confers effective field level control of bacterial spot disease in tomato. PLoS One 7:
e42036
Huang Y, Nordeen RO, Di M, Owens LD, Mc Beth JH (1997) Expression of an engineered
cecropin gene cassette in transgenic tobacco plants confers disease resistance to Pseudomonas
syringae pv. tabaci. Phytopathology 87:494–499
Kamala S, Makeshkumar T (2015) Transcriptome profiling of in vitro lines propagated from corm
bud tip of elephant foot yam (Amorphophallus paeoniifolius) approves complete potyviruses
elimination. Eur J Plant Pathology 143(2):363–371
Ingwell LL, Eigenbrode SD, Bosque-Pérez NA (2012) Plant viruses alter insect behavior to enhance
their spread. Sci Rep 2:578
Ilardi V, Tavazza M (2015) Biotechnological strategies and tools for Plum pox virus resistance:
trans-, intra-, cis-genesis, and beyond. Front Plant Sci 6:379
Iwanami T, Shimizu T, Ito T, Hirabayashi T (2004) Tolerance to citrus mosaic virus in transgenic
trifoliate orange lines harboring capsid polyprotein gene. Plant Dis 88(8):865–868
Jan PS, Huang HY, Chen HM (2010) Expression of a synthesized gene encoding cationic peptide
cecropin B in transgenic tomato plants protects against bacterial diseases. Appl Environ
Javaid S, Amin I, Jander G, Mukhtar Z, Saeed NA, Mansoor S (2016) A transgenic approach to
control hemipteran insects by expressing insecticidal genes under phloem-specific promoters.
Sci Rep 6:34706
Jha S, Tank HG, Prasad BD, Chattoo BB (2009) Expression of Dm-AMP1 in rice confers resistance
to Magnaporthe oryzae and Rhizoctonia solani. Transgenic Res 18:59–69
Ji X, Zhang H, Zhang Y, Wang Y, Gao C (2015) Establishing a CRISPR–Cas-like immune system
conferring DNA virus resistance in plants. Nat Plants 1:1–4
Jia H, Orbovic V, Jones JB, Wang N (2016) Modification of the PthA4 effector binding elements in
type I Cs LOB 1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit
alleviating XccΔpthA4: dCs LOB 1.3 infection. Plant Biotechnol J 14:1291–1301
Jo KR, Kim CJ, Kim SJ, Kim TY, Bergervoet M, Jongsma MA, Vossen JH (2014) Development of
late blight resistant potatoes by cisgene stacking. BMC Biotechnol 14:50
Jo KM, Jo Y, Choi H, Chu H, Lian S, Yoon JY, Choi SK, Kim KH, Cho WK (2015) Development
of genetically modified chrysanthemums resistant to Chrysanthemum stunt viroid using sense
and antisense RNAs. Sci Hortic 195:17–24
Johnson R (1981) Durable resistance: definition of, genetic control, and attainment in plant
breeding. Phytopathology 71:567–568
Jones RAC (2014) Plant virus ecology and epidemiology: historical perspectives, recent progress
and future prospects. Ann Appl Biol 164:320–347
Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323
Jones JD, Witek K, Verweij W, Jupe F, Cooke D, Dorling S, Foster S (2014) Elevating crop disease
resistance with cloned genes. Philos Trans R Soc B 369:0087
Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants.
Annu Rev Plant Biol 57:19–53
Joubert DA, Kars I, Wagemakers L, Bergmann C, Kemp G, Vivier MA, van Kan JA (2007) A
polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the
endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without
any evidence for in vitro interaction. Mol Plant Microbe Interact 20:392–402
Kamachi S, Mochizuki A, Nishiguchi M, Tabei Y (2007) Transgenic Nicotiana benthamiana plants
resistant to cucumber green mottle mosaic virus based on RNA silencing. Plant Cell Rep
26:1283–1288
Kawazu Y, Fujiyama R, Sugiyama K, Sasaya T (2006) A transgenic lettuce line with resistance to
both lettuce big-vein associated virus and mirafiori lettuce virus. J Am Soc Horticult Sci
131:760–763
Kachroo A, Robin GP (2013) Systemic signaling during plant defense. Curr Opin Plant Biol
16:527–533
Kaur J, Sagaram US, Shah D (2011) Can plant defensins be used to engineer durable commercially
useful fungal resistance in crop plants. Fungal Biol Rev 25:128–135
Kertbundit S, Pongtanom N, Ruanjan P, Chantasingh D, Tanwanchai A, Panyim S, Juříček M
(2007) Resistance of transgenic papaya plants to papaya ringspot virus. Biol Plant 51:333–339
Kerr CH, Wang QS, Keatings K, Khong A, Allan D, Yip CK, Jan E (2015) The 50 untranslated
region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus
modulates infection. J Virol 89:5919–5934
Khoury J, Singh R, Boucher A, Coombs D (1988) Concentration and distribution of mild and severe
strains of potato spindle tuber viroid in cross-protected tomato plants. Phytopathology 78:1331–
1336
Khan RS, Sjahril R, Nakamur I (2008) Production of transgenic potato exhibiting enhanced
resistance to fungal infections and herbicide applications. Plant Biotechnol Rep 2:13–20
Kim SJ, Paek KH, Kim BD (1995) Delay of disease development in transgenic petunia plants
expressing cucumber mosaic virus I17N-satellite RNA. J Am Soc Hortic Sci 120:353–359
Kim SJ, Lee SJ, Kim BD, Paek KH (1997) Satellite-RNA-mediated resistance to cucumber mosaic
virus in transgenic plants of hot pepper (Capsicum annuum cv. Golden Tower). Plant Cell Rep
16:825–830
Kim JK, Jang IC, Wu R, Zuo WN, Boston RS, Lee YH, Ahn IP, Nahm BH (2003) Co-expression of
a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice
plants confers enhanced resistance to sheath blight. Transgenic Res 12:475–484
Kim SH, Qi D, Ashfield T, Helm M, Innes RW (2016) Using decoys to expand the recognition
specificity of a plant disease resistance protein. Science 351:684–687
Kishimoto K, Nishizawa Y, Tabei Y, Hibi T, Nakajima M, Akutsu K (2002) Detailed analysis of
rice chitinase gene expression in transgenic cucumber plants showing different levels of disease
resistance to gray mold (Botrytis cinerea). Plant Sci 162:655–662
Koo JC, Asurmendi S, Bick J, Woodford-Thomas T, Beachy RN (2004) Ecdysone agonist
inducible expression of a coat protein gene from tobacco mosaic virus confers viral resistance
in transgenic Arabidopsis. Plant J 37:439–448
Kollàr À, Dalmay T, Burgyàn J (1993) Defective interfering RNA-mediated resistance against
cymbidium ringspot tombusvirus in transgenic plants. Virology 193:313–318
Koseoglou E (2017) The study of SlPMR4 CRISPR/Cas9-mediated tomato allelic series for
resistance against powdery mildew (Doctoral dissertation, Wageningen University and
Research, Wageningen)
Kouassi NK, Chen L, Siré C, Bangratz-Reyser M, Beachy RN, Fauquet CM, Brugidou C (2006)
Expression of rice yellow mottle virus coat protein enhances virus infection in transgenic plants.
Arch Virol 151:2111–2122
Krubphachaya P, Juricek M, Kertbundit S (2007) Induction of RNA-mediated resistance to papaya
ringspot virus type W. J Biochem Mol Biol 40:404–411
Kung YJ, Bau HJ, Wu YL, Huang CH, Chen TM, Yeh SD (2009) Generation of transgenic papaya
with double resistance to papaya ringspot virus and papaya leaf-distortion mosaic virus.
Kung YJ, Yu TA, Huang CH, Wang HC, Wang SL, Yeh SD (2010) Generation of hermaphrodite
transgenic papaya lines with virus resistance via transformation of somatic embryos derived
from adventitious roots of in vitro shoots. Transgenic Res 19:621–635
Langenberg WG, Zhang L, Giunchedi L, Mitra A (1997) Transgenic tobacco plants expressing the
bacterial mc gene resist virus infection. Mol Breed 3:391–399
Lacombe S, Rougon-Cardoso A, Sherwood E, Peeters N, Dahlbeck D, Van Esse HP, Jones JD
(2010) Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum
bacterial resistance. Nat Biotechnol 28:365
Langenbach C, Schultheiss H, Rosendahl M, Tresch N, Conrath U, Goellner K (2016) Interspecies
gene transfer provides soybean resistance to a fungal pathogen. Plant Biotechnol J 14:699–708
Lapidot M, Gafny R, Ding B, Wolf S, Lucas WJ, Beachy RN (1993) A dysfunctional movement
protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus
spread in transgenic plants. Plant J 4:959–970
Lee SC, Hwang IS, Choi HW, Hwang BK (2008) Involvement of the pepper antimicrobial protein
CaAmp1 gene in broad spectrum disease resistance. Plant Physiol 148:1004–1020
Lee YH, Jung M, Shin SH, Lee JH, Choi SH, Her NH, Lee JH, Ryu KH, Paek KY, Harn CH (2009)
Transgenic peppers that are highly tolerant to a new CMV pathotype. Plant Cell Rep 28:223–
232
Lee G, Shim HK, Kwon MH, Son SH, Kim KY, Park EY, Yang JK, Lee TK, Auh CK, Kim D Kim
YS (2013) RNA virus accumulation is inhibited by ribonuclease activity of 3D8 scFv in
transgenic Nicotiana tabacum. Plant Cell Tissue Organ Cult 115:189–197
Lee HA, Kim S, Kim S, Choi D (2017) Expansion of sesquiterpene biosynthetic gene clusters in
pepper confers nonhost resistance to the Irish potato famine pathogen. New Phytol
215:1132–1143
Leibman D, Wolf D, Saharan V, Zelcer A, Arazi T, Yoel S, Gaba V, Gal-On A (2011) A high level
of transgenic viral small RNA is associated with broad potyvirus resistance in cucurbits. Mol
Plant Microbe Interact 24:1220–1238
Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012) High-efficiency TALEN-based gene editing
produces disease-resistant rice. Nat Biotechnol 30:390
Li X, Shin S, Heinen S, Dill-Macky R, Berthiller F, Nersesian N, Clemente T, McCormick S
Muehlbauer GJ (2015) Transgenic wheat expressing a barley UDP-glucosyltransferase
detoxifies deoxynivalenol and provides high levels of resistance to Fusarium graminearum.
Mol Plant Microbe Interact 28:1237–1246
Lionetti V, Fabri E, De Caroli M, Hansen AR, Willats WG, Piro G, Bellincampi D (2017) Three
pectin methylesterase inhibitors protect cell wall integrity for Arabidopsis immunity to Botrytis.
Liu XH, Zhang HW, Liu X, Liu XJ, Tan ZB, Rong TZ (2005) Isolation of the capsid protein gene of
maize dwarf mosaic virus and its transformation in maize. Sheng wu gong cheng xue bao Chin J
Liu X, Tan Z, Li W, Zhang H, He D (2009) Cloning and transformation of SCMV CP gene and
regeneration of transgenic maize plants showing resistance to SCMV strain MDB. Afr J
Liu N, Zhang X, Sun Y, Wang P, Li X, Pei Y, Hou Y (2017) Molecular evidence for the
involvement of a polygalacturonase-inhibiting protein, GhPGIP1, in enhanced resistance to
Verticillium and Fusarium wilts in cotton. Sci Rep 7:39840
Liu H, Soyars CL, Li J, Fei Q, He G, Peterson BA, Wang X (2018) CRISPR/Cas9-mediated
resistance to cauliflower mosaic virus. Plant Direct 2:e00047
Lin CY, Ku HM, Chiang YH, Ho HY, Yu TA, Jan FJ (2012) Development of transgenic
watermelon resistant to cucumber mosaic virus and Watermelon mosaic virus by using a single
chimeric transgene construct. Transgenic Res 21:983–993
Lin KY, Hsu YH, Chen HC, Lin NS (2013) Transgenic resistance to Bamboo mosaic virus by
expression of interfering satellite RNA. Mol Plant Pathol 14:693–707
Lindbo JA, Dougherty WG (1992a) Pathogen-derived resistance to a potyvirus: immune and
resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein
nucleotide sequence. Mol Plant Microbe Interact 5(2):144–153
Lindbo JA, Dougherty WG (1992b) Untranslatable transcripts of the tobacco etch virus coat protein
gene sequence can interfere with tobacco etch virus replication in transgenic plants and
protoplasts. Virology 189(2):725–733
Lindbo JA, Dougherty WG (2005) Plant pathology and RNAi: A brief history. Ann Rev Phytopath
43:191–204
Li Q, Lawrence CB, Xing HY, Babbitt RA, Bass WT, Maiti IB, Everett NP (2001) Enhanced
disease resistance conferred by expression of an antimicrobial magainin analogue in transgenic
tobacco. Planta 212:635–639
Li H, Conner RL, Chen Q, Graf RJ, Laroche A, Ahmad F, Kuzyk AD (2005) Promising genetic
resources for resistance to wheat streak mosaic virus and the wheat curl mite in wheat-
Thinopyrum partial amphiploids and their derivatives. Genet Resour Crop Evol 51:827–835
Liao LJ, Pan IC, Chan YL, Hsu YH, Chen WH, Chan MT (2004) Transgene silencing in
Phalaenopsis expressing the coat protein of Cymbidium mosaic virus is a manifestation of
RNAmediated resistance. Mol Breed 13:229–242
Lim HS, Ko TS, Hobbs HA, Lambert KN, Yu JM, McCoppin NK, Korban SS, Hartman GL,
Domier LL (2007) Soybean mosaic virus helper component-protease alters leaf morphology and
reduces seed production in transgenic soybean plants. Phytopathology 97:366–372
Lopez-Ochoa L, Ramirez-Prado J, Hanley-Bowdoin L (2006) Peptide aptamers that bind to a
geminivirus replication protein interfere with viral replication in plant cells. J Virol
80:5841–5853
Lopez C, Cervera M, Fagoaga C, Moreno P, Navarro L, Flores R, Pena L (2010) Accumulation of
transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza
virus in transgenic Mexican lime. Mol Plant Pathol 11:33–41
Lodge JK, Kaniewski WK, Tumer NE (1993) Broad-spectrum virus resistance in transgenic plants
expressing pokeweed antiviral protein. Proc Natl Acad Sci 90:7089–7093
Loeza-Kuk E, Gutiérrez-Espinosa MA, Ochoa-Martínez DL, Villegas-Monter Á, Mora-Aguilera G,
Palacios-Torres EC, Pérez-Molphe-Balch E (2011) Análisis de la resistencia en pomelo y limón
mexicano transformados con el gen p25 del Citrus tristeza virus. Agrociencia 45(1):55–65
Lucioli A, Noris E, Brunetti A, Tavazza R, Ruzza V, Castillo AG, Bejarano ER, Accotto GP
Tavazza M (2003) Tomato yellow leaf curl Sardinia virus rep-derived resistance to homologous
and heterologous geminiviruses occurs by different mechanisms and is overcome if virus-
mediated transgene silencing is activated. J Virol 77:6785–6798
Ludlow EJ, Mouradva A, Spangenberg G (2009) Post transcriptional gene silencing as an efficient
tool for engineering resistance to white clover mosaic virus in white clover. J Pl Physiol
166:1557–1567
Ma J, Song Y, Wu B, Jiang M, Li K, Zhu C, Wen F (2011) Production of transgenic rice new
germplasm with strong resistance against two isolations of Rice stripe virus by RNA interfer-
ence. Transgenic Res 20:1367–1377
Ma Z, Song T, Zhu L, Ye W, Wang Y, Shao Y, Tyler BM (2015) A Phytophthora sojae glycoside
hydrolase 12 protein is a major virulence factor during soybean infection and is recognized as a
PAMP. Plant Cell 27:2057–2072
Ma J, Chen J, Wang M, Ren Y, Wang S, Lei C, Cheng Z (2018) Disruption of OsSEC3A increases
the content of salicylic acid and induces plant defense responses in rice. J Exp Bot 69:1051–
1064
Maiti IB, Von Lanken C, Hong Y, Dey N, Hunt AG (1999) Expression of multiple virus-derived
resistance determinants in transgenic plants does not lead to additive resistance properties. J
Plant Biochem Biotechnol 8(2):67–73
Malnoy M, Venisse JS, Brisset MN, Chevreau E (2003) Expression of bovine lactoferrin cDNA
confers resistance to Erwinia amylovora in transgenic pear. Mol Breed 12:231–244
Makeshkumar T, Varma A, Singh KK, Malathi VG, Duttagupta M (2002) Coat protein mediated
resistance against potato virus Y in transgenic tobacco. Ind Phytopath 55(2):187–194
Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ,
Charpentier E, Haft DH, Horvath P (2015) An updated evolutionary classification of CRISPR–
Cas systems. Nat Rev Microbiol 13:722–736
Mäki-Valkama T, Valkonen JP, Lehtinen A, Pehu E (2001) Protection against potato virus Y (PVY)
in the field in potatoes transformed with the PVY P1 gene. Am J Potato Res 78:209–214
Matoušek J, Trněná L, Rakouský S, Riesner D (1994) Inhibition of potato spindle tuber viroid
(PSTVd) infectivity with antisense RNA transcripts. J Phytopathol 141:10–24
Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, Velasco R, Nagamangala Kanchiswamy C
(2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9
ribonucleoproteins. Front Plant Sci 7:1904
Manfredini C, Sicilia F, Ferrari S, Pontiggia D, Salvi G, Caprari C, De Lorenzo G (2005)
Polygalacturonase-inhibiting protein 2 of Phaseolus vulgaris inhibits BcPG1, a
polygalacturonase of Botrytis cinerea important for pathogenicity, and protects transgenic

plants from infection. Physiol Mol Plant Pathol 67:108–115
Mayee CD, Singh P, Dongre AB, Rao MRK, Raj S (2003) Transgenic Bt cotton. CICR Technical
Bulletin No.22. Nagpur. Central Institute for Cotton Research
Mawassi M, Gera A (2012) Controlling plant response to the environment: viral diseases. In:
Altman A, Hasegawa PM (eds) Plant biotechnology and agriculture: prospects for the 21st
century, pp 343–352
Macovei A, Sevilla NR, Cantos C, Jonson GB, Slamet-Loedin I, Čermák T, Voytas DF, Choi IR,
Chadha-Mohanty P (2018) Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted
mutagenesis confer resistance to Rice tungro spherical virus. Plant Biotechnol J 16(11):1918–
1927
McCue KF, Ponciano G, Rockhold DR, Whitworth JL, Gray SM, Fofanov Y, Belknap WR (2012)
Generation of PVY coat protein siRNAs in transgenic potatoes resistant to PVY. Am J Potato
Res 89:374–383
Mehta R, Radhakrishnan T, Kumar A, Yadav R, Dobaria JR, Thirumalaisamy PP, Jain RK,
Chigurupati P (2013) Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea
L.) to peanut stem necrosis disease through agrobacterium-mediated genetic transformation.
Indian J Virol 24:205–213
Mentag R, Lukevich M, Morency MJ, Seguin A (2003) Bacterial disease resistance of transgenic
hybrid poplar expressing the synthetic antimicrobial peptide D4E1. Tree Physiol 23:405–411
Minoia S, Carbonell A, Di Serio F, Gisel A, Carrington JC, Navarro B, Flores R (2014) Specific
argonautes selectively bind small RNAs derived from potato spindle tuber viroid and attenuate
viroid accumulation in vivo. J Virol 88:11933–11945
Missiou A, Kalantidis K, Boutla A, Tzortzakaki S, Tabler M, Tsagris M (2004) Generation of
transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Mol
Breed 14:185–197
Mitra A, Higgins DW, Langenberg WG, Nie H, Sengupta DN, Silverman RH (1996) A mammalian
2-5A system functions as an antiviral pathway in transgenic plants. Proc Natl Acad Sci
93:6780–6785
Moravčíková J, Matušíková I, Libantova J, Bauer M, Mlynárová LU (2004) Expression of a
cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic
potato plants. Plant Cell Tissue Organ Cult 79:161–168
Moravčíková J, Libantová J, Heldák J, Salaj J, Bauer M, Matušíková I, Gálová Z, Mlynárová Ľ
(2007) Stress-induced expression of cucumber chitinase and Nicotiana plumbaginifolia β-1,3-
glucanase genes in transgenic potato plants. Acta Physiol Plant 29:133–141
Mlotshwa S, Verver J, Sithole-Niang I, Prins M, Van Kammen AB, Wellink J (2002) Transgenic
plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene
results in resistance. Virus Genes 25:45–57
Mundembe R, Matibiri A, Sithole-Niang I (2009) Transgenic plants expressing the coat protein
gene of cowpea aphid-borne mosaic potyvirus predominantly convey the delayed symptom
development phenotype. Afr J Biotechnol 8:2682–2690
Muniz FR, De Souza AJ, Stipp LCL, Schinor E, Freitas W, Harakava R, Stach-Machado DR,
Rezende JAM, Mourão Filho FAA, Mendes BMJ (2012) Genetic transformation of Citrus
sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to
CTV infection. Biol Plant 56:162–166
Namukwaya B, Tripathi L, Tripathi JN, Arinaitwe G, Mukasa SB, Tushemereirwe WK (2012)
Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt
disease. Transgenic Res 21:855–865
Navarro B, Gisel A, Rodio ME, Delgado S, Flores R, Di Serio F (2012) Viroids: how to infect a host
and cause disease without encoding proteins. Biochimie 94:1474–1480
Nekrasov V, Wang C, Win J, Lanz C, Weigel D, Kamoun S (2017) Rapid generation of a transgene-
free powdery mildew resistant tomato by genome deletion. Sci Rep 7:482
Nelson RS, McCormick SM, Delannay X, Dubé P, Layton J, Anderson EJ, Fraley RT (1988) Virus
tolerance, plant performance of transgenic tomato plants expressing coat protein from tobacco
mosaic virus. Biotechnology 6:403
Niblett CL, Dickson E, Fernow KH, Horst RK, Zaitlin M (1978) Cross protection among four
viroids. Virology 91:198–203
Nickel H, Kawchuk L, Twyman RM, Zimmermann S, Junghans H, Winter S, Fischer R, Prüfer D
(2008) Plantibody-mediated inhibition of the potato leafroll virus P1 protein reduces virus
accumulation. Virus Res 136:140–145
Nishizawa Y, Saruta M, Nakazono K, Nishio Z, Soma M, Yoshida T, Nakajima E, Hibi T (2003)
Characterization of transgenic rice plants over-expressing the stress-inducible β-glucanase gene
Gns1. Plant Mol Biol 51:143–152
Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH (2006) Expression of artificial
microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420
Nobori T, Velásquez AC, Wu J, Kvitko BH, Kremer JM, Wang Y, Tsuda K (2018) Transcriptome
landscape of a bacterial pathogen under plant immunity. Proc Natl Acad Sci 115:E3055–E3064
Norelli JL, Mills JZ, Momol MT, Aldwinkle HS (1998) Effect of cercropintype transgenes on fire
blight resistanc of apple. Acta Hortic 489:273–278
Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Schweizer P (2010) HIGS: host-
induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell
22:3130–3141
Nomura K, Ohshima K, Anai T, Uekusa H, Kita N (2004) RNA silencing of the introduced coat
protein gene of turnip mosaic virus confers broad-spectrum resistance in transgenic Arabidopsis.
Ntui VO, Azadi P, Thirukkumaran R, Chin DP, Nakamura I, Mii M (2011) Increased resistance to
Fusarium wilt in transgenic tobacco co-expressing chitinase and wasabi defensin genes. Plant
Pathol 60:221e231
Ntui VO, Kong K, Khan RS, Igawa T, Janavi GJ, Rabindran R, Nakamura I, Mii M (2015)
Resistance to Sri Lankan cassava mosaic virus (SLCMV) in genetically engineered cassava
cv. KU50 through RNA silencing. PLoS One 10:e0120551
Nyaboga NE, Ateka ME, Bulimo DW (2008) Serological detection of virus diseases of sweet potato
in Kenya. J Appl Biosci 7:222–229
Ogwok E, Odipio J, Halsey M, Gaitán-Solís E, Bua A, Taylor NJ, Fauquet CM, Alicai T (2012)
Transgenic RNA interference (RNAi)-derived field resistance to cassava brown streak disease.
Mol Plant Pathol 13:1019–1031
Okada Y, Saito A, Nishiguchi M, Kimura T, Mori M, Hanada K, Sakai J, Miyazaki C, Matsuda Y,
Murata T (2001) Virus resistance in transgenic sweetpotato [Ipomoea batatas L.(lam)]
expressing the coat protein gene of sweet potato feathery mottle virus. Theor Appl Genet
103:743–751
Okada Y, Saito A (2008) Evaluation of resistance to complex infection of SPFMVs in transgenic
sweet potato. Breed Sci 58:243–250
Okada Y, Yoshinaga M (2010) Advanced resistance to Sweetpotato feathery mottle virus (SPFMV)
in transgenic sweetpotato. Sweetpotato Res Front 23:4
Okubara P, Blechl A, McCormick S, Alexander N, Dill-Macky R, Hohn T (2002) Engineering
deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene
acetyltransferase gene. Theor Appl Genet 106:74–83
Ordiz MI, Magnenat L, Barbas CF, Beachy RN (2010) Negative regulation of the RTBV promoter
by designed zinc finger proteins. Plant Mol Biol 72:621–630
Ortigosa A, Gimenez-Ibanez S, Leonhardt N, Solano R (2019) Design of a bacterial speck resistant
tomato by CRISPR/Cas9-mediated editing of SlJAZ2. Plant Biotechnol J 17(3):665–673
Ortigosa A, Gimenez-Ibanez S, Leonhardt N, Solano R (2018) Design of a bacterial speck resistant
tomato by CRISPR/Cas9-mediated editing of SlJAZ2. Plant Biotechnol J 17:665–673
Osusky M, Osuska L, Hancock RE, Kay WW, Misra S (2004) Transgenic Potatoes Expressing a
Novel Cationic Peptide are Resistant to Late Blight and Pink Rot. Transgenic Res 13:181–190
Ouibrahim L, Mazier M, Estevan J, Pagny G, Decroocq V, Desbiez C, Caranta C (2014) Cloning of

the Arabidopsis rwm1 gene for resistance to Watermelon mosaic virus points to a new function
for natural virus resistance genes. Plant J 79:705–716
Pack IS, Kim YJ, Youk ES, Lee WK, Yoon WK, Park KW, Kim CG, Harn CH, Kim HM, Jeong SC
(2012) A molecular framework for risk assessment of a virus-tolerant transgenic pepper line. J
Crop Sci Biotechnol 15:107–115
Park SM, Lee JS, Jegal S, Jeon BY, Jung M, Park YS, Han SL, Shin YS, Her NH, Lee JH, Lee MY
(2005) Transgenic watermelon rootstock resistant to CGMMV (cucumber green mottle mosaic
virus) infection. Plant Cell Rep 24:350–356
Park HM, Choi MS, Kwak DY, Lee BC, Lee JH, Kim MK, Kim YG, Shin DB, Park SK, Kim YH
(2012) Suppression of NS3 and MP is important for the stable inheritance of RNAimediated
Rice stripe virus (RSV) resistance obtained by targeting the fully complementary RSV-CP gene.
Mol Cells 33:43–51
Panopoulos NJ, Hatziloukas E, Afendra AS (1996) Transgenic crop resistance to bacteria. Field
Crops Res 45:85–97
Pang SZ, Jan FJ, Tricoli DM, Russell PF, Carney KJ, Hu JS, Fuchs M, Quemada HD, Gonsalves D
(2000) Resistance to squash mosaic comovirus in transgenic squash plants expressing its coat
protein genes. Mol Breed 6:87–93
Pavan S, Jacobsen E, Visser RG, Bai Y (2010) Loss of susceptibility as a novel breeding strategy for
durable and broad-spectrum resistance. Mol Breed 25:1
Peng HC, Mantelin S, Hicks GR, Takken FL, Kaloshian I (2016) The conformation of a plasma
membrane-localized somatic embryogenesis receptor kinase complex is altered by a potato
aphid-derived effector. Plant Physiol 171:2211–2222
Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Zou X (2017) Engineering canker-resistant plants
through CRISPR/Cas9 targeted editing of the susceptibility gene Cs LOB 1 promoter in citrus.
Plant Biotechnol J 15:1509–1519
Prasad K, Bhatnagar-Mathur P, Waliyar F, Sharma KK (2013) Overexpression of a chitinase gene
in transgenic peanut confers enhanced resistance to major soil borne and foliar fungal
pathogens. J Plant Biochem Biotechnol 22:222–233
Pratap D, Kumar S, Raj SK, Sharma AK (2011) Agrobacterium-mediated transformation of
eggplant (Solanum melongena L.) using cotyledon explants and coat protein gene of cucumber
mosaic virus. Indian J Biotechnol 10:19–24
Pratap D, Raj SK, Kumar S, Snehi SK, Gautam KK, Sharma AK (2012) Coat protein-mediated
transgenic resistance in tomato against a IB subgroup cucumber mosaic virus strain.
Phytoparasitica 40:375–382
Praveen S, Kushwaha CM, Mishra AK, Singh V, Jain RK, Varma A (2005) Engineering tomato for
resistance to tomato leaf curl disease using viral rep gene sequences. Plant Cell Tissue Org Cult
83:311–318
Prins M, Laimer M, Noris E, Schubert J, Wassenegger M, Tepfer M (2008) Strategies for antiviral
resistance in transgenic plants. Mol Plant Pathol 9:73–83
Prins M, Resende RD, Anker C, van Schepen A, de Haan P, Goldbach R (1996) Engineered RNA-
mediated resistance to tomato spotted wilt virus is sequence specific. Mol Plant Microbe Interact
9:416–418
Prins M, Kikkert M, Ismayadi C, De Graauw W, De Haan P, Goldbach R (1997) Characterization of
RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants expressing
NSM gene sequences. Plant Mol Biol 33:235–243
Pumplin N, Voinnet O (2013) RNA silencing suppression by plant pathogens: defence, counter-
defence and counter-counter-defence. Nat Rev Microbiol 11:745–760
Pyott DE, Sheehan E, Molnar A (2016) Engineering of CRISPR/Cas9-mediated potyvirus resis-
tance in transgene-free Arabidopsis plants. Mol Plant Pathol 17:1276–1288
Rachman DS, McGeachy K, Reavy B, Štrukelj B, Žel J, Barker H (2001) Strong resistance to potato
tuber necrotic ringspot disease in potato induced by transformation with the coat protein gene
sequences from an NTN isolate of potato virus Y. Ann Appl Biol 139:269–275
Ram MN, Mohandas S (2003) Transformation of African violet (Saintpaulia ionantha) with
glucanase-chitinase genes using Agrobacterium tumefaciens. Acta Hort 624:471–478
Raj SK, Singh R, Pandey SK, Singh B (2005) Agrobacterium-mediated tomato transformation and
regeneration of transgenic lines expressing tomato leaf curl virus coat protein gene for resistance
against TLCV infection. Curr Sci 88:1674–1679
Reyes CA, De Francesco A, Peña EJ, Costa N, Plata MI, Sendin L, Castagnaro AP, García ML
(2011) Resistance to citrus psorosis virus in transgenic sweet orange plants is triggered by coat
protein–RNA silencing. J Biotechnol 151:151–158
Ren W, Wang K, Liao P, Yang G, Zhao Y, Zhou Y (2016) The regulation of innate immunity by
nutritional factors. Biomed Res Int 2016:5138706
Reyes CA, Peña EJ, Zanek MC, Sanchez DV, Grau O, García ML (2009) Differential resistance to
Citrus psorosis virus in transgenic Nicotiana benthamiana plants expressing hairpin RNA
derived from the coat protein and 54K protein genes. Plant Cell Rep 28:1817–1825
Rodriguez-Hernabdez AM, Gosalvez B, Sempere RN, Burgos L, Aranda MA, Truniger V (2012)
Melon RNA interference (RNAi) lines silenced for cm-eIF4E show broad virus resistance. Mol
Rodríguez-Leal D, Lemmon ZH, Man J, Bartlett M, Lippman ZB (2017) Engineering quantitative
trait variation for crop improvement by genome editing. Cell 171:470–480
Rojas CM, Senthil-Kumar M, Wang K, Ryu CM, Kaundal A, Mysore KS (2012) Glycolate oxidase
modulates reactive oxygen species–mediated signal transduction during nonhost resistance in
Nicotiana benthamiana and Arabidopsis. Plant Cell 24:336–352
Rubio T, Borja M, Scholthof HB, Feldstein PA, Morris TJ, Jackson AO (1999) Broad-spectrum
protection against tombusviruses elicited by defective interfering RNAs in transgenic plants. J
Virol 73:5070–5078
Rubio J, Montes C, Castro Á, Alvarez C, Olmedo B, Munoz M, Miccono M (2015) Genetically
engineered Thompson Seedless grapevine plants designed for fungal tolerance: selection and
characterization of the best performing individuals in a field trial. Transgenic Res 24:43–60
Rudolph C, Schreier PH, Uhrig JF (2003) Peptide-mediated broad-spectrum plant resistance to
tospoviruses. Proc Natl Acad Sci 100:4429–4434
Rushton PJ, Reinstädler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters
containing defined regulatory elements provide novel insights into pathogen-and wound-
induced signaling. Plant Cell 14:749–762
Ryabov EV, Keane G, Naish N, Evered C, Winstanley D (2009) Densovirus induces winged
morphs in asexual clones of the rosy apple aphid, Dysaphis plantaginea. Proc Natl Acad Sci
U S A 106:8465–8470
Sano T, Nagayama A, Ogawa T, Ishida I, Okada Y (1997) Transgenic potato expressing a double-
stranded RNA-specific ribonuclease is resistant to potato spindle tuber viroid. Nat Biotechnol
15:1290–1294
Savenkov EI, Valkonen JP (2002) Silencing of a viral RNA silencing suppressor in transgenic
plants. J Gen Virol 83:2325–2335
Sarowar S, Kim YJ, Kim KD, Hwang BK, Ok SH, Shin JS (2009) Overexpression of lipid transfer
protein (LTP) genes enhances resistance to plant pathogens and LTP functions in longdistance
systemic signaling in tobacco. Plant Cell Rep 28:419–427
Saur IM, Kadota Y, Sklenar J, Holton NJ, Smakowska E, Belkhadir Y, Zipfel C, Rathjen JP (2016)
NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicoti-
ana benthamiana. Proc Natl Acad Sci 113:3389–3394
Saharan V, Jain D, Pareek S, Pal A, Kumaraswamy RV, Jakhar SK, Singh M (2016) Viral, fungal
and bacterial disease resistance in transgenic plants. In: Advances in plant breeding strategies:
agronomic, abiotic and biotic stress traits. Springer, Cham, pp 627–656
Sanford JC, Johnston SA (1985) The concept of parasite-derived resistance—deriving resistance
genes from the parasite’s own genome. J Theor Biol 113:395–405
Schouten HJ, Krens FA, Jacobsen E (2006) Cisgenic plants are similar to traditionally bred plants:
international regulations for genetically modified organisms should be altered to exempt
cisgenesis. EMBO Rep 7:750–753
Schwind N, Zwiebel M, Itaya A, Ding B, Wang MB, Krczal G, Wassenegger M (2009) RNAi-
mediated resistance to potato spindle tuber viroid in transgenic tomato expressing a viroid
hairpin RNA construct. Mol Plant Pathol 10:459–469
Schoonbeek HJ, Wang HH, Stefanato FL, Craze M, Bowden S, Wallington E, Ridout CJ (2015)
Arabidopsis EF-Tu receptor enhances bacterial disease resistance in transgenic wheat. New
Phytol 206:606–613
Schüttelkopf AW, Gros L, Blair DE, Frearson JA, van Aalten DM, Gilbert IH (2010)
Acetazolamide-based fungal chitinase inhibitors. Bioorgan Med Chem 18:8334–8340
Scorza R, Callahan AM, Damsteegt V, Levy L, Ravelonandro M (1997) Transferring potyvirus coat
protein genes through hybridization of transgenic plants to produce plum pox virus resistant
plums (Prunus domestica L.). In: In XVII international symposium virus and virus-like diseases
of temperate fruit crops, vol 472, pp 421–428
Senthil-Kumar M, Mysore KS (2013) Nonhost resistance against bacterial pathogens:
retrospectives and prospects. Annu Rev Phytopathol 51:407–427
Senthilkumar R, Cheng CP, Yeh KW (2010) Genetically pyramiding protease-inhibitor genes for
dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco. Plant
Biotechnol J 8:65–75
Sengoda VG, Tsai WS, De La Peña RC, Green SK, Kenyon L, Hughes J (2012) Expression of the
full-length coat protein gene of tomato leaf curl Taiwan virus is not necessary for recovery
phenotype in transgenic tomato. J Phytopathol 160:213–219
Seybold H, Trempel F, Ranf S, Scheel D, Romeis T, Lee J (2014) Ca2+ signalling in plant immune
response: from pattern recognition receptors to Ca2+ decoding mechanisms. New Phytol
204:782–790
Seo GJ, Kincaid RP, Phanaksri T, Burke JM, Pare JM, Cox JE, Hsiang TY, Krug RM, Sullivan CS
(2013) Reciprocal inhibition between intracellular antiviral signaling and the RNAi machinery
in mammalian cells. Cell Host Microbe 14:435–445
Shepherd DN, Mangwende T, Martin DP, Bezuidenhout M, Kloppers FJ, Carolissen CH, Monjane
AL, Rybicki EP, Thomson JA (2007) Maize streak virus-resistant transgenic maize: a first for
Africa. Plant Biotechnol J 5:759–767
Shekhawat UK, Ganapathi TR, Hadapad AB (2012) Transgenic banana plants expressing small
interfering RNAs targeted against viral replication initiation gene display high-level resistance
to banana bunchy top virus infection. J Gen Virol 93:1804–1813
Shin R, Han JH, Lee GJ, Peak KH (2002) The potential use of a viral coat protein gene as a
transgene screening marker and multiple virus resistance in pepper plants coexpressing coat
proteins of cucumber mosaic virus and tomato mosaic virus. Transgenic Res 11:215–219
Shin S, Mackintosh CA, Lewis J, Heinen SJ, Radmer L, Dill-Macky R, Baldridge GD, Zeyen RJ,
Muehlbauer GJ (2008) Transgenic wheat expressing a barley class II chitinase gene has
enhanced resistance against Fusarium graminearum. J Exp Bot 59:2371–2378
Shimizu T, Nakazono-Nagaoka E, Uehara-Ichiki T, Sasaya T, Omura T (2011) Targeting specific
genes for RNA interference is crucial to the development of strong resistance to Rice stripe
virus. Plant Biotechnol J 9:503–512
Shimizu T, Nagaoka EK, Akita F, Wei T, Sasaya T, Omura T, Ichiki TU (2012) Haripin RNA
derived from the gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus, confers
strong resistance to virus infection in transgenic rice plants. J of Biotech 157:421–427
Shimizu T, Yoshii M, Wei T, Hirochika H, Omura T (2009) Silencing by RNAi of the gene for
Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic
rice plants to the virus. Plant Biotechnol J 7:24–32
Shimizu T, Ogamino T, Hiraguri A, Nakazono-Nagaoka E, Uehara-Ichiki T, Nakajima M, Akutsu
K, Omura T, Sasaya T (2013) Strong resistance against Rice grassy stunt virus is induced in
transgenic rice plants expressing double-stranded RNA of the viral genes for nucleocapsid or
movement proteins as targets for RNA interference. Phytopathology 103:513–519
Sijen T, Wellink J, Hendriks J, Verver J, van Kammen A (1995) Replication of cowpea mosaic
virus RNA1 and RNA2 is specifically blocked in transgenic Nicotiana benthamiana plants
expressing the full-length replicase or movement protein genes. Mol Plant-Microbe Interact
8:340–347
Silva Y, Portieles R, Pujol M, Terauchi R, Matsumura H, Serrano M, Borrás-Hidalgo O (2013)
Expression of a microbial serine proteinase inhibitor gene enhances the tobacco defense against
oomycete pathogens. Physiol Mol Plant Pathol 84:99–106
Silva MS, Arraes FBM, de Araújo CM, Grossi-de-Sa M, Fernandez D, de Souza CE, Grossi-de-Sa
MF (2018) Potential biotechnological assets related to plant immunity modulation applicable in
engineering disease-resistant crops. Plant Sci 270:72–84
Simon AE, Roossinck MJ, Havelda Z (2004) Plant virus satellite and defective interfering RNAs:
new paradigms for a new century. Annu Rev Phytopathol 42:415–437
Simón-Mateo C, García JA (2011) Antiviral strategies in plants based on RNA silencing. BBA-
Gene Regul Mech 1809:722–731
Singh A, Chaudhary S, Shankar A, Prasad V (2019) Enhanced resistance to fungal pathogens
through selective utilization of useful microbial genes. In: New and future developments in
microbial biotechnology and bioengineering. Elsevier, Amsterdam, pp 87–93
Singh VB, Haq QMR, Malathi VG (2013) Antisense RNA approach targeting Rep gene of
Mungbean yellow mosaic India virus to develop resistance in soybean. Arch Phytopathol
Plant Protect 46:2191–2207
Sivamani E, Brey CW, Talbert LE, Young MA, Dyer WE, Kaniewski WK, Qu R (2002) Resistance
to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene.
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene
expression: total silencing by intron-spliced hairpin RNAs. Nature 407:319
Stafford CA, Walker GP, Ullman DE (2011) Infection with a plant virus modifies vector feeding
behavior. Proc Natl Acad Sci 108:9350–9355
Stanley J, Frischmuth T, Ellwood S (1990) Defective viral DNA ameliorates symptoms of
geminivirus infection in transgenic plants. Proc Natl Acad Sci 87:6291–6295
Stenger DC (1994) Strain-specific mobilization and amplification of a transgenic defective-
interfering DNA of the geminivirus beet curly top virus. Virology 203:397–402
Stam M, Mol JN, Kooter JM (1997) The silence of genes in transgenic plants. Ann Bot 79:3–12
Sridevi G, Parameswari C, Sabapathi N, Raghupathy V, Veluthambi K (2008) Combined expres-
sion of chitinase and β-1, 3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance
against Rhizoctonia solani. Plant Sci 175:283–290
Soler N, Plomer M, Fagoaga C, Moreno P, Navarro L, Flores R, Peña L (2012) Transformation of
Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes
coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to
the virus. Plant Biotechnol J 10:597–608
Souza JMT, Nickel O, Gonsalves D (2005) Development of virus resistant transgenic papayas
expressing the coat protein gene from a Brazilian isolate of papaya ringspot virus. Fitopatol Bras
30:357–365
Swaney S, Powers H, Goodwin J, Rosales LS, Dougherty WG (1995) RNA-mediated resistance
with nonstructural genes from the tobacco etch virus genome. Mol Plant Microbe Interact
8:1004–1011
Tavert-Roudet G, Ravelonandro M, Bachelier JC, Dunez J (1998) Transgenic Nicotiana
benthamiana plants containing the P1 gene of plum pox virus are resistant to virus challenge.
Tavladoraki P, Benvenuto E, Trinca S, De Martinis D, Cattaneo A, Galeffi P (1993) Transgenic
plants expressing a functional single-chain Fv antibody are specifically protected from virus
attack. Nature 366:469–472
Tacke E, Salamini F, Rohde W (1996) Genetic engineering of potato for broad-spectrum protection
against virus infection. Nat Biotechnol 14:1597
Tai TH, Dahlbeck D, Clark ET, Gajiwala P, Pasion R, Whalen M, Staskawicz BJ (1999) Expression
of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato. Proc Natl Acad Sci
96:14153–14158
Tang X, Xie M, Kim YJ, Zhou J, Klessig DF, Martin GB (1999) Overexpression of Pto activates
defense responses and confers broad resistance. Plant Cell 11:15–29
Tamarzizt HB, Chouchane SG, Lengliz R, Maxwell DP, Marrakchi M, Fakhfakh H, Gorsane F
(2009) Use of tomato leaf curl virus (TYLCV) truncated Rep gene sequence to engineer
TYLCV resistance in tomato plants. Acta Virol 53:99–104
Tinoco MLP, Dias BB, Dall’Astta RC, Pamphile J, Aragão FJ (2010) In vivo trans-specific gene
silencing in fungal cells by in planta expression of a double-stranded RNA. BMC Biol 8:27
Tran DT, Cho S, Hoang PM, Kim J, Kil EJ, Lee TK, Rhee Y, Lee S (2016) A codon-optimized
nucleic acid hydrolyzing single-chain antibody confers resistance to Chrysanthemums against
Chrysanthemum stunt viroid infection. Plant Mol Biol Report 34:221–232
Trifonova EA, Sapotsky MV, Komarova ML, Scherban AB, Shumny VK, Polyakova AM,
Lapshina LA, Kochetov AV, Malinovsky VI (2007) Protection of transgenic tobacco plants
expressing bovine pancreatic ribonuclease against tobacco mosaic virus. Plant Cell Rep
26:1121–1126
Trifonova EA, Romanova AV, Sangaev SS, Sapotsky MV, Malinovsky VI, Kochetov AV (2012)
Inducible expression of the gene of Zinnia elegans coding for extracellular ribonuclease in
Nicotiana tabacum plants. Biol Plant 56:571–574
Tougou M, Furutani N, Yamagishi N, Shizukawa Y, Takahata Y, Hidaka S (2006) Development of
resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus.
Plant Cell Rep 25:1213–1218
Tripathi L, Mwaka H, Tripathi JN, Tushemereirwe WK (2010) Expression of sweet pepper Hrap
gene in banana enhances resistance to Xanthomonas campestris pv. musacearum. Mol Plant
Pathol 11:721–731
Tripathi L, Tripathi JN, Shah T, Muiruri KS, Katari M (2019) Molecular basis of disease resistance
in banana progenitor Musa balbisiana against Xanthomonas campestris pv. musacearum. Sci
Rep 9:1–17
Tripathi L, Tripathi JN, Kiggundu A, Korie S, Shotkoski F, Tushemereirwe WK (2014) Field trial
of Xanthomonas wilt disease-resistant bananas in East Africa. Nat Biotechnol 32:868–870
Tougou M, Yamagishi N, Furutani N, Shizukawa Y, Takahata Y, Hidaka S (2007) Soybean dwarf
virus-resistant transgenic soybeans with the sense coat protein gene. Plant Cell Rep 26:1967–
1975
Tundo S, Kalunke R, Janni M, Volpi C, Lionetti V, Bellincampi D, D’Ovidio R (2016) Pyramiding
PvPGIP2 and TAXI-III but not PvPGIP2 and PMEI enhances resistance against Fusarium
graminearum. Mol Plant-Microbe Interact 29:629–639
Uma B, Rani TS, Podile AR (2011) Warriors at the gate that never sleep: non-host resistance in
plants. J Plant Physiol 168:2141–2152
van der Vossen EA, Gros J, Sikkema A, Muskens M, Wouters D, Wolters P, Pereira A, Allefs S
(2005) The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring
broad-spectrum late blight resistance in potato. Plant J 44:208–222
Vanderschuren H, Moreno I, Anjanappa RB, Zainuddin IM, Gruissem W (2012) Exploiting the
combination of natural and genetically engineered resistance to cassava mosaic and cassava
brown streak viruses impacting cassava production in Africa. PLoS One 7:e45277
van Vu T, Choudhury NR, Mukherjee SK (2013) Transgenic tomato plants expressing artificial
microRNAs for silencing the pre-coat and coat proteins of a begomovirus, tomato leaf curl New
Delhi virus, show tolerance to virus infection. Virus Res 172:35–45
van Schie C, Takken FL (2014) Susceptibility genes 101: how to be a good host. Annu Rev
Vaskin MFS, Vidal MS, Alves ED, Farinelli L, de Oliveira DE (2001) Co-suppression mediated
virus resistance in transgenic tobacco plants harboring the 3-untranslated region of Andean
potato mottle virus. Transgenic Res 10:489–499
Verma V, Sharma S, Devi SV, Rajasubramaniam S, Dasgupta I (2012) Delay in virus accumulation
and low virus transmission from transgenic rice plants expressing Rice tungro spherical virus
RNA. Virus Genes 45:350–359
Vidya CS, Manoharan M, Kumar CR, Savtthri HS, Sita GL (2000) Agrobacterium-mediated
transformation of tomato (Lycopersicon esculentum var. Pusa Ruby) with coat-protein gene of
Physalis mottle tymovirus. J Plant Physiol 156:106–110
Vincelli P (2016) Genetic engineering and sustainable crop disease management: opportunities for
case-by-case decision-making. Sustainability 8:495
Voinnet O (2008) Use, tolerance and avoidance of amplified RNA silencing by plants. Trends Plant
Sci 13:317–328
Wally O, Jayaraj J, Punja Z (2009) Comparative resistance to foliar fungal pathogens in transgenic
carrot plants expressing genes encoding for chitinase, β-1, 3-glucanase and peroxidise. Eur J
Wang M-B, Abbott DC, Waterhouse PM (2000) A single copy of a virus-derived transgene
encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol 1
(6):347–356
Wang X, Eggenberger AL, Nutter FW Jr, Hill JH (2001) Pathogen-derived transgenic resistance to
soybean mosaic virus in soybean. Mol Breed 8:119–127
Wang HZ, Zhao PJ, Xu JC, Zhao H, Zhang HS (2003) Virus resistance in transgenic watermelon
plants containing a WMV-2 coat protein gene. Acta Genet Sin 30:70–75
Wang X, Zhu X, Tooley P, Zhang X (2013) Cloning and functional analysis of three genes
encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic
CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens. Plant Mol
Biol 81:379–400
Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (2014) Simultaneous editing of three
homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat
Biotechnol 32:947
Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Zhao K (2016) Enhanced rice blast resistance by
CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One
11:e0154027
Wagaba H, Beyene G, Aleu J, Odipio J, Okao-Okuja G, Chauhan RD, Munga T, Obiero H, Halsey
ME, Ilyas M, Raymond P, Bua A, Taylor NJ, Miano D Alicai T (2017) Field level
RNAimediated resistance to cassava brown streak disease across multiple cropping cycles and
diverse east African agro-ecological locations. Front Plant Sci 7:2060
Wang B, Sumit R, Sahu BB, Ngaki MN, Srivastava SK, Yang Y, Bhattacharyya MK (2018)
Arabidopsis novel glycine-rich plasma membrane PSS1 protein enhances disease resistance in
transgenic soybean plants. Plant Physiol 176:865–878
Watanabe Y, Ogawa T, Takahashi H, Ishida I, Takeuchi Y, Yamamoto M, Okada Y (1995)
Resistance against multiple plant viruses in plants mediated by a double stranded-RNA specific
ribonuclease. FEBS Lett 372:165–168
Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in plants can
be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci
95:13959–13964
Waterhouse PM, Helliwell CA (2003) Exploring plant genomes by RNA-induced gene silencing.
Nat Rev Genet 4:29
Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence against viruses.
Nature 411:834–842
Wako T, Terami F, Hanada K, Tabei Y (2001) Resistance to Zucchini yellow mosaic virus (ZYMV)
in transgenic cucumber plants (Cucumis sativus L.) harboring the coat protein gene of ZYMV.
Bull Natl Res Inst Veg Ornament Plant Tea (Japan) 16:175–186
Wegener CB (2002) Induction of defence responses against Erwinia soft rot by an endogenous
pectate lyase in potatoes. Physiol Mol Plant Pathol 60(2):91–100
Westwood JH, Groen SC, Du Z, Murphy AM, Anggoro DT, Tungadi T, Smith AG (2013) A trio of
viral proteins tunes aphid-plant interactions in Arabidopsis thaliana. PLoS One 8:e83066
Weiberg A, Wang M, Bellinger M, Jin H (2014) Small RNAs: a new paradigm in plant-microbe
interactions. Annu Rev Phytopathol 52:495–516
Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott
D, Stoutjesdijk PA, Robinson SP (2001) Construct design for efficient, effective and high
throughput gene silencing in plants. Plant J 27(6):581–590
Whitfield AE, Rotenberg D (2015) Disruption of insect transmission of plant viruses. Curr Opin
Insect Sci 8:79–87
Wu HW, Yu TA, Raja JA, Wang HC, Yeh SD (2009) Generation of transgenic oriental melon
resistant to Zucchini yellow mosaic virus by an improved cotyledon-cutting method. Plant Cell
Rep 28(7):1053–1064
Wu HW, Yu TA, Raja J, Christopher SJ, Wang SL, Yeh SD (2010) Double-virus resistance of
transgenic oriental melon conferred by untranslatable chimeric construct carrying partial coat
protein genes of two viruses. Plant Dis 94(11):1341–1347
Wu Y, Zhou JM (2013) Receptor-Like kinases in plant innate immunity. J Integr Plant Biol 55
(12):1271–1286
Xu BL, Shi GY, Xue YY (2005) Plant regeneration of transgenic Huanghemi (Cucumis melo) and
identification of its resistance to virus disease. J Fruit Sci 22:734–736
Xu W, Yang S, Bhadury P, He J, He M, Gao L, Song B (2011) Synthesis and bioactivity of novel
sulfone derivatives containing 2, 4-dichlorophenyl substituted 1, 3, 4-oxadiazole/thiadiazole
moiety as chitinase inhibitors. Pestic Biochem Physiol 101:6–15
Xu G, Yuan M, Ai C, Liu L, Zhuang E, Karapetyan S, Dong X (2017) uORF-mediated translation
allows engineered plant disease resistance without fitness costs. Nature 545:491
Yu TA, Chiang CH, Wu HW, Li CM, Yang CF, Chen JH, Chen YW, Yeh SD (2011) Generation of
transgenic watermelon resistant to Zucchini yellow mosaic virus and papaya ringspot virus type
W. Plant Cell Rep 30(3):359–371
Yan F, Kuang Y, Ren B, Wang J, Zhang D, Lin H, Zhou H (2018) Highly efficient AT to GC base
editing by Cas9n-guided tRNA adenosine deaminase in rice. Mol Plant 11:631–634
Yadav JS, Ogwok E, Wagaba H, Patil BL, Bagewadi B, Alicai T, Gaitan-Solis E, Taylor NJ,
Fauquet CM (2011) RNAi-mediated resistance to cassava brown streak Uganda virus in
transgenic cassava. Mol Plant Pathol 12(7):677–687
Yang Y, Sherwood TA, Patte CP, Hiebert E, Polston JE (2004) Use of tomato yellow leaf curl virus
(TYLCV) Rep gene sequences to engineer TYLCV resistance in tomato. Phytopathology 94
(5):490–496
Yang X, Wang X, Li X, Zhang B, Xiao Y, Li D, Xie C, Pei Y (2008) Characterization and
expression of an nsLTPs-like antimicrobial protein gene from motherwort (Leonurus
japonicus). Plant Cell Rep 27(4):759–766
Yang X, Yie Y, Zhu F, Liu Y, Kang L, Wang X, Tien P (1997) Ribozyme-mediated high resistance
against potato spindle tuber viroid in transgenic potatoes. Proc Natl Acad Sci 94(10):4861–4865
Yang X, Niu L, Zhang W, Yang J, Xing G, He H (2018) RNAi-mediated SMV P3 cistron silencing
confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic
soybean. Plant Cell Rep 37:103–114
Yassaie M, Afsharifar AR, Niazi A, Salehzadeh S, Izadpanah K (2011) Induction of resistance to
barley yellow dwarf virus (PAV) in bread wheat using post-transcriptional gene silencing
(PTGS). Iran J Plant Pathol 47:67–82
Yuan Y, Zhong S, Li Q, Zhu Z, Lou Y, Wang L, Wang J, Wang M, Li Q, Yang D, He Z (2007)
Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue
conferring disease resistance with enhanced herbivore susceptibility. Plant Biotechnol J 5
(2):313–324
Yuan M, Wang S (2013) Rice MtN3/saliva/SWEET family genes and their homologs in cellular
organisms. Mol Plant 6:665–674
Yuan M, Chu Z, Li X, Xu C, Wang S (2010) The bacterial pathogen Xanthomonas oryzae
overcomes rice defenses by regulating host copper redistribution. Plant Cell 22:3164–3176
Zaccomer B, Cellier F, Boyer JC, Haenni AL, Tepfer M (1993) Transgenic plants that express genes
including the 30 untranslated region of the turnip yellow mosaic virus (TYMV) genome are
partially protected against TYMV infection. Gene 136(1-2): 87–94
Zaidi SSEA, Mukhtar MS, Mansoor S (2018) Genome editing: targeting susceptibility genes for
plant disease resistance. Trends Biotechnol 36:898–906
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Koonin
EV (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell
163:759–771
Zhang H, Tian Y, Zhou Y, Dang B, Lan H, Song G (1999) Introduction of pokeweed antiviral
protein cDNA into Brassica napus and acquisition of transgenic plants resistant to viruses. Chin
Sci Bull 4:701–704
Zhang P, Vanderschuren H, Fütterer J, Gruissem W (2005) Resistance to cassava mosaic disease in
transgenic cassava expressing antisense RNAs targeting virus replication genes. Plant
Biotechnol J 3(4):385–397
Zhang YY, Li HX, Ouyang B, Ye ZB (2006) Regulation of eukaryotic initiation factor 4E and its
isoform: implications for antiviral strategy in plants. J Integr Plant Biol 48:1129–1139
Zhang ZY, Fu FL, Gou L, Wang HG, Li WC (2010) RNA interference-based transgenic maize
resistant to maize dwarf mosaic virus. J Plant Biol 53:297–305
Zhang ZY, Yang L, Zhou SF, Wang HG, Li WC, Fu FL (2011) Improvement of resistance to maize
dwarf mosaic virus mediated by transgenic RNA interference. J Biotechnol 153:181–187
Zhang B, Oakes AD, Newhouse AE, Baier KM, Maynard CA, Powell WA (2013) A threshold level
of oxalate oxidase transgene expression reduces Cryphonectria parasitica-induced necrosis in a
transgenic American chestnut (Castanea dentata) leaf bioassay. Transgenic Res 22(5):973–982
Zhang M, Li Q, Liu T, Liu L, Shen D, Zhu Y (2015) Two cytoplasmic effectors of Phytophthora
sojae regulate plant cell death via interactions with plant catalases. Plant Physiol 167(1):164–
175
Zhang T, Zheng Q, Yi X, An H, Zhao Y, Ma S, Zhou G (2018) Establishing RNA virus resistance in
plants by harnessing CRISPR immune system. Plant Biotechnol J 16:1415–1423
Zhao B, Ardales EY, Raymundo A, Bai J, Trick HN, Leach JE, Hulbert SH (2004) The avrRxo1
gene from the rice pathogen Xanthomonas oryzae pv. oryzicola confers a nonhost defense
reaction on maize with resistance gene Rxo1. Mol Plant-Microbe Interact 17:771–779
Zhao B, Lin X, Poland J, Trick H, Leach J, Hulbert S (2005) A maize resistance gene functions
against bacterial streak disease in rice. Proc Natl Acad Sci 102:15383–15388
Zhao Y, Yang X, Zhou G, Zhang T (2019) Engineering plants virus resistance: from RNA silencing
to genome editing strategies. Plant Biotechnol J 18(2):328–336
Zhou Y, Yuan Y, Yuan F, Wang M, Zhong H, Gu M, Liang G (2012) RNAi-directed down-
regulation of RSV results in increased resistance in rice (Oryza sativa L.). Biotechnol Lett
34:965–972
Zhou J, Peng Z, Long J, Sosso D, Liu B, Eom JS, Huang S, Liu S, Vera Cruz C, Frommer WB,
White FF (2015) Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for
bacterial blight of rice. Plant J 82:632–643
Zhu YX, Ou-Yang WJ, Zhang YF, Chen ZL (1996) Transgenic sweet pepper plants from
agrobacterium mediated transformation. Plant Cell Rep 16:71–75
Zhu H, Xu X, Xiao G, Yuan L, Li B (2007) Enhancing disease resistances of super hybrid rice with
four antifungal genes. Sci China Life Sci 50:31–39
Zhu YJ, McCafferty H, Osterman G, Lim S, Agbayani R, Lehrer A, Schenck S, Komor E (2011)
Genetic transformation with untranslatable coat protein gene of sugarcane yellow leaf virus
reduces virus titers in sugarcane. Transgenic Res 20(3):503–512
Zipfel C, Felix G (2005) Plants and animals: a different taste for microbes. Curr Opin Plant Biol
8:353–360
Zrachya A, Kumar PP, Ramakrishnan U, Levy Y, Loyter A, Arazi T, Lapidot M, Gafni Y (2007)
Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its
expression and resistance to the virus. Transgenic Res 16:385–398
RNAi Technology: A Novel Platform in Crop
Protection 24
Munmi Borah and Naga Charan Konakalla
Abstract
Since long, plant breeding has been the sole option and traditional method for
developing resistant cultivar with gene manipulation against different crop pests.
Various strategies have been put forward to render plants resistant to fungi,
bacteria, viruses, insects and nematodes. In the recent years, RNA interference
(RNAi) has become a highly effective and commanding tool of functional
genomics for silencing the gene expression for crop enhancement. RNAi-
mediated gene suppression approaches have opened up a new path in the devel-
opment of eco-friendly biotech approaches for crop improvement by knocking-
out the specific genes for better stress tolerance and integrating novel traits in
various plant species including insect, pest, pathogen resistance and also
enhanced nutritional status. RNAi or RNA silencing is a sequence-specific
post-transcriptional gene silencing (PTGS) mechanism induced by double-
stranded RNA (dsRNA). DsRNA molecules have been shown to play a key
role by protecting plants from invasive nucleic acids. The approach could repre-
sent a simple and environmentally safe way for controlling plant pathogens and
pests. In this chapter, we review RNAi applications in plants to acquire resistance
against biotic stress such as viruses, fungi, bacteria and insect pests.
M. Borah (*)
Plant Virology Laboratory, Department of Plant Pathology, Assam Agricultural University, Jorhat,
Assam, India
e-mail: munmi.borah@aau.ac.in
N. C. Konakalla
Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp,
Sweden

https://doi.org/10.1007/978-981-15-6275-4_24
562 M. Borah and N. C. Konakalla
24.1 Introduction
India is known as a growing economic giant but the benefits of its progress are
mostly restricted to urban or semi-urban areas in India. Modern high input
monocropping-based intensive agriculture has resulted in loss of biodiversity, out-
break of pests and diseases and degradation of soil and water, which has ultimately
led to stagnation of agricultural production and productivity. Climatic changes and
plant health management are becoming major factors in the present scenario (Kumar
2013). Effective control of plant pathogens on economically important crop species
is the major challenge for sustainable agricultural production in India. Although
plant breeding has been the traditional method of manipulating a plant genome to
develop a resistant cultivar for controlling plant diseases, the introduction of genetic
engineering technology provides an entirely new approach. Presently, the cultivated
area of genetically modified crops that are resistant to diseases is less compared with
that of crops for tolerance to herbicide, or resistant to insects. Various strategies have
been put forward to render plants resistant to fungi, bacteria, viruses and nematodes.
In modern-day biotechnology-based approaches in plant disease management,
pathogen-derived resistance is considered as one of the most important approach.
RNAi-mediated gene suppression approach has opened new avenues in the devel-
opment of eco-friendly biotech approaches for crop protection by knocking-out the
specific genes. RNA interference (RNAi) technology has appeared to be a promising
and efficient technology. The advancement of RNAi as a novel non-transgenic gene
therapy against fungal, viral and bacterial infection in plants lies in the fact that it
controls gene expression via mRNA degradation, repression of translation and by
chromatin remodeling through small non-coding RNAs. RNA silencing mechanisms
are guided by processing the products of dsRNA degradation by Dicer-like proteins,
which are known as small interfering RNAs (siRNAs) and microRNAs (miRNAs).
The application of technologies like RNAi to silence several genes simultaneously
enhances the researcher’s capability to protect the agriculturally important crop
varieties against destructive pathogens and pests. The discovery of RNAi has
transformed the research areas of plant breeding and serves as a tool to recognize
the expression pattern of plant genomes and as a toolbox to control plant gene
expression quantitatively and qualitatively (Hirai and Kodama 2008), controlling
pathogenicity of plant parasites (Runo 2011) and enhancing resistance against biotic
(Wani et al. 2010) and abiotic stresses (Jagtap et al. 2011).
RNAi has revolutionized the chances of making custom “knock-downs” of
cistron activity. RNAi operates in each plant and animal and uses double-stranded
RNA (dsRNA) as a trigger that targets homologous mRNAs for degradation or
inhibition of their transcription or translation, whereby vulnerable genes are often
suppressed. This RNA-mediated cistron management technology has provided new
technologies for developing eco-friendly molecular tools for crop enhancement by
suppressing particular genes responsible for numerous stresses as well as disease
resistance. This chapter updates the current state on the use of RNAi, molecular
principles underlying the biology of this phenomenon and development of RNAi
24 RNAi Technology: A Novel Platform in Crop Protection 563
technologies in relation to plants and discusses strategies and applications of this

technology in plant disease and pest management.
24.2 The Concept of RNAi
‘RNA interference’ or RNA silencing, is an endogenous machinery, existing in all

eukaryotes, that controls gene expression at the post-transcriptional level in several
developmental events and contribute to immunity of a plant against invading nucleic
acids. It has most likely been evolved as a potent mechanism for cells to suppress
foreign genes. The combining features of this phenomena include the production of
small RNAs (21–26 nucleotides (nts) that act as sequence-specific determinants for
down-regulating gene expression (Waterhouse et al. 2001; Hannon 2002; Pickford
and Cogoni 2003) and the requirement of one or additional members of the
Argonaute proteins (Hammond et al. 2001). RNAi operates by triggering the action
of dsRNA intermediates, which are processed into RNA duplexes of 21–24 nts by a
ribonuclease III-like enzyme called Dicer (Fire et al. 1998; Bernstein et al. 2001).
The products of dicing are double-stranded small interfering RNAs (siRNAs), which
are of 21–24 nt in length, where the guide strand (ssRNA) is subsequently
incorporated into the RNA-induced silencing complex (RISC), including Argonaute
and other related proteins (RNase H enzymes), guiding the cognate RNA molecules
for degradation (Hammond et al. 2000; Tang et al. 2003). The host genome encodes
small RNAs called miRNAs that are responsible for endogenous gene silencing. The
microRNAs (miRNAs) are naturally existing small non-coding RNA molecules
(containing about 22 nucleotides) found in plants, animals and some viruses. The
main function of miRNA is to regulate gene expression. MicroRNAs have been part
of the organism over evolutionary time, so the organisms have adapted with the
presence of the miRNA. miRNAs are products of dsRNAs encoded in genes of the
host genome. miRNAs are endogenous. The dsRNAs triggering gene silencing on
the other hand can be initiated by several external sources such as invading nucleic
acids such as transposons and viruses conferring plant immunity through expression
of endogenous or transgenic antisense sequences, expression of inverted repeat
sequences or RNA synthesis during viral replication. The cascade leading to the
generation of mature siRNA begins with transcription by RNA polymerase II
(in animals), RNA polymerase III (from a shRNA template), or RNA polymerase
IV (in plants), forming double stranded RNA (dsRNA) (Voinnet 2005). The remark-
able feature of RNA silencing in plants is that once it is triggered in a certain cell, a
mobile signal is formed and spread through the whole plant causing the entire plant
to be silenced (Dunoyer et al. 2007). After triggering the RNA silencing mechanism,
the mobile signaling molecules can be spread over or amplified via production of
dsRNAs on the primary cleavage of product templates or by their cleavage into
secondary siRNAs. Accumulation of siRNAs is considered an indicator or reliable
marker of RNAi (Hutvagner and Zamore 2002; Tang et al. 2003). Moreover,
production of the secondary siRNAs leads to the increasing activity of silencing
via its spread from the first activated cell to the neighbouring cells, and systemically
through the system (Himber et al. 2003). The invention of RNA-binding protein
(PSRP1) in the plant phloem and its capability to bind 25 nts sRNA species add
further to the argument that siRNAs (24–26 nts) are the main and unique
components for the systemic silencing signal (Xie and Guo 2006). The extent of
cell-to-cell movements is dependent on the levels of siRNAs produced at the site of
silencing initiation but is not dependant on the presence of siRNA target transcripts
in either source or recipient cell (Li and Ding 2006).
24.3 RNAi in Plants
RNA-mediated gene control technology has provided new platforms for developing
environmentally friendly molecular tools for crop improvement (Umesh et al. 2012).
Two main categories of small regulatory RNAs are distinguished in plants, based on
their formation and function: (miRNAs) and (siRNAs). MiRNAs and siRNAs have
been shown to be highly conserved, important regulators of gene expression in
plants (Jones-Rhoades and Bartel 2006; Axtell and Bowman 2008). The modes of
action by which small RNAs control gene expression at the transcriptional and post-
transcriptional levels are now being evolved into tools for plant molecular biology
research. However, consequent work has shown that RNA silencing works on at
least three different levels in plants, first is the cytoplasmic silencing by dsRNA that
results in cleavage of mRNA and is known as PTGS. Secondly, endogenous mRNAs
are silenced by miRNAs, which negatively regulate gene expression by base pairing
to specific mRNAs, resulting in either RNA cleavage or arrest of protein translation.
Third, RNA silencing is associated with sequence-specific methylation of DNA and
the consequent suppression of transcription (TGS) (Mansoor et al. 2006). There are
evidences indicating that miRNAs can participate in biotic stress responses in plants.
The first such role of miRNAs in plants was described by Jones-Rhoades and Bartel
(2006). A number of miRNAs have been linked to biotic stress responses in plants,
and the role of these miRNAs in plants infected by pathogenic bacteria, viruses,
nematodes and fungi has been reported (Ruiz-Ferrer and Voinnet 2009; Katiyar and
Jin 2010). Additionally, miRNAs are also important in regulating plant–microbe
interactions during nitrogen (N) fixation by Rhizobium and tumour formation by
Agrobacterium species (Katiyar and Jin 2010). Moreover, Mishra et al. (2009)
detected a significant increase in the GC content of stress-regulated miRNA
sequences, which in turn supports the view that miRNAs act as ubiquitous regulators
under stress conditions. The GC content may also be considered a critical parameter
for predicting stress-regulated miRNAs in plants. The first plant-endogenous siRNA
that was found to be involved in plant biotic stress was nat-siRNAATGB2, which
regulates R-gene mediated effector triggered immunity (Katiyar et al. 2006). A
unique class of endogenous siRNA, the long siRNAs (lsiRNAs), is 30–40 nt long
and is prompted by bacterial infection or specific growth conditions, such as cell
suspension culture (Katiyar and Jin 2007). However, it may be considered that
generation of small RNAs is a mechanism which allows plants to modulate gene
expression programmes necessary for adaptation to stressful environments. Small
RNAs may facilitate the flexibility in environmental adaptation. The reason that
small RNAs have a high complexity in plants may be justified by the fact that plant
growth and reproduction are generally confined to many diverse and extreme
habitats.
24.4 Approaches to Induce RNAi in Plants
A major challenge for scientists in RNAi research is to induce/suppress a specific

target gene. Genes are induced by various methods. Most successful methods are
virus-induced gene silencing (VIGS), agro inoculation and particle bombardment.
Fenselau et al. (2012) have reported VIGS as the most successful method for
inducing gene activity in plants; different RNA and DNA viruses have been
modified to serve as vectors for gene expression. Replication of plant viruses
produces dsRNA replication intermediates very effectively as well as efficiently
because of a type of RNA silencing gene called VIGS (Senthilkumar and Mysore
2011). When viruses incorporated into plants, they trigger a post transcriptional gene
silencing (PTGS) response by generating dsRNAs as replicative intermediates of
viral RNAs. Similarly when transgenes are incorporated into plants, they trigger a
PTGS response where dsRNAs generated as deviant transgene coded RNAs (Tyagi
et al. 2008). Viral RNAs not only trigger PTGS, but they also serve as targets.
Cleavage of viral RNA results in reduction of virus titres in local and distant leaves
and the plant recovery phenotype (Godge et al. 2008). At the same time, all RNA
virus-derived expression vectors will not be useful as silencing vectors because
many have potent anti-silencing proteins, which directly interfere with the host
silencing machinery (Diaz-pendon and Ding 2008). Similarly, DNA viruses have
not been used extensively as expression vectors due to their size constraints for
movement (Wani and Sanghera 2010). Another one is agro inoculation; it is a
powerful method to study processes connected with RNAi. The injection of
agrobacterium carrying similar DNA constructs into the intracellular spaces of
leaves for triggering RNA silencing is known as agro inoculation or agro infiltration
(Hily and Liu 2007). In most cases, agro inoculation is used to initiate systemic
silencing or to monitor the effect of suppressor genes. In plants, cytoplasmic RNAi
can be induced efficiently by agro inoculation, similar to a strategy for transient
expression of T-DNA vectors after delivery by Agrobacterium tumefaciens
(Usharani et al. 2005; Karthikeyan et al. 2011). One of the important
non-biologistical methods is particle bombardment. As an alternative tool, protoplast
transformation was first described as a method for the production of transgenic plants
in 1987 (Sanford et al. 1987). Unique advantages of this methodology are discussed
in terms of the range of species and genotypes that have been engineered and
with high transformation frequencies. In plant research, the major applications of
biolistics include transient gene expression studies, production of transgenic plants
and inoculation of plants with viral pathogens (Taylor and Faquet 2002). In
this method, a linear or circular template is transferred into the nucleus by
microbombardment. Synthetic siRNAs are delivered into plants by biolistic pressure
Table 24.1 Exogenous application of naked dsRNA for RNAi-mediated protection against a range of viruses/viroids on different plants
566
dsRNA
Virus/ dsRNA target and expression
viroid size technique Host Virus inoculation Efficiency Reference
PMMoV Replicase gene In vitro N. tabacum cv. Co-inoculation No lesions observed Tenllado and
(977 bp) Xanthi, C. chinense Diaz-Ruiz
(2001)
PMMoV Replicase gene In vitro N. benthamiana Co-inoculation 18% infected Tenllado and
(977, 596 and Diaz-Ruiz
315 bp) (2001)
AMV RNA 3 (1124 bp) In vitro N. benthamiana Co-inoculation 0% infected Tenllado and
Diaz-Ruiz
(2001)
TEV HC-Pro gene In vitro N. tabacum cv. Co-inoculation 0% infected Tenllado and
(1483 bp) Xanthi Diaz-Ruiz
(2001)
PMMoV Replicase gene Bacterial N. benthamiana Co-inoculation; sprayed dsRNA Days 1–5: 0% infected Tenllado
(977 bp) HT115 and challenged, 3, 5, and 7 days Day 7: 80% infected et al. (2003a)
expression post-spray
PMMoV CP gene Bacterial N. benthamiana Co-inoculation; sprayed dsRNA CP: 27% infected Tenllado
(1081 bp) HC-Pro HT115 and challenged 5 days post-spray HC-Pro: 17.6% infected et al. (2003b)
gene (1492 bp) expression
CEVd Less than full- In vitro Gynuraaurantiaca, Co-inoculation 50% infected Carbonell
length dsRNA tomato et al. (2008)
PSTVd 180 bp In vitro Tomato Co-inoculation 100% infected, some Carbonell
(nucleotide plants showed delay in et al. (2008)
position 1–179) symptoms
CChMVd Less than full- In vitro Chrysanthemum Co-inoculation 50% infected Carbonell
length dsRNA et al. (2008)
M. Borah and N. C. Konakalla
24
TMV CP gene (480 bp) Bacterial Tobacco Co-inoculation 50% infected Yin et al.
M-JM109 (2009)
lacY
expression
SCMV CP gene (CP1: Bacterial Maize Co-inoculation. Sprayed dsRNA Co-inoculation Gan et al.
147 bp, HT115 and challenged 1, 3, 5, 7 and 9 days CP-1: 20% infected (2010)
CP2:140 bp) expression post-spray CP-2: 30% infected
Day 1: 0% infected
Day 3: 4% infected
Day 5: 12% infected
Day 7: 43.3% infected
Day 9: 72% infected
PVY NIb gene Bacterial Tobacco Co-inoculation NIb-1: 34% infected Sun et al.
(3 different M-JM109 NIb-2: 66% infected (2010a)
dsRNAs, all lacY NIb-3: 52% infected
500 bp) expression
PVY HC-Pro gene, Bacterial Tobacco Co-inoculation NIb: 28% infected Sun et al.
Nibgene CP gene HT115 HC-Pro: 54% infected (2010b)
(all 600 bp each) expression CP: 44% infected
RNAi Technology: A Novel Platform in Crop Protection
TMV MP gene, CP Bacterial Tobacco Co-inoculation MP: 34% infected Sun et al.
gene, RP gene (all HT115 CP: 52% infected (2010b)
480 bp each) expression RP: 66% infected
RNA: 60% infected
PRSV CP gene (279 bp) Bacterial Papaya Co-inoculation. Sprayed dsRNA 35% infected. Shen et al.
M-JM109 and challenged 1, 2, 3 and 5 days All others: 100% (2014)
lacY post-spray infected
expression
PSbMV CP gene (500 bp) In vitro Pea cv. Raman dsRNA sprayed and co-inoculated All 100% infected, Safarova
with virus. dsRNA was sprayed reduced viral titre et al. (2014)
after 1, 2 and 21 days post-
inoculation.
567
(continued)
568
dsRNA
Virus/ dsRNA target and expression
viroid size technique Host Virus inoculation Efficiency Reference
CymMV CP gene (237 bp) Bacterial Orchid Co-inoculation 20% infected Lau et al.
HT115 (2014)
expressions
TMV p126 (666 bp), CP In vitro N. tabacum cv. Co-inoculation p126: 35% infected Konakalla
gene (480 bp) Xanthi CP: 50% infected et al. (2016)
ZYMV HC-Pro, CP gene In vitro Cucumber, Co-inoculation HC-Pro (cucumber)- Kaldis et al.
watermelon and 82% (2018)
squash plants HC-Pro (watermelon) –
50%
HC-Pro (squash) – 18%
CP (cucumber) – 70%
CP (watermelon) – 43%
CP (squash) – 16%
AMV Alfalfa mosaic virus, CChMVd Chrysanthemum chlorotic mottle viroid, CEVd Citrus exocortis viroid, CP Coat protein, RP Replicase protein, CymMV
Cymbidium mosaic virus, HC-Pro Helper component protein, NIb Nuclear inclusion b, MP Movement Protein, PMMoV Pepper mild mottle virus, PPV Plum
pox virus, PRSV Papaya ringspot virus, PSbMV Pea seed-borne mosaic virus, PSTVd Potato spindle tuber viroid, PVY Potato virus Y, p126 Protein 126, RP
Replicase protein, SCMV Sugarcane mosaic virus, TEV Tobacco etch virus, TMV Tobacco mosaic virus, ZYMV Zucchini yellow mosaic virus
M. Borah and N. C. Konakalla
Table 24.2 RNAi against fungal pathogens

Pathogen Targeted region Reference
Magnaporthe oryzae eGFP Kadotani et al. (2003)
Cladosporium falvum cgl1 and cgl2 Segers et al. (1999)
F. oxysporum f. sp. conglutinans FOW2, FRP1 and OPR Zongli et al. (2015)
Blumeriagraminis f.sp. tritici Rnr Dimitar et al. (2014)
Blumeria graminis Mlo Schweizer et al. (2000)
Venturia inaequalis Multiple inverted repeats Fitzgerald et al. (2004)
to cause silencing of green florescent protein expression. Bombarding cells with

particles coated with dsRNA, siRNA or DNA that encode hairpin constructs as well
as sense or antisense RNA activates the RNAi pathway (Shabhir et al. 2010).
24.5 RNA Interference for Engineering Resistance Against Plant

Diseases
The effects of gene silencing in plants were used in efforts to develop resistance to
diseases caused by viruses, fungi and bacteria. This “pathogen-derived resistance”
was achieved by transforming plants with genes, or sequences, derived from the
pathogen, with the aim of blocking a specific step in the life or infection cycle of the
pathogen.
24.5.1 RNAi Against Plant Viruses
Plant viruses are responsible for a significant proportion of crop diseases and are
very difficult to combat due to the scarcity of effective counter measures, placing
them among the most important agricultural pathogens. RNAi application has
resulted in successful control of many economically important viral diseases in
plants (Francisco et al. 2004, Cakir and Tor 2010). The effectiveness of RNAi
technology for generating virus resistance in plants was first demonstrated in
1998. VIGS is one of the commonly used RNA silencing methods to control plant
viruses (Senthilkumar and Mysore 2011). (Refer Table 24.1).
24.5.2 Application of RNAi for Fungal Resistance Development
RNA interference is a powerful and versatile genetic tool that can be applied to
filamentous fungi of agricultural importance. It is shown that gene silencing plays an
important role in plant defence against multicellular microbial pathogens, such as
vascular fungi belonging to the Verticillium genus. Several components of RNA
silencing pathways were tested, of which many were found to affect Verticillium
defence. It is speculated that the gene silencing mechanisms affect regulation of
Verticillium-specific defence responses (Ellendorff et al. 2009). An early successful

application of the RNAi system using sense and antisense RNA was reported for the
pathogenic fungus Cryptococcus neoformans (Liu et al. 2002). The efficacy of RNAi
was demonstrated in Magnaporthe oryzae, Venturia inaequalis, Phytophthora
infestans, Histoplasma capsulatum and Blastomyces dermatitidis by expression of
the GFP gene in fungus and then silencing by RNAi. Rust fungi cause devastating
diseases of wheat and other cereal species globally. Gene fragments from the rust
fungus, Puccinia striiformis f. sp. tritici or P. graminis f. sp. tritici, were delivered to
plant cells through the Barley stripe mosaic virus (BSMV) system and some reduced
the expression of the corresponding genes in the rust fungus. The ability to detect
suppression was associated with the expression patterns of the fungal genes because
reduction was only detected in transcripts with relatively high levels of expression in
fungal haustoria. The results indicate that in plants the RNAi approach can be used in
functional genomics research for rust fungi and that it could potentially be used to
engineer durable resistance (Yin et al. 2011). The below examples are the RNAi
strategies used against different fungal species (Table 24.2).
24.5.3 RNA Silencing-Mediated Resistance to Plant Pathogenic

Bacteria
Very few researches have been appeared on the use of gene silencing against plant
pathogenic bacteria. Escobar et al. (2001) for the first-time documented RNAi
application for engineering resistance in plant against bacterial pathogen triggering
crown gall disease. In the particular disease, iaaM and ipt oncogenes were found
responsible for gall formation and these genes are pre-requisite for development of
galls. Therefore, for management of the disease these oncogenes were targetted.
With the help of RNAi technology, they showed that transgenic plants (Arabidopsis
thaliana and Lycopersicon esculentum) containing modified construct of these two
bacterial genes (s) showed resistance against crown gall. The transgenic genes shut
down the expression of iaaM and ipt oncogenes of the incoming bacterial pathogen,
thereby disturbing the hormonal production and ultimately, tumourogenesis or gall
formation process after infection. Dunoyer et al. 2007 also reported that plants
lacking the modified oncogenes were hyper-susceptible to A. tumefaciens. Another
example is the RNAi-mediated enhanced resistance to Xanthomonas oryzae, the leaf
blight bacterium due to successful knockdown of a rice homolog of OsSSI2 (Jiang
et al. 2009). Zhai et al. (2011) and Li et al. (2012) studied the function of several
miRNA target gene families of plant innate immune receptors (NBS-LRR) in
legumes and solanaceae, respectively. They gave a new insight into viral and
bacterial infection in plants that suppresses miR482- mediated silencing of R
genes. Considering the findings from different researchers (Zhai et al. 2011 and Li
et al. 2012), a general understanding can be drawn that miRNA can either act as up-
or down-regulators of bacterial invasion. Identification and characterization of
pathogen-responsive miRNAs that induced positive regulators of bacterial resistance
will open a flood gate to enhancement of transgenic plants that will involve the
constitutive over-expression of miRNA.
24.6 RNAi and Insect Pest Control in Agriculture
RNAi is a powerful tool for gene function studies and control of insect pests. Several
research groups have recently explored the possibility of conducting RNAi in insects
through different application methods. There is a wide range of target insects from
different insect orders, target genes and feeding methods, demonstrating the richness
in application of dsRNA and the potential of RNAi. Despite having been considered
for many years, application of RNAi technology to give resistance to herbivorous
insects has only just been realized. The key to the success of this approach would be
(a) insect species and its life stages; (b) the type of exogenous RNA: dsRNA, siRNA,
miRNA, etc.; (c) the dose and method of application; (d) the type of target gene and
its expression profile; (e) gene function and the type of tissue; (f) nucleotide
sequence and length of dsRNA; (g) persistence of the silencing effect and (h) gut
physiology.
Several crop insect pests belonging to different orders were tested for their
possible control by RNAi. In these insects, RNAi knockdown has been developed
for various genes encoding developmental proteins, salivary gland proteins, proteins
involved in host–insect interaction, hormone receptors and gut enzymes. Baum et al.
(2007) provided evidence for the potential use of RNAi to control insect pests in crop
protection and demonstrated the fact that it is possible to silence genes in insects
when they consume plant material expressing hairpin dsRNA constructs against
well-chosen target genes. They reported the reduction of corn root damage in
transgenic maize plants producing vacuolar H + ATPase dsRNA after infestation
of the plant with the western corn rootworm. In another report, the model plants
Nicotiana tabacum and Arabidopsis thaliana were modified with the cytochrome
P450 gene of Helicoverpa armigera. When the cotton bollworm larvae were fed
transgenic leaves, the levels of cytochrome P450 mRNA were reduced and larval
growth retarded (Mao et al. 2007). Bautista et al. (2009) studied the influence of
silencing the cytochrome P450 gene CYP6BG1 that is over-expressed in a
permethrin-resistant diamondback moth (Plutella xylostella) strain. When the gene
was silenced after consumption of a droplet of dsRNA solution, the moths became
significantly more sensitive to the pyrethroid insecticide. Another significant devel-
opment employing RNAi is that the susceptibility of insect pests to Bt toxins could
be enhanced by silencing the genes involved in Bt resistance development.
24.7 Conclusion
Application of RNAi in management of biotic stress will prove to be an incredible

revolution in the field of functional genomics and a breakthrough in plant molecular
genetics. If RNAi technology is developed successfully and employed for the
management of major diseases on a commercial scale, it can prove to be an

eco-friendly and biologically safe technology. Moreover, this technique eliminates
the risk associated with development of transgenics, and it will also have enormous
potential for engineering control of gene expression. An agronomically superior
cultivar can be engineered for additional plant fitness by using RNAi technology.
However, selection of targeting sequence and delivery of siRNA are major
challenges for plant molecular biologists. More understanding and exploration in
the field of RNAi promoting resistance is needed. Therefore, further molecular
research is needed to unfurl the factors affecting RNAi-mediated resistance and
solve all the challenges in delivering siRNA to the host system and identifying the
targeted region to effectively overcome the pathogen and promote crop
improvement.
References
Axtell MJ, Bowman JL (2008) Evolution of plant micro RNAs and their targets. Trends Plant Sci
13:343–349
Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plaetinck G,
Munyikwa T, Pleau M, Vaughn T, Roberts J (2007) Control of coleopteran insect pests through
RNA interference. Nat Biotechnol 25:1322–1326
Bautista MA, Miyata T, Miura K, Tanaka T (2009) RNA interference-mediated knockdown of
acytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval
resistance to permethrin. Insect Biochem Mol Biol 39:38–46
Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the
initiation step of RNA interference. Nature 409:363–366
Cakir C, Tor M (2010) Factors influencing barley stripe mosaic virus-mediated gene silencing in
wheat. Physiol Mol Plant Pathol 74:246–253
Carbonell A, de Alba ÁE, Flores R, Gago S (2008) Double-stranded RNA interferes in a sequence-
specific manner with the infection of representative members of the two viroid families.
Virology 371:44–53
Diaz-Pendon JA, Ding SW (2008) Direct and indirect roles of viral suppressors of RNA silencing in
pathogenesis. Annu Rev Phytopathol 46:303–326
Dimitar D, Stefanie L, Annika J, Daniela Axel H, Jeya-raman R, Nils S, Rajiv S, Benjamin K,
Patrick S (2014) Discovery of genes affecting resistance of barley to adapted and non-adapted
powdery mildew fungi. Genome Biol 15:518
Dunoyer P, Himber C, Ruiz-Ferrer V, Alioua A, Voinnet O (2007) Intra- and intercellular RNA
interference in Arabidopsis thaliana requires components of the micro RNA and heterochro-
matic silencing pathways. Nat Genet 39:848–856
Ellendorff U, Fradin EF, de Jonge R, Thomma BPHJ (2009) RNA silencing is required for
Arabidopsis defense against Verticillium wilt disease. J Exp Bot 60:591–602
Escobar MA, Civerolo EL, Summerfelt KR, Dandekar AM (2001) RNA immediated oncogene
silencing confers resistance to crown gall tumorigenesis. PNAS 98:13437–13442
Fenselau F, Jiawei W, Detlef W (2012) MIGS: miRNA-induced gene silencing. Plant J 70:541–547
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific
genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
Fitzgerald A, Van Kha JA, Plummer KM (2004) Simultaneous silencing of multiple genes in the
apple scab fungus Venturia inaequalis, by expression of RNA with chimeric inverted repeats.
Fungal Genet Biol 41:963–971
Francisco T, Cesar L, Ramon L, Diaz-Ruiz (2004) RNA interference as a new biotechnological tool
for the control of virus diseases in plants. Virus Res 102:85–96
Gan D, Zhang J, Jiang H, Jiang T, Zhu S, Cheng B (2010) Bacterially expressed dsRNA protects
maize against SCMV infection. Plant Cell Rep 29:1261–1268
Godge MR, Purkayastha A, Dasgupta I, Kumar PP (2008) Virus-induced gene silencing for
functional analysis of selected genes. Plant Cell Rep 27:209–219
Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates
post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296
Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001) Argonaute2, a link
between genetic and biochemical analyses of RNAi. Science 293(5532):1146–1150
Hannon GJ (2002) RNA interference. Nature 418:244–251
Hily JM, Liu Z (2007) An overview of small RNAs. In: Regulation of gene expression in plants
2007. Springer, Boston, MA, pp 123–147
Himber C, Dunoyer P, Moissiard G, Ritzenthaler C, Voinnet O (2003) Transitivity dependent and
independent cell-to-cell movement of RNA silencing. EMBO J 22:4523–4533
Hirai S, Kodama H (2008) RNAi vectors for manipulation of gene expression in higher plants.
Open Plant Sci J 2:21–30
Hutvagner G, Zamore PD (2002) A micro RNA in a Multiple-turnover RNAi enzyme complex.
Science 297:2056–2060
Jagtap UB, Gurav RG, Bapat VA (2011) Role of RNA interference in plant improvement.
Naturwissenschaften 98:473–492
Jiang CJ, Shimono M, Maeda S, Inoue H, Mori M, Hasegawa M, Sugano S, Takatsuji H (2009)
Suppression of the rice fatty-acid desaturase gene Os-SSI2 enhances resistance to blast and leaf
blight diseases in rice. Mol Plant-Microbe Interact 22:820–829
Jones-Rhoades MW, Bartel DP (2006) MicroRNAs and their regulatory roles in plants. Annu Rev
Plant Biol 57:19–53
Kadotani N, Nakayashiki H, Tosa Y, Mayama S (2003) RNA silencing in the pathogenic fungus
Magnaporthe oryzae. Mol Plant-Microbe Interact 16:769–776
Kaldis A, Berbati M, Melita O, Reppa C, Holeva M, Otten P, Voloudakis A (2018) Exogenously
applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome
move systemically and protect cucurbits against ZYMV. Mol Plant Pathol 19:883–895
Karthikeyan A, Pandian SK, Ramesh M (2011) Transgenic indica rice cv. ADT 43 expressing a Δ
1-pyrroline-5-carboxylate synthetase (P5CS) gene from Vigna aconitifolia demonstrates salt
tolerance. Plant Cell Tissue Organ Cult 107:383–395
Katiyar AS, Jin H (2007) Discovery of pathogen – regulated small RNAs in plants. Methods
Enzymol 427:215–227
Katiyar AS, Jin H (2010) Role of small RNAs in host-microbe interactions. Annu Rev Phytopathol
48:225
Katiyar AS, Morgan R, Dahlbeck D, Borsani O, Villegas A, Zhu JK, Staskawicz BJ, Jin H (2006) A
pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci USA
103:18002–18007
Konakalla NC, Kaldis A, Berbati M, Masarapu H, Voloudakis AE (2016) Exogenous application of
double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV
in tobacco. Planta 244:961–969
Kumar S (2013) Plant disease management under changing climatic scenario. J Mycol Plant Pathol
42:149–154
Lau SE, Mazumdar P, Hee TW, Song AL, Othman RY, Harikrishna JA (2014) Crude extracts of
bacterially-expressed dsRNA protect orchid plants against Cymbidium mosaic virus during
transplantation from in vitro culture. J Hortic Sci Biotechnol 89:569–576
Li F, Ding SW (2006) Virus counter defense: diverse strategies for evading the RNA silencing
immunity. Ann Rev Microbiol 60:503–531
Li X, Wang X, Zhang S, Liu D, Duan Y, Dong W (2012) Identification of soybean microRNAs

involved in soybean cyst nematode infection by deep sequencing. PLoS One. https://doi.org/10.
1371/journal.pone.0039650
Liu H, Cottrell TR, Pierini LM, Goldman WE, Doering TL (2002) RNA interference in the
pathogenic fungus Cryptococcus neoformans. Genetics 160:463–470
Mansoor M, Imran A, Mazhar H, Yusuf Z, Briddon RW (2006) Engineering novel traits in plants
through RNA interference. Trends Plant Sci 11:559–565
Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY (2007) Silencinga
cotton bollworm P450 monooxygenase gene by plant- mediated RNAi impairs larval tolerance
of gossypol. Nat Biotechnol 25:1307–1313
Mishra AK, Agarwal S, Jain CK, Rani V (2009) High GC content: critical parameter for predicting
stress regulated miRNAs in Arabidopsis thaliana. Bioinformation 4:151–154
Pickford AS, Cogoni C (2003) RNA-mediated gene silencing. Cell Mol Life Sci 60:871–882
Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev
Plant Biol 60:485–510
Runo S, Alakonya A, Machuka J, Sinha N (2011) RNA interference as a resistance mechanism
against crop parasites in Africa: a ‘Trojan horse’ approach. Pest Manag Sci 67:129–136
Safarova D, Brazda P, Navratil M (2014) Effect of artificial dsRNA on infection of pea plants by
pea seed-borne mosaic virus. Czech J Genet Plant Breed 50:105–108
Sanford JC, Klein TM, Wolf ED, Allen N (1987) Delivery of substances into cells and tissues using
a particle bombardment process. J Par Sci Technol 5:27–37
Schweizer P, Pokorny P, Schulze-Lefert P, Dudler R (2000) Double stranded RNA interference
with gene functions at the single cell in cereals. Plant J 24:895–903
Segers GC, Hamada W, Oliver RP, Spanu PD (1999) Isolation and characterisation of five different
hydrophobin-encoding cDNA from the fungal tomato pathogen Cladosporium fulvum. Mol Gen
Genet 261:644–652
Senthilkumar M, Mysore KS (2011) Virus-induced gene silencing can persist for more than two
years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato. Plant
Biotechnol J 9:797–806
Shabhir HW, Gulzar SS, Singh NB (2010) Biotechnology and plant disease control-role of RNA
interference. Am J Plant Sci 1:55–68
Shen W, Yang G, Chen Y, Yan P, Tuo D, Li X, Zhou P (2014) Resistance of non-transgenic papaya
plants to papaya ringspot virus (PRSV) mediated by intron-containing hairpin dsRNAs
expressed in bacteria. Acta Virol 58:261–266
Sun ZN, Yin GH, Song YZ, An HL, Zhu CX, Wen FJ (2010a) Bacterially expressed double-
stranded RNAs against hot-spot sequences of tobacco mosaic virus or potato virus Y genome
have different ability to protect tobacco from viral infection. Appl Biochem Biotechnol
162:1901–1914
Sun ZN, Song YZ, Yin GH, Zhu CX, Wen FJ (2010b) HpRNAs derived from different regions of
the NIb gene have different abilities to protect tobacco from infection with Potato virus Y. J
Tang G, Reinhart BJ, Bartel D, Zamore PD (2003) A Biochemical framework for RNA silencing in
plants. Genes Dev 17:49–63
Taylor NJ, Fauquet CM (2002) Microparticle bombardment as a tool in plant science and agricul-
tural biotechnology. DNA Cell Biol 21:963–977
Tenllado F, Dıaz-Ruız JR (2001) Double-stranded RNA-mediated interference with plant virus
infection. J Virol 75:12288–12297
Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR (2003a) Crude extracts of bacterially
expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol 3:1–1
Tenllado F, Barajas D, Vargas M, Atencio FA, González-Jara P, Díaz-Ruíz JR (2003b) Transient
expression of homologous hairpin RNA causes interference with plant virus infection and is
overcome by a virus encoded suppressor of gene silencing. Mol Plant-Microbe Interact
16:149–158
Tyagi H, Rajasubramaniam S, Rajam MV, Dasgupta I (2008) RNA interference in rice against Rice
Tungro Bacilliform virus results in its decreased accumulation in inoculated rice plants.
Umesh BJ, Ranjit GG, Vishwas AB (2012) Role of RNA interference in plant improvement.
Naturwissenschaften 1:1913–1919
Usharani KS, Surendranath B, Haq QMR, Malathi VG (2005) Infectivity analysis of a soybean
isolate of Mungbean yellow mosaic India virus by agro inoculation. J Gen Plant Pathol
71:230–237
Voinnet O (2005) Non-cell autonomous RNA silencing. FEBS Letters 579:5858–5871
Wani SH, Sanghera GS (2010) Genetic engineering for viral disease management in plants. Not Sci
Biol 2:20–28
Wani SH, Sanghera GS, Singh NB (2010) Biotechnology and plant disease control-role of RNA
interference. Am J Plant Sci 1:55–68
Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defense against viruses.
Nature 411:834–842
Xi Q, Guo H (2006) Systemic antiviral silencing in plants. Virus Res 118:1–6
Yin G, Sun Z, Liu N, Zhang L, Song Y, Zhu C, Wen F (2009) Production of double-stranded RNA
for interference with TMV infection utilizing a bacterial prokaryotic expression system. Appl
Yin C, Jurgenson JE, Hulbert SH (2011) Development of a host-induced RNAi system in the wheat
stripe rust fungus Puccinia striiformis f. sp. tritici. MPMI 24:554–561
Zhai J, Jeong DH, DePaoli E, Park S, Rosen BD, Li Y (2011) Micro RNAs as master regulators of
the plant NB-LRR defense gene family via the production of phased, transacting siRNAs. Genes
Dev 25:2540–2553
Zongli H, Urvi P, Natsumi M, Yuri T, Jose RB (2015) Down regulation of Fusarium oxysporum
endogenous genes by host-delivered RNA interference enhances disease resistance. Front Chem
3:1–10
Genome Editing for Plant Disease
Resistance 25
Rajeev Singh
Abstract
Plant diseases severely affect crop yield and quality, and this poses a huge threat
to global food security. Plant pathogens are a hazard for agriculture. Mostly
phytopathogens are known to misuse the dominantly inherited genes, called
susceptibility (S) genes, to facilitate their proliferation. Genetic disruption of
these genes has been one of the successful ways to combat the pathogens and
induce a durable disease resistance. Novel genome-editing technologies offer
opportunities to control viral, bacterial, fungal pathogens, etc., and implement a
pathogen resistance in plants. Site-directed mutagenesis, zinc finger nucleases
(ZFNs), transcription activator-like effector nucleases (TALENs), and clustered
regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated
protein 9 (Cas9) are some of the most important genetic tools witnessed in the
recent years. CRISPR/Cas9 has been reported as an effective tool since it is
versatile, less expensive, easier to design and implement, and has a higher success
rate. In this chapter, we focus on the use of the genome-editing techniques for the
development of transgene-free and durable disease-resistant crop varieties.
Keywords
Gene editing · CRISPR/Cas9 · Plant pathology · Plant diseases · Agriculture
R. Singh (*)
Center for Tumor Biology and Immunology, Philipps Universität Marburg, Marburg, Hessen,
Germany

https://doi.org/10.1007/978-981-15-6275-4_25
578 R. Singh
25.1 Introduction
Agriculture is one of the prime sources of survival and development of the society.
Due to the increasing population and requirement of high crop yield, we aim at
sustainable agriculture, which provides food security, whilst reducing the environ-
mental pressure. Plant breeding has been the most successful approach for develop-
ing new crop varieties and hence coping up with the increasing demand for food.
Crops are prone to pathogens (fungi, bacteria, viruses), which greatly affects the crop
yield (20–40% loss) and hence the intensifying the economic losses. The resulting
plant diseases adversely affect plant growth, impeding the quality and also affecting
the long-term storage of crops, which leads to a slow agricultural development
(Borrelli et al. 2018; Yin and Qiu 2019). In most parts of the world, the use of
chemical pesticides to curb plant diseases is one of the most common methods.
Although, the reports suggest that these pesticides pose a greater threat to the
environment, as they are not specific, and they may or may not be harmful to the
humans in the longer run. But, the high evolutionary potential of various pathogens
leads to different kinds of mutations and recombination, hence developing resistance
against different pesticides due to natural selection. This can have a greater geo-
graphical impact, as it leads to an increase in the development of resistant genotypes
and can easily spread to other locations (Damalas and Eleftherohorinos 2011). New
fumigation methods, improved diagnostic protocols, improved trade standards
among countries, and high throughput screening technologies are some of the
many initiatives being taken but looking at the current situation, we need a more
stagnant solution.
Conventional resistance breeding was based on the incorporation of the identified
natural and induced mutant allele in a preferred genotype through breeding
techniques. Although the different traditional genetic approaches to disease control
have mostly yielded positive results for decades, they have several limitations too.
These approaches can only be performed within the plants having enough genetic
variation and can mate with each other. These approaches can also be imprecise and
uncertain as they can transfer many different traits (large genome regions) instead of
just the resistance trait (single gene insertion). Also, genetic crossing and progeny
selection can be impeding resources like time and labor. Therefore, in the current
world, conventional techniques have to keep pace with the evident availability of
genome and transcriptome sequences, changing pathogens, and increasing global
food demand particularly during an era of global climate change (Gao 2018).
25.2 Genome Editing Technology
Genetic association studies, utilizing single nucleotide polymorphisms (SNPs) and

different other molecular markers identified from linkage studies, are becoming
increasingly helpful in determining the identification of quantitative trait loci
(QTL). These QTLs are becoming a part of the new breeding programs, as they
provide quantitative resistance to pathogens by using resistance (R) genes
introduced into varieties with agricultural characteristics.
25 Genome Editing for Plant Disease Resistance 579
Genome editing (also known as gene editing) is a group of technologies that

allows genetic material to be added, removed, or altered at specific locations in the
genome of a living organism. New breeding techniques (NBT) encompass current
and precise molecular approaches for genetic modification of single and multiple
gene targets (Nelson et al. 2018). The core processing of genome editing is based on
DNA double-stranded break (DSB) repair mechanics. DSB breaks are produced
using sequence-specific nucleases for recognizing specific DNA sequences. Two
major pathways repair DSBs: nonhomologous end joining (NHEJ) pathway and
homologous recombination (HR) pathway (Voytas and Gao 2014). NHEJ uses
different enzymes to repair the DSBs, whereas HR is more specific and uses a
homologous sequence as a template to regenerate the missing DNA sequences at
the breakpoint. Although the NHEJ pathway is error-prone and can result in inser-
tion or deletion mutations, most cells use this pathway to repair DSBs. However, in
the presence of a donor DNA template, the HR pathway is mostly used, which
results in precise and specific changes. Four different types of sequence-specific
nucleases are used in genome editing: meganucleases (MNs), zinc finger nucleases
(ZFNs), transcription activator-like effector nucleases (TALENs), and clustered
regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated pro-
tein (Cas9). In recent years, CRISPR/Cas9 editing is very effective and useful for
improving the agronomic traits in crops (Fig. 25.1) (Mohanta et al. 2017). CRISPR/
Cas9, unlike another gene editing (GE) technologies, is independent of protein
Fig. 25.1 A brief overview of the CRISPR/Cas9 system. Engineered CRISPR/Cas9 system
depends on RNA-guided nuclease, Cas9 to introduce double-stranded breaks in target DNA. A
single guide RNA, whose 20 nucleotides match the target DNA and a PAM (NGG or NAG, where
N is any nucleotide) are essentially required for cleavage of the DNA in a sequence-dependent
manner. Cas9 cleavage generates DSBs, which can be repaired through NHEJ or the HR pathway.
(Source: Mushtaq et al., Frontiers in Plant Science 2019)
580 R. Singh
Fig. 25.2 Different applications of CRISPR technology. (Source: Ahmad et al., J Cell Physiol.
2019: 1–17)
engineering steps and sequence alteration of the single-guide RNA is enough for
generate new DNA sequences. This technique requires a duplex-RNA structure
which includes CRISPR RNA (crRNA): trans-activating crRNA (tracrRNA), and
this guides the Cas9 nucleases to target DNA. For efficient usage of this technology,
the dual crRNA: tracrRNA structure is modified/engineered into single-guide RNA
(sgRNA) and is targeted to specific genomic loci (Fig. 25.2) (Mushtaq et al. 2018;
Ahmad et al. 2020).
25.3 Genome Editing for Resistance Against Bacterial

Pathogens
The diversity of bacterial pathogens, high multiplication rate, and increase in the
epidemics have led to an overall increase in bacterial diseases. Phytopathogenic
bacteria can spread in a lot of different ways and are difficult to control due to
undetected asymptomatic infections and paucity of specific agrochemicals. To target
the bacterial diseases more specifically, a lot of research has been conducted in
elucidating the molecular pathways in connection with various host–bacterial path-
ogen interactions. Many host plant genes have been identified, which also includes
some S genes which participate in this complex process. S genes have become one of
the popular targets for breeding crops that are resistant to bacterial diseases via
genome editing.
Phytopathogenic bacteria can be grouped as crop specific, such as Clavibacter
michiganensis, causal agent of tomato bacterial ring rot; polyphagous specific, such
Table 25.1 CRISPR/Cas9 applications for bacterial resistance

Plant Target
species Fungus gene Gene function Strategy
Oryza Bacterial blight SWEET13 Sucrose transporter Agrobacterium-mediated
sativa (Xanthomonas gene transformation of
oryzae embryogenic callus with
pv. oryzae) Cas9/gRNA expression
plasmid vectors and
TALEN
Citrus Citrus canker LOB1 Susceptibility (S) Agrobacterium-mediated
paradisi (Xanthomonas gene-promoting transformation of epicotyl
citri subspecies pathogen growth with Cas9/gRNA
citric) and pustule expression plasmid
formation vectors
Citrus Citrus canker LOB1 Susceptibility (S) Agrobacterium-mediated
sinensis (Xanthomonas gene-promoting transformation of epicotyl
Osbeck citri subspecies pathogen growth with Cas9/gRNA
citric) and pustule expression plasmid
formation vectors
Malus Fire blight DIPM-1 Susceptibility PEG-mediated protoplast
domestica (Erwinia DIPM- factor involved in transformation with
amylovora) 2 DIPM-4 fire blight disease CRISPR
ribonucleoproteins
Source: Borelli et al., Frontiers in Plant Science (2019)
CsLOB1 Lateral Organ Boundaries 1, DIPM DspE-interacting proteins of Malus
as Ralstonia solanacearum, which causes disease in multiple monocot and dicot

species; and “kingdom crosser,” such as Dickeya dadantii, which is an entomo-
phytopathogen and can affect plants and animals.
Few studies have been published where the application of the CRISPR/Cas
system to counteract crop bacterial diseases has been discussed (Table 25.1).
CRISPR/Cas9 mutagenesis of OsSWEET13 in rice has been performed to achieve
resistance against the bacterial blight disease caused by γ-proteobacterium
Xanthomonas oryzae pv. oryzae (Zhou et al. 2015). OsSWEET13 is an S gene that
encodes a sucrose transporter involved in plant–pathogen interaction. X. oryzae
produces an effector protein, PthXo2, inducing OsSWEET13 expression in the
host and the consequent condition of susceptibility. In a previous work which
concerns OsSWEET14 promoter mutagenesis adopting a TALEN approach, the
disruption of this gene rendered the X. oryzae effector unable to bind OsSWEET14
and it ultimately resulted in disease resistance (Li et al. 2012). Similarly, Zhou et al.
(2015) obtained a null mutation in OsSWEET13 for exploring PthXo2-dependent
disease susceptibility, and thereby the resultant mutants were resistant to bacterial
blight. In the future, genome-editing strategies for multiplexed recessive resistance,
using a combination of the major effectors and other resistance (R) genes, would be
the next step toward achieving bacterial blight resistance.
Citrus canker is another devastating disease that is caused by the bacterium
Xanthomonas citri ssp. citri (Xcc). CsLOB1 is a member of the lateral organ
582 R. Singh
boundaries domain (LBD) family of transcription factors, which was previously

identified as an S gene for Xcc (Hu et al. 2014). TheCsLOB1 promoter contains an
effector-binding element (EBE), which is recognized by the Xcc effector PthA4,
which activates CsLOB1 expression to facilitate canker advancement. In one study,
the CsLOB1 promoter EBE was targeted, whereas another study targeted the coding
region of CsLOB1 using CRISPR/Cas9. Both studies showed that
editingCsLOB1provided resistance to Xcc (Jia et al. 2017; Peng et al. 2017).
Although the potential negative effect of mutatingCsLOB1 on plant growth has yet
to be determined, the growth status of CsLOB1 null mutant was similar to the wild-
type plants (Jia et al. 2017), suggesting thatCsLOB1 is an ideal promoter for
engineering canker resistance in elite citrus varieties.
Although tomato is one of the most economically important crops throughout the
world, the crop yield and quality are still limited by several major pathogens,
including Pseudomonas syringae, Phytophthora spp., and Xanthomonas spp.
(Schwartz et al. 2015). A recent study has shown that mutation of a single gene in
Arabidopsis, DMR6 (downy mildew resistance 6), led to increased salicylic acid
levels and resistance to some plant pathogens, which includes bacteria and
oomycetes (Zeilmaker et al. 2015). The tomato orthologue SlDMR6–1 is also
upregulated in response to infection by P. syringae pv. tomato and Phytophthora
capsici. Null mutants ofSlDMR6–1, generated using the CRISPR/Cas9 system
showed resistance to P. syringae, P. capsica, and Xanthomonas spp. without
detrimental effects on tomato growth and development. Taken together, these results
suggest that knocking out DMR6 might be a good strategy to establish broad-
spectrum disease resistance to plants.
25.4 Genome Editing Resistance Against Fungal Pathogens
Fungal pathogens are prominently responsible for various plant diseases such as
mildew smut, rust, and others. These diseases have a drastic effect on the quality of
the crop yield and thereby greater economic losses. Due to increased genetic
diversity, these pathogens are steady with invading new hosts and compromising
the R gene-mediated resistance and thereby assuring the resistance to fungicides
(Doehlemann et al. 2017). Also due to the production of secondary metabolites such
as mycotoxins via mycotoxigenic fungi, these pathogens pose a greater threat to
humans and the livestock, which are exposed to contaminated feed. The evolving
knowledge of molecular mechanisms in the field of plant–pathogen interaction has
led to different strategies in the area of disease control. Modification of potential host
S genes is the prime target for editing via CRISPR/Cas9 technology.
Powdery mildew is one of the most common fungal diseases that affect a wide
variety of plants. Developing resistant varieties is one of the most effective
approaches to combat the disease. Hybridization of the resistance-induced R genes
from foreign species into the elite species was the traditional approach used against
this disease. With evolving generations of wheat powdery mildew, the resistance
genes are slowly lost and thereby we need more broad-spectrum and resistant
varieties.
The discovery of barley mildew resistance locus o (MLO) mutants was a signifi-
cant step in generation disease-resistant varieties (Büschges et al. 1997). The MLO
gene was cloned in 1997 and it encodes a protein with seven transmembrane
domains localized in the plasma membrane and is evolutionarily conserved in
monocots and dicots. It has been reported that MLO were S genes and homozygous
loss-of-function mutants had shown increased resistance against powdery mildew in
barley, Arabidopsis, and tomato (Piffanelli et al. 2004; Consonni et al. 2006; Bai
et al. 2008). Bread wheat is an allohexaploid that has three orthologues of barley
MLO (TaMlo-A1, B1, and D1). Using CRISPR/Cas9 technology the MLO genes
were modified and it showed improved resistance against Blumeria graminis f. sp.
tritici (Bgt) infection, demonstrating an important role of TaMlo genes in powdery
mildew disease (Wang et al. 2014). This shows that genome editing plays a greater
role in modifying targets within polyploidy genomes. In tomato, MLO knockout
mutants are generated by targeting SIMlo1, which is identified as one of the most
important of 16 SIMlo genes. SIMlo was targeted at two sites and a 48 bp deletion
was obtained, which generated plants that were self-pollinated to obtain CRISPR/
Cas cassette-free individuals. As a result, the new non-transgenic variety, “Tomello”
was fully resistant to Oidium neolycopersici, a tomato powdery mildew fungus
(Nekrasov et al. 2017).
Enhanced disease resistance 1 (EDR1) in Arabidopsis is highly conserved across
plant species and is also known to negatively regulate the resistance against Erysiphe
cichoracearum and thereby it becomes an important target for improving powdery
mildew. Wheat EDR1 has three homologs that were targeted by the CRISPR/Cas9
system leading to the generation of Taedr1 wheat plants. Taedr1 mutant plants have
shown to have resistance against Bgt, but without mildew-induced cell death (Zhang
et al. 2017).
Rice blast is one of the most devastating diseases caused by Magnaporthe oryzae,
and it affects the rice production drastically worldwide. To improve the adaptation of
rice during biotic or abiotic stresses, ethylene responsive factors (ERFs) play a major
role and these factors belong to APETELA2/ERF (AP2/ERF) superfamily.
M. oryzae is known to induce the expression of OsERF922and the plants resistant
to rice blast disease are generated by disrupting OsERF922and OsSEC3A genes in
rice using CRISPR/Cas9 technology (Wang et al. 2016). Overall, these examples put
forward the positive implications of the CRISPR/Cas9 system for crop improvement
as regards fungal disease resistance (Table 25.2).
25.5 Genome-Editing Resistance Against Viral Pathogens
Plant viruses are a serious threat to a lot of economically important crops and this is
because of the rapid evolvement of the viruses and involvement of the insect vectors.
Plant viruses are classified according to their genome structure into six categories:
(1) double-stranded DNA (dsDNA) viruses, (2) single-stranded DNA (ssDNA)
584 R. Singh
Table 25.2 CRISPR/Cas9 applications for fungal resistance

Target
Plant species Fungus gene Gene function Strategy
Triticum Powdery MLO-A1 Susceptibility Particle bombardment of
aestivum mildew (S) gene immature wheat embryos
(Blumeria involved in with Cas9/gRNA
graminis f. sp. powdery expression plasmid vectors
tritici) mildew disease
Solanum Powdery MLO1 Major Agrobacterium-mediated
lycopersicum mildew responsible for transformation of
(Oidium powdery cotyledons with Cas9/
neolycopersici) mildew gRNA expression plasmid
vulnerability vectors
Vitis vinifera Powdery MLO-7 Susceptibility PEG-mediated protoplast
mildew (S) gene transformation with
(Erysiphe involved in CRISPR
necator) powdery ribonucleoproteins
mildew disease
Vitis vinifera Gray mold WRKY52 Transcription Agrobacterium-mediated
(Botrytis factor involved transformation of
cinerea) in response to proembryonal masses with
biotic stress Cas9/gRNA expression
binary vectors
Theobroma Black pod NPR3 Regulator of the Agrobacterium-mediated
cacao disease immune system transient transformation of
(Phytophthora stage C leaves with Cas9/
tropicalis) gRNA expression binary
vectors
Oryza sativa Rice blast SEC3A Subunit of the Protoplast transformation
L. japonica disease exocyst with Cas9/gRNA
(Magnaporthe complex expression binary vectors
oryzae)
Oryza sativa Rice blast ERF922 Transcription Agrobacterium-mediated
L. japonica disease factor transformation of
(Magnaporthe implicated in embryogenic calli with
oryzae) multiple stress Cas9/gRNA expression
responses binary vectors
MLO, MILDEW RESISTANT LOCUS; NPR3, non-expressor of pathogenesis-related 3; ERF922,
ethylene responsive factor
viruses, (3) reverse-transcribing viruses, (4) double-stranded RNA (dsRNA) viruses,

(5) negative-sense single-stranded RNA (ssRNA–) viruses, and (6) positive-sense
single-stranded RNA (ssRNA+) viruses (Roossinck, Martin and Roumagnac 2015).
Remedies to combat the plant viral diseases have been growing and various novel
methods targeting the pathogen-derived resistance have been implemented. Genome
editing adds to novel tools that are being used against viral pathogens (Table 25.3).
Geminiviruses are a large group of plant DNA viruses which comprise of
360 species or more. They are a huge threat to plant families such as Cucurbitaceae,
Table 25.3 CRISPR/Cas9 applications for viral resistance

Target Gene
Plant species Virus gene function Strategy
Nicotiana BeYDV CP, rep, RCA Agrobacterium-mediated
benthamiana and IR mechanism transformation of leaves with
and Arabidopsis Cas9/gRNA expression plasmid
thaliana vectors
Nicotiana BSCTV LIR and RCA Agrobacterium-mediated
benthamiana rep/RepA mechanism transformation of leaves with
Cas9/gRNA expression plasmid
vectors
Nicotiana TYLCV CP, rep, RCA Agrobacterium-mediated
benthamiana BCTV and IR mechanism transformation of leaves with a
MeMV TRV vector in Cas9
Nicotiana TuMV GFP1, Replication Agrobacterium-mediated
benthamiana GFP2, mechanism transformation of leaves with a
HC-Pro, TRV vector in Cas13a
CP overexpressing plants
Nicotiana CMV ORF1, Replication Agrobacterium-mediated
benthamiana TMV 2, 3, CP mechanism transformation of leaves with
and Arabidopsis and FnCas9/gRNA expression binary
thaliana 30 UTR vectors floral dipping for
Arabidopsis
Cucumis sativus CVYV eIF4E Host factor Agrobacterium-mediated
ZYMV for RNA transformation of cut cotyledons
PRSV- viruses (without embryo) with Cas9/
W translation gRNA binary vectors
Arabidopsis TuMV eIF(iso) Host factor Agrobacterium-mediated
thaliana 4E for RNA transformation with Cas9/gRNA
viruses recombinant plasmid binary
translation vectors (floral dipping)
Oryza sativa RTSV eIF4G Host factor Agrobacterium-mediated
L. japonica for RNA transformation of immature
viruses embryos with Cas9/gRNA
translation expression plasmid vectors
Euphorbiaceae, Solanaceae, Malvaceae, and Fabaceae (Zaidi et al. 2016). These

viruses have a circular single-stranded DNA which is replicated through a rolling-
circle amplification mechanism via a double-stranded DNA (dsDNA) or by
recombination-mediated replication (Hanley-Bowdoin et al. 2013). The first two
genome-editing studies involving CRISPR/Cas9 approach on developing resistance
against geminiviruses focused on beet severe curly top virus (BSCTV) and bean
yellow dwarf virus (BeYDV) in N. benthamiana and Arabidopsis (Baltes et al. 2015)
(Ji et al. 2015). Begomoviruses which are a genus of geminiviruses are known to
infect dicotyledonous plants. Their genome is either monopartite or bipartite with a
common region of 220 bp (Gilbertson et al. 2015). To develop resistance against
begomoviruses, CRISPR/Cas9 system was expressed in the host cell nucleus to
586 R. Singh
target and cleave the virus during replication. This system was also tested against the
monopartite beet curly top virus (BCTV) and bipartite Merremia mosaic virus
(MeMV) geminiviruses. The results showed attenuated symptoms of both the
viruses (Ali et al. 2015, 2016).
Host-translation machinery is responsible for the synthesis of viral proteins. For
the translation of the viral proteins, a multicomponent translation complex consisting
of eukaryotic translation initiation factor 4E (eIF4E) and its isoforms, recruits
ribosomes to the 50 untranslated regions (UTRs) of mRNAs, because viruses do
not harbor ribosomes (Sanfaçon 2015). Loss of function mutation in the eIF(iso)4E
gene has shown to confer resistance against Turnip mosaic virus (TuMV) in
Arabidopsis mutants and does not affect the plant vigor. This makes eIF4E genes
an ideal target for generating broad-spectrum virus resistance (Lellis et al. 2002).
CRISPR/Cas9-generated mutations in eIF4E genes in cucumber led to resistance
against cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus
(ZYMV), and papaya ring spot mosaic virus-W (PRSV-W) (Chandrasekaran et al.
2016). Also, in cassava, where only two (novel cap-binding protein-1 (nCBP-1) and
nCBP-2) out five genes encoding eIF4E proteins can associate with viral genome-
linked proteins (VPgs), CRISPR/Cas9-generated ncbp-1/ncbp-2 double mutants
showed attenuated symptoms after infection with Cassava brown streak virus
(CBSV) (Gomez et al. 2019). With the advancement of the CRISPR/Cas9 system,
it is possible to target the plant viruses with RNA genomes eventually leading to the
production of RNA virus-resistant plants.
BeYDV, bean yellow dwarf virus; BSCTV, beet severe curly top virus; TYLCV,
tomato yellow leaf curl virus; BCTV, beet curly top virus; MeMV, Merremia mosaic
virus; TRV, tobacco rattle virus; CLCuKoV, cotton leaf curl Kokhran virus; TuMV,
turnip mosaic virus; CMV, cucumber mosaic virus; TMV, tobacco mosaic virus;
CVYV, cucumber vein yellowing virus; ZYMV, zucchini yellow mosaic virus;
PRSV-W, papaya ring spot mosaic virus-W; PVX, potato virus X; TCV, turnip
crinkle virus; CMV, cucumber mosaic virus; RTSV, rice tungro spherical virus; CP,
coat protein; Rep, replication association protein; IR, intergenic region; RCA,
rolling-circle amplification; LIR, long intergenic region; GFP1, green fluorescent
protein 1; GFP2, green fluorescent protein 2; HC-Pro, helper component proteinase
silencing suppressor; ORF, open reading frame; UTR, untranslated terminal repeat;
eIF4E, eukaryotic translation initiation factor 4E; eIF4G, eukaryotic translation
initiation factor 4G.
25.6 Future Perspectives
The present situation demands the reduction in the use of chemicals in pesticides,
improvement in crop yield, and development of disease-resistant varieties to ensure
food security around the globe. Genome editing could be one of the most useful tools
to target the upcoming challenges in agriculture. With the help of genome editing,
we can also overcome the limitations of conventional breeding for disease resistance.
Genome editing is specific and does not introduce any further changes than the target
site and it also bypasses genetic crosses and progeny selection. The use of the
CRISPR/Cas9 system is more and more justified as the knowledge of the metabolic
pathways has increased considerably and mostly disease resistance can be obtained
by modification of a single gene using this system. Also with the help of targeted
mutagenesis using the CRISPR system, susceptibility genes can be inactivated
which would lead to disease resistance. Although it is expected that additional S
genes will be discovered, hence proving more targets for editing. Nonetheless, as S
genes are responsible for plant growth and development, generating S gene mutants
can also have some detrimental effects. Before implementing the editing
technologies, we need to take into account the challenges that we would face.
We need to look into the feasible implementation of the concept from regulated
environments to the crop field conditions, which are certainly more dynamic. Due to
variable conditions, with time it is difficult to devise a stable method. We would also
need to test the fitness of the edited crop in the field, and thereby regular field tests
become a necessity. As discussed before, the downside of editing a gene could be a
disrupted physiological effect. For example, the triple knockouts of wheat TaMLO
were resistant to powdery mildew but also showed leaf chlorosis (Wang et al. 2014).
On the contrary, ethyl methanesulfonate (EMS)-induced triple mutants with non-
conservative point mutations did not show any phenotypic effects (Acevedo-Garcia
et al. 2017). Thereby to rule out the negative effects, regular checks of the agronomic
varieties which are generated in labs or greenhouses should be done and all the
parameters should be measured and statistically recorded to find if there exists a
discrepancy.
Secondly, we need to take into account the durability of the conferred disease
resistance. It is an important point if we have to maintain the resistance such that the
public use of the crop variety is sustained. To upscale the durability of the disease
resistance, we can stack up to different resistance genes via different modes of
action, we can focus on more stable systems, and we can perform good agricultural
practices, such as crop rotations and use of biocontrol agents. With the help of
CRISPR technology, we have created the knockout of TaMLO (Wang et al. 2014)
and TaEDR1 (Zhang et al. 2017) against the same disease, powdery mildew. In this
way, we can stack the resistance genes and can also create multiple resistances in a
single generation by multiplexing.
Thirdly, we need to overcome the limitations generated by targeted mutagenesis.
True genome editing of crop plants would introduce predetermined base changes at
one or several specific positions in a gene, whereas in targeted mutagenesis, random
mutations are introduced.
There are three major parts of plant pathology – pathogen, host, and a favorable
environment. Plant diseases occur with the presence of all three parts. By using
genome editing we could constrain any one part of the system and achieve
interrupted plant-pathogen interaction and thereby control the disease. To attain a
long-term success with CRISPR/Cas9 technology, we need to increase our knowl-
edge of molecular pathways, to know more about the specific genes, signaling
588 R. Singh
molecules, and receptor proteins that can be targeted for disease resistance. Alto-
gether, genome editing has become an important tool for molecular plant–microbe
interactions and disease-resistance breeding. With the ongoing development and
research in this area, environmentally sustainable agriculture would improve and
thereby accelerate the quality and quantity of the crop yield.
References
Acevedo-Garcia J, Spencer D, Thieron H, Reinstädler A, Hammond-Kosack K, Phillips AL
Panstruga R (2017) mlo-based powdery mildew resistance in hexaploid bread wheat generated
by a non-transgenic TILLING approach. Plant Biotechnol J 15:367–378
Ahmad N, Rahman MU, Mukhtar Z, Zafar Y, Zhang B (2020) A critical look on CRISPR-based
genome editing in plants. J Cell Physiol 235:666–682
Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM (2015) CRISPR/Cas9-mediated
viral interference in plants. Genome Biol 16:238
Ali Z, Ali S, Tashkandi M, Zaidi SS, Mahfouz MM (2016) CRISPR/Cas9-mediated immunity to
geminiviruses: differential interference and evasion. Sci Rep 6:26912
Bai Y, Pavan S, Zheng Z, Zappel NF, Reinstädler A, Lotti C, De Giovanni C, Ricciardi L,
Lindhout P, Visser R, Theres K, Panstruga R (2008) Naturally occurring broad-spectrum
powdery mildew resistance in a central American tomato accession is caused by loss of mlo
function. Mol Plant-Microbe Interact 21:30–39
Baltes NJ, Hummel AW, Konecna E, Cegan R, Bruns AN, Bisaro DM, Voytas DF (2015)
Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system.
Nat Plants 1:15145
Borrelli VMG, Brambilla V, Rogowsky P, Marocco A, Lanubile A (2018) The enhancement of
plant disease resistance using CRISPR/Cas9 technology. Front Plant Sci 9:1245–1245
Büschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, van Daelen R, van der
Lee T, Diergaarde P, Groenendijk J, Töpsch S, Vos P, Salamini F, Schulze-Lefert P (1997) The
barley Mlo gene: a novel control element of plant pathogen resistance. Cell 88:695–705
Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T,
Gal-On A (2016) Development of broad virus resistance in non-transgenic cucumber using
CRISPR/Cas9 technology. Mol Plant Pathol 17:1140–1153
Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V,
Kemmerling B, Schulze-Lefert P, Somerville SC, Panstruga R (2006) Conserved requirement
for a plant host cell protein in powdery mildew pathogenesis. Nat Genet 38:716–720
Damalas CA, Eleftherohorinos IG (2011) Pesticide exposure, safety issues, and risk assessment
indicators. Int J Environ Res Public Health 8:1402–1419
Doehlemann G, Ökmen B, Zhu W, Sharon A (2017) Plant pathogenic fungi. Microbiol Spectr
5:703–726
Gao C (2018) The future of CRISPR technologies in agriculture. Nat Rev Mol Cell Biol
19:275–276
Gilbertson RL, Batuman O, Webster CG, Adkins S (2015) Role of the insect supervectors Bemisia
tabaci and Frankliniella occidentalis in the emergence and global spread of plant viruses. Annu
Rev Virol 2:67–93
Gomez MA, Lin ZD, Moll T, Chauhan RD, Hayden L, Renninger K, Beyene G, Taylor NJ,
Carrington JC, Staskawicz BJ, Bart RS (2019) Simultaneous CRISPR/Cas9-mediated editing
of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom
severity and incidence. Plant Biotechnol J 17:421–434
Hanley-Bowdoin L, Bejarano ER, Robertson D, Mansoor S (2013) Geminiviruses: masters at
redirecting and reprogramming plant processes. Nat Rev Microbiol 11:777–788
Hu Y, Zhang J, Jia H, Sosso D, Li T, Frommer WB, Yang B, White FF, Wang N, Jones J (2014)
Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease.
Proc Natl Acad Sci U S A 111:E521–E529
Ji X, Zhang H, Zhang Y, Wang Y, Gao C (2015) Establishing a CRISPR–Cas-like immune system
conferring DNA virus resistance in plants. Nat Plants 1:15144
Jia H, Zhang Y, OrbovićV XJ, White FF, Jones JB, Wang N (2017) Genome editing of the disease
susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnol J
15:817–823
Lellis AD, Kasschau KD, Whitham SA, Carrington JC (2002) Loss-of-susceptibility mutants of
Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection. Curr Biol
12:1046–1051
Li T, Liu B, Spalding M, Weeks DP, Yang B (2012) High-efficiency TALEN-based gene editing
produces disease-resistant rice. Nat Biotechnol 30:390–392
Mohanta TK, Bashir T, Hashem A, Abd Allah E, Bae H (2017) Genome editing tools in plants.
Genes (Basel) 8(12):399
Mushtaq M, Bhat JA, Mir ZA, Sakina A, Ali S, Singh AK, Tyagi A, Salgotra RK, Dar AA, Bhat R
(2018) CRISPR/Cas approach: a new way of looking at plant-abiotic interactions. J Plant
Physiol 224-225:156–162
Nekrasov V, Wang C, Win J, Lanz C, Weigel D, Kamoun S (2017) Rapid generation of a transgene-
free powdery mildew resistant tomato by genome deletion. Sci Rep 7:482–490
Nelson R, Wiesner-Hanks T, Wisser R, Balint-Kurti P (2018) Navigating complexity to breed
disease-resistant crops. Nat Rev Genet 19:21–33
Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Yao L, Zou X (2017) Engineering canker-resistant
plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in
citrus. Plant Biotechnol J 15:1509–1519
Piffanelli P, Ramsay L, Waugh R, Benabdelmouna A, D’Hont A, Hollricher K, Jørgensen JH,
Schulze-Lefert P, Panstruga R (2004) A barley cultivation-associated polymorphism conveys
resistance to powdery mildew. Nature 430:887–891
Roossinck MJ, Martin D, Roumagnac P (2015) Plant virus metagenomics: advances in virus
discovery. Phytopathology 105:716–727
Sanfaçon H (2015) Plant translation factors and virus resistance. Viruses 7:3392–3419
Schwartz AR, PotnisN TS, Wilson M, Patané J, Martins J, Minsavage GV, Dahlbeck D,
Akhunova A, Almeida N, Vallad GE, Barak JD, White FF, Miller SA, Ritchie D, Goss E,
Bart RS, Setubal JC, Jones JB, Staskawicz BJ (2015) Phylogenomics of Xanthomonas field
strains infecting pepper and tomato reveals diversity in effector repertoires and identifies
determinants of host specificity. Front Microbiol 6:535–549
Voytas DF, Gao C (2014) Precision genome engineering and agriculture: opportunities and
regulatory challenges. PLoS Biol 12:e1001877
Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (2014) Simultaneous editing of three
homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat
Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu YG, Zhao K (2016) Enhanced rice blast
resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene
OsERF922. PLoS One 11:e0154027
Yin K, Qiu JL (2019) Genome editing for plant disease resistance: applications and perspectives.
Philos Trans R Soc Lond Ser B Biol Sci 374:20180322
Zaidi SS, Tashkandi M, Mansoor S, Mahfouz MM (2016) Engineering plant immunity: using
CRISPR/Cas9 to generate virus resistance. Front Plant Sci 7:1673
Zeilmaker T, Ludwig NR, Elberse J, Seidl MF, BerkeL VDA, Schuurink RC, Snel B, Van den
Ackerveken G (2015) DOWNY MILDEW RESISTANT 6 and DMR6-LIKE OXYGENASE
1 are partially redundant but distinct suppressors of immunity in Arabidopsis. Plant J
81:210–222
590 R. Singh
Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D (2017) Simultaneous modification of three
homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat.
Plant J 91:714–724
Zhou J, Peng Z, Long J, Sosso D, Liu B, Eom JS, Huang S, Liu S, Vera Cruz C, Frommer WB,
White FF, Yang B (2015) Gene targeting by the TAL effector PthXo2 reveals cryptic resistance
gene for bacterial blight of rice. Plant J 82:632–643
Green Nanotechnology and Its Application
in Plant Disease Management 26
V. B. Nargund, J. U. Vinay, K. N. Basavesha, S. Chikkanna,
S. Jahagirdar, and R. R. Patil
Abstract
Nanotechnology is a new approach for the management of plant diseases.

Varieties of microorganisms and plant extracts have been used for the effective
synthesis of green metal nanoparticles. Thus, green synthesis of nanoparticles is
the most convenient, simple, and environmentally friendly way, and by avoiding
the use of toxic chemicals and the generation of harmful/dangerous by-products it
minimizes the side effects of chemical and physical processes. The use of
nanoparticles due to their superior properties is common and has been intensively
studied in recent years. The physical and in vitro effects of antimicrobial,
antioxidative and non-toxic nanoparticles obtained by green synthesis are becom-
ing increasingly important. Metal nanoparticles have both antifungal and
antibacterial activity, hence these are future weapons to combat plant diseases.
For effective disease management, nanoparticles must be incorporated as one of
the components in integrated disease management. Future studies will probably
focus on obtaining nanoparticles with antimicrobial effects at the maximum level
and toxicity at the minimum level. Because of this reason, synthesizing metallic
nanoparticles, especially by non-toxic green synthesis methods, which are used in
many application fields.
V. B. Nargund (*)
College of Agriculture, University of Agricultural Sciences, Dharwad, Karnataka, India
J. U. Vinay · K. N. Basavesha · S. Chikkanna · S. Jahagirdar · R. R. Patil
Department of Plant Pathology, College of Agriculture, University of Agricultural Sciences,
Dharwad, Karnataka, India

https://doi.org/10.1007/978-981-15-6275-4_26
592 V. B. Nargund et al.
26.1 Introduction
Plant pathogens cause significant crop loss as observed in several crop species. In
agriculture, annual crop losses due to pre- and post-harvest fungal diseases exceed
200 billion euros (Fernandez et al. 2010). Nearly one by fourth of food crops
worldwide are affected by mycotoxins such as aflatoxins, ergot toxins, Fusarium
toxins, patulin and tenuazonic acid (Schneider and Ullrich 1994). Horticultural
product waste is estimated around 20–30% in developing countries, so even if we
manage to reduce this amount by 5–10%, huge saves will be obtained. Reducing
these losses can not only improve farmers’ incomes but could also encourage more
consumption of this highly nutritious fruit in a region where per capita consumption
is only half of the recommended level. Now, increasing production efficiency and
decreasing post-harvest wastage of products by using novel sciences such as bio-
technology and nanotechnology could be counted as the best solution to this
problem.
Nano is a metric measure of one billionth of a meter and covers a width of
10 atoms. In terms of comparison with real objects, an example that hair is 150,000
nanometres may be given. The term “nano” has found, in the last decade, a wider
application in different fields of knowledge and sciences. Nano-science, nano-
technology, nano-particles and nano-chemistry are only a few of the new nano-
containing terms that occur frequently in scientific reports, popular books and
newspapers. The word nano comes from the ancient Greek through the Latin word
nanus, which means literally dwarf or very small. According to the convention of
International System of Units (SI), nano is used to indicate a reduction factor of 109
times. Hence, the word nano is typically used in nanometers (1 nm is equal to
109 m), and it encompasses systems whose size is greater than molecular
dimensions and below macroscopic sizes (generally >1 nm and <100 nm). Nano-
technology is the science of the small and the very small. It is the application and
manipulation of matter at a nanoscale. At this size, atoms and molecules act
differently and provide a variety of novel and interesting properties and uses. The
rapidly developing nanotechnology is the inter-disciplinary research and develop-
ment in the field of biology, chemistry, physics, food, medicine, electronics, aero-
space, agriculture and so on, which examines the synthesis or manufacture,
assembly, characterization of materials smaller than 100 nanometres in scale and
the application of miniature functional systems derived from these materials (Pearce
2012).
26.2 Nanoparticles and Their Properties
The process of removing toxic and waste metals from the environment through
microorganisms, plants and other biological structures is achieved by means of
oxidation, reduction or catalysis of metals with metallic nanoparticles. Metallic
nanoparticles produced by biological or green methods are used in the biomedical
field for purposes such as protection from harmful microorganisms, bio-imaging,
26 Green Nanotechnology and Its Application in Plant Disease Management 593
drug transport, cancer treatment, medical diagnosis and sensor construction because
of their unique properties such as insulating, optical, antimicrobial, antioxidant and
anti-metastasis properties and biocompatibility, stability and manipulability. Metal-
lic nanoparticles can be used in the industrial field due to their catalytic activity
(Singh et al. 2016). The reason for the great interest of scientists nowadays in
nanotechnology is that nanoparticles can exhibit different novel properties and
functions compared to normal bulk materials. The most important factors that enable
production of nanostructures are the desired size, shape and properties. These enable
their usage in various fields of bio-medical, agricultural and other life sciences. Other
reasons for the different behaviour of nanoparticles in physical, chemical, optical,
electrical and magnetic properties include the limitation of load carriers, size-
dependent electronic structures, increased surface/volume ratios, and other factors
incurred by the unique properties of atoms (Shah et al. 2015).
26.3 Methods of Nanoparticle Synthesis
The synthesis of nanoparticles can be natural or synthetic in origin, and they exhibit
unique properties at the nanoscale. Two basic approaches that include various
preparation methods are known from the past research. The first approach is “top-
down” which means breaking down of solid materials into small pieces by applying
external force/pressure. In this approach, many physical, chemical and thermal
techniques are used to provide the necessary energy for nanoparticle formation.
The second approach, known as “bottom-up”, is based on gathering and combining
atoms or molecules. These two approaches have advantages and disadvantages
relative to each other. The top-down approach is costlier to implement, and it is
impossible to obtain perfect surfaces and edges due to cavities and roughness that
can occur in nanoparticles in this method. Excellent nanoparticle synthesis results
can be obtained by the bottom-up approach. In the bottom-up approach, there are no
waste materials that need to be removed and nanoparticles having a smaller size can
be obtained with better control of their sizes.
26.4 Green Synthesis of Nanoparticles
The biological or green method of nanoparticle synthesis is represented as an

alternative to chemical and physical methods because it provides an environmentally
friendly way of synthesizing nanoparticles. Moreover, green synthesis does not
require expensive, harmful and toxic chemicals. Metallic nanoparticles with various
shapes, sizes, contents and physicochemical properties can be synthesized through
the biological methods which are actively used in recent years. Green synthesis can
be done using biological organisms such as fungi, bacteria, actinobacteria, yeasts,
molds and algae and plants and their products. Molecules in plants or
microorganisms such as proteins, enzymes, phenolic compounds, amines, alkaloids
and pigments are used as reducing agents in nanoparticle synthesis (Nadaroglu et al.
2017). In traditional chemical and physical methods, reducing agents involved in the
reduction of metal ions and stabilizing agents used to prevent undesired agglomera-
tion of the produced nanoparticles carry a risk of toxicity to the environment and to
the cell. Besides, the contents of the produced nanoparticles are thought to be toxic in
terms of the shape, size and surface chemistry. In the green synthesis method in
which nanoparticles with biocompatibility are produced, these agents are naturally
present in the employed biological organisms (Hussain et al. 2016). Because of rapid
development, affordable culturing costs and easy control and manipulation of the
growth environment, bacteria are clearly targets in the production of nanoparticles.
At the same time, it is known that some species of bacteria have special mechanisms
to suppress the toxicity of metals or heavy metals. Bacteria preferred for these
properties can perform nanoparticle synthesis in-situ and ex-situ. Through the use
of biochemical pathways and reducing agents such as proteins, enzymes, etc., which
present in the bacteria, metal ions can be reduced and precipitated for nanoparticle
production (Korbekandi et al. 2009).
Plants have great potential for detoxification, reduction and accumulation of
metals. These are promising, fast and economical in removing metal-borne
pollutants. Metallic nanoparticles having various morphological characteristics can
be produced intra-cellularly and extra-cellularly. The synthesis process is initiated by
addition of extracts obtained from plant parts such as leaves, roots and fruits into the
aqueous solution of metal ions. The materials present in plant extracts viz., sugar,
flavonoid, protein, enzyme, polymer and organic acid, which act as reducing agents,
result in bio-induction of metal ions into nanoparticles (Park et al. 2016). Synthesis
of nanoparticles can be done extra-cellularly or intra-cellularly with enzymes by
simply employing cultured and fast-breeding eukaryotic yeasts and molds. The
incubation conditions and the metallic ion solutions used in the synthesis influence
the size and distribution of the nanoparticles produced (Moghaddam et al. 2015).
The shape and size of the nanoparticles mainly depend on the variation in the
composition and concentration of active biomolecules in plants or microorganisms
and their interaction with the metal ion aqueous solution of the precursor. In case of
chemical and biological synthesis of nanoparticles, the aqueous metal ions from
metal salt precursor aqueous solutions are reduced, which leads to a change in the
colour of the reaction mixture and which provides qualitative indication of nanopar-
ticle formation. The nanoparticles synthesized from green reducing agents may
exhibit general toxicity, engendering serious concern for developing eco-friendly
processes. The process of the formation of nanoparticles begins by mixing a metal–
salt aqueous solution with plant or microbial extracts. During the synthesis of
nanoparticles, biochemical reduction of the metallic salt solution starts immediately
after reaction with green reducing agents and the change in the colour of the reaction
mixture indicates the preliminary confirmation of the formation of nanoparticles.
During synthesis, initially there is an activation period process, and in this period
metal ions are converted to zero-valent state from their mono or divalent oxidation
states, and hence the nucleation of reduced metal atoms occurs (Yu et al. 2016).
Further, the process of nanoparticle synthesis is followed by the integration of
smaller adjacent particles to form larger nanoparticles, which are thermodynamically
stable. Finally, the metal ions are reduced biologically. In this manner, growth
progression and nanoparticle aggregation occur to form a variety of shapes of
nanoparticles, such as spheres, cubes, triangles, rods, wires, hexagons and
pentagons. In the final stage of the process, the ability of plant or microbial extracts
to stabilize the nanoparticles determines the stable morphology of synthesized
nanoparticles. Significantly, the size and morphology of the nanoparticles are
influenced by properties of plant or microbial extracts (Niederberger and
Garnweitner 2006).
Different kinds of copper (Cu) and copper oxide (CuO) nanoparticles have been
synthesized from plant and microbial extracts. For example, Cu nanoparticles were
biologically synthesized using magnolia leaf extracts as the reducing agent and
stable nanoparticles sized from 40 to 100 nm were developed. Further, Cu
nanoparticles have shown potential antibacterial activity against Escherichia coli
(Harikumar and Aravind 2016). Syzygium aromaticum (clove) extracts were used in
the synthesis of Cu nanoparticles. Synthesized nanoparticles were spherical to
granular in morphology and their mean particle size was 40 nm (Tran et al. 2013).
Cu nanoparticles were synthesized by using the stem latex of Euphorbia nivulia
(common milk hedge). These nanoparticles were stabilized by peptides and
terpenoids which were present in latex. Also Procumbens, Calotropis procera,
Tinospora cordifolia and Euphorbia milii extracts were used to synthesize the
smallest spherical Cu nanoparticles with the size ranging from 43 to 342 nm (Rai
et al. 2009). Salem et al. (2016a, b) synthesized sulphur nanoparticles (SNPs) from
sodium thiosulphate in the presence of Punica granatum peel aqueous extracts at
room temperature. Sodium thiosulphate pentahydrate (Na2S2O35H2O) was
dissolved in Punica granatum peel extracts under mild stirring for 10 min. at room
temperature and then diluted with deionized water. Then the precipitation was
centrifuged to obtain suspended sulphur nanoparticles. Awwad et al. (2015)
synthesized SNPs from sodium thiosulphate in the presence of Albizia julibrissin
fruit extracts at room temperature. Sodium thiosulphate was dissolved in Albizia
julibrissin fruit extracts under stirring for 5 minutes at room temperature and then
diluted with sterile distilled water. Further, the precipitation was centrifuged and
nano pellets were dried.
Wei et al. (2009) synthesized AgNPs by reducing silver nitrate salts using
non-toxic and biodegradable chitosan as a green reducing agent. Green synthesis
of AgNPs was done by stirring and heating AgNO3 in chitosan solution. A change in
the colour from colourless to yellowish brown gave the preliminary confirmation of
AgNPs. Ponarulselvum et al. (2012) synthesized AgNPs by the reaction of silver
nitrate with the leaf extracts of Catheranthus roseus. Nanoparticle formation was
confirmed by change in the colour. Banerjee et al. (2014) synthesized AgNPs by
using M. balbisiana, Azadirachta indica and Ocimum tenuiflorum leaf extracts.
Similarly, Jalaluddin et al. (2016) synthesized AgNPs (~30.5 nm) using Aloe vera
leaf extract. Aloe vera leaf extract was added into the aqueous solution of silver
nitrate and incubated in the dark overnight at room temperature. Complete reduction
of AgNO3 to Ag+ ions was confirmed by the change in colour from colourless to
colloidal brownish yellow.
26.5 Characterization of Nanoparticles
Nanoparticles are characterized by their size, morphology and surface charge using
advanced microscopy techniques such as scanning electron microscopy (SEM),
transmission electron microscopy (TEM) and atomic force microscopy (AFM).
The average particle diameter and their size distribution and charge affect the
stability and distribution of the nanoparticles. Electron microscopy techniques are
very useful in retrieving the shape of polymeric nanoparticles. The surface charge of
the nanoparticles affects the physical stability and dispensability of the polymer
dispersion and their performance.
Particle size, distribution and morphology are the most important parameters of
characterization of nanoparticles. The morphology and size are measured by electron
microscopy. Recently, the fastest and most popular method of determining the
particle size has been dynamic light scattering (DLS). DLS is widely used to
determine the size of Brownian nanoparticles in colloidal suspensions in nano and
submicron ranges. Passing of monochromatic light (laser) into a solution of
nanoparticles causes a Doppler shift when the light hits the moving particle and
changes the wavelength of the incoming light. Scanning electron microscopy (SEM)
is a revealing morphological examination with direct visualization. The electron
microscopy techniques have several advantages in morphological and sizing analy-
sis. However, they provide limited information about the size distribution and true
population average. For SEM characterization, nanoparticle solutions should be first
converted into a dry powder, which is then mounted on a sample holder followed by
coating with a conductive metal, such as gold using a sputter coater. The sample is
then scanned with a focused fine beam of electrons (Jores et al. 2004). The surface
characteristics of the sample are obtained from the secondary electrons emitted from
the sample surface. The average size obtained by SEM is comparable with results
obtained by dynamic light scattering. Moreover, these techniques are time consum-
ing, costly and frequently need complementary information about sizing distribution
(Molpeceres et al. 2000).
TEM operates on a different principle than SEM. The sample preparation for
TEM is complex and time consuming because of its requirement to be ultra thin for
electron transmittance. The nanoparticles are deposited onto support grids or films.
To make nanoparticles withstand the instrument vacuum and facilitate handling,
they are fixed using either a negative staining material, such as phosphotungstic acid
or derivatives, uranyl acetate, etc., or by plastic embedding. The surface
characteristics of the sample are obtained when a beam of electrons is transmitted
through an ultra-thin sample, interacting with the sample as it passes through
(Molpeceres et al. 2000). Atomic force microscopy (AFM) offers ultra-high resolu-
tion in particle size measurement and is based on a physical scanning of samples at
the sub-micron level using a probe tip of atomic scale (Muhlen et al. 1996). The
instrument provides a topographical map of the sample based on forces between the
tip and the sample surface. Samples are usually scanned in contact or non-contact
mode depending on their properties. In contact mode, the topographical map is
generated by tapping the probe onto the surface across the sample and the probe
hovers over the conducting surface in non-contact mode. The prime advantage of
AFM is its ability to image non-conducting samples without any specific treatment,
thus allowing imaging of delicate biological and polymeric nano and
microstructures. AFM provides the most accurate description of size. Moreover,
the particle size obtained by the AFM technique provides a real picture, which helps
understand the effect of various biological conditions (Polakovic et al. 1999).
Green-synthesized (Catharanthus roseus) silver nanoparticles were characterized
using UV-Vis spectrophotometry and XRD. The result confirmed that silver
nanoparticles were successfully synthesized with an average size of 35–55 nm and
were crystalline in nature with face-centred cubic structure (Ponarulselvum et al.
2012). Banerjee et al. (2014) characterized the green-synthesized silver
nanoparticles by SEM, which showed an average size of 35–55 nm and XRD
showed that the particles were crystalline in nature. AgNPs synthesized using
aqueous leaf extracts of Urtica dioica (Linn.) were characterized by UV-vis spec-
troscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and trans-
mission electron microscopy (TEM). The results of characterization revealed that
AgNPs were in the size range of 20–30 nm and crystallized in face-centred cubic
structure (Jyoti et al. 2016). The synthesized sulphur nanoparticles were
characterized by X-ray diffraction (XRD) and scanning electron microscopy
(SEM). The average particle diameter was found to be 20 4 nm. SEM analysis
showed that the nanoparticles are crystalline in nature with a spherical shape (Salem
et al. 2016a, b). The list of published research papers on synthesis and characteriza-
tion of nanoparticles by various instruments is depicted in the below table
(Table 26.1).
26.6 Evaluation of Nanoparticles Against Plant Pathogens
Plants in nature are attacked by various kinds of pathogens which suppress their
growth and productivity. In view of the huge crop losses inflicted by pathogens,
various methods are used by farmers to check the pathogen attack, but none of them
offer perfect holistic control of the disease. Innovative and advanced methods are
needed to be integrated with conventional methods to enhance the efficiency of
disease management modules. Hence, nanoparticles have great scope in the man-
agement of plant diseases in future. Singh et al. (2013) reported that among the tested
15 nano-micronutrients, CuSO4 and Na2B4O7 were found to be most effective in
controlling rust disease of field peas. El-Hai et al. (2009) reported that the nano form
of manganese and zinc suppressed spread of damping-off and charcoal rot diseases
in sunflower. Metallic nanoparticles provide protection to plants through different
mechanisms. The simplest and most obvious treatment of nanoparticles is direct
application into the soil, on the seeds or foliage to protect plants from pathogen
invasion. Nanoparticles such as carbon tubes, cups, rods and clays can also be used
as carriers of some highly reactive chemicals such as pheromones, systemic acquired
resistance (SAR) inducing chemicals, polyamine synthesis inhibitors or even
concentrated active ingredients of pesticides for their controlled release, especially
Table 26.1 Synthesis and characterization of NPs by various instruments
598
Instruments
TEM/
Sl. UV-Vis PSA SEM/ HR-
No. Precursor Reducing agent (λ) nm nm XRD nm FTIR AFM FESEM TEM References
1 ZnCl2 Chitosan and 200–400 – Crystalline √ – Shape: – Vaseeharan
NaOH ZnO hexagonal et al. (2015)
Size: Size:
30–60 nm 100–200 nm
2 Zn(NO3)26H2O Chitosan 360 – – – Shape: – – Vinay et al.
spherical (2016)
to
irregular
3 Zn Chlamydomonas – – √ √ – Size: – Rao and
(O2CCH3)2(H2O)2 reinhardtii 55–80 nm Gautam (2016)
extract Shape:
Nanorod
4 Zn Passiflora 380 – Zn– √ √ Shape: – Santhoshkumar
(O2CCH3)2(H2O)2 caerulea extract 75.36% spherical et al. (2017)
O–22.36% Size: 70 nm
Size:
37.67 nm
5 ZnO Pseudomonas 365 67 – – Shape: – – Vinay et al.
fluorescens spherical (2017)
extract to
irregular
6 ZnSO4 Fusarium 261 – √ – Size: – – Harishkumar
sp. extract 20–60 nm and Savalgi
(2017)
7 Zn Glycosmis 351 – Crystalline √ – Shape: Size: Vijayakumar
(O2CCH3)2(H2O)2 pentaphylla ZnO spherical 36 nm et al. (2018)
V. B. Nargund et al.
26
Size: Size:
30 nm 32–40 nm
8 CuSO4.5H2O Phyllanthus 285 – 5–10 – – Size: Acharyulu et al.
amarus leaf 50 nm (2014)
9 CuSO4.5H2O Vitis vinifera leaf 384 – – – – – – Angrasan and
Subbaiya
(2014)
10 CuSO4.5H2O Nerium oleander 325–370 – – – – – – Gopinath et al.
leaf (2014)
11 CuSO4.5H2O Penicillium 265 80–179 – – – Shape: Shape: Honary et al.
aurantiogriseum, spherical spherical (2012)
P. citrinum and
P. waksmanii
12 Na2S2O3 Melia azedarach – – – – Shape: – Salem et al.
leaf spherical (2016a, b)
Size:
5–80 nm
13 FeCl36H2O Padina pavonica – √ √ – – Size: – El-Kassas et al.
extract 10–27.4 nm (2017)
Shape:
spherical
14 Na2S2O3 Sophora japonica – – √ √ – Shape: – Awwad et al.
pods spherical (2014)
Size: 80 nm
15 Na2S2O3 Albizia julibrissin – – √ √ – Shape: – Awwad et al.
Green Nanotechnology and Its Application in Plant Disease Management
pods spherical (2015)

Size:
10–100 nm
599
under flooded conditions. Hence, to reveal the potentiality of nanotechnology in

plant disease management, the effects can be discussed through two major goals and
perceptions. The goals are understanding the direct effect of nanoparticles (NPs) on
microorganisms/pathogens and the use of nanomaterials in formulating the
pesticides, i.e. nanopesticides.
Jo et al. (2009) reported that various forms of silver ions and nanoparticles
inhibited the plant pathogenic fungi, Bipolaris sorokiniana and Magnaporthe grisea.
Effective concentrations of AgNPs in inhibiting the colonization by 50% (EC50)
were higher for B. sorokiniana than for M. grisea. Inoculation assays in growth
chamber again confirmed that both ionic and nanoparticles of silver significantly
reduced the diseases caused by the above two fungi on perennial rye grass (Lolium
perenne). Kasprowicz et al. (2010) recorded a significant reduction in the mycelial
growth of Fusarium culmorum when spores were incubated with silver
nanoparticles. Safavi et al. (2011) reported that nano silver had good antibacterial
potential for removing bacterial contaminants in the tobacco plant tissue culturing
process.
The effect of silver nanoparticles at different concentrations was evaluated
against Golovinomyces cichoracearum or Sphaerotheca fusca on cucumber, melon
and pumpkin under field conditions. The silver nanoparticle treatment (100 ppm)
resulted in greater suppression of the fungal diseases when applied on the initiation
of the cucurbit powdery mildew disease. The treatment also resulted in maximum
growth inhibition of fungal hyphae and conidial germination in vivo (Lamsal et al.
2010). Krishnaraj et al. (2012) studied the effect of silver nanoparticles (AgNPs) on
various plant pathogenic fungi viz., Alternaria alternata, Sclerotinia sclerotiorum,
Macrophomina phaseolina, Rhizoctonia solani, Botrytis cinerea and Curvularia
lunata. The result revealed that 15 mg/L concentration of AgNPs greatly inhibited
the growth of all the tested plant pathogenic fungi. Kim et al. (2012) showed the
antifungal effect of AgNPs against various plant pathogenic fungi viz., Alternaria
alternata, A. brassicicola, Botrytis cinerea, Cladosporium cucumerinum,
Corynespora cassiicola, Cylindrocarpon destructans, Didymella bryoniae, Fusar-
ium oxysporum f. sp. cucumerinum, F. oxysporum f. sp. lycopersici, F. oxysporum,
F. solani, Glomerella cingulata, Monosporascus cannonballus, Pythium
aphanidermatum, Pythium spinosum and Stemphylium lycopersici under in vitro
conditions.
Al-Zubaidi et al. (2019) reported the antifungal activity of AgNPs against various
pathogenic fungi. The biosynthesized AgNPs inhibited the growth of three different
pathogenic fungi, including Fusarium oxysporum, Aspergillus flavus and Penicil-
lium digitatum. Hence, AgNPs could be considered as excellent broad-spectrum
antifungal agents. The minimum inhibitory concentration (MIC) of AgNPs was
estimated to be 0.5–10.0 μg/mL, which was lower than that of the standard antibiotic
Nystatin. Arciniegas-Grijalba et al. (2017) reported the antifungal efficacy of
ZnONPs on pink disease of coffee caused by Erythricium salmonicolor. The inhibi-
tory effect on the growth and morphological change includes thinning of the fibres of
the hyphae and a clumping tendency of the fungus. TEM analysis noticed the
changes in the ultra structure of pathogen, which include liquefaction of the
cytoplasmic content, making it less electron-dense, with the presence of a number of

vacuoles and significant detachment of the cell wall.
Zinc oxide nanoparticles (ZnONPs) showed antifungal activity against Botrytis
cinerea and Penicillium expansum at 12 mmol l1. ZnONPs significantly inhibited
the growth of B. cinerea and P. expansum. SEM images and Raman spectra
indicated that ZnONPs caused deformation in fungal hyphae and restricted the
development of conidiophores and conidia (He et al. 2011). Rao and Paria (2013)
reported the fungicidal efficacy of sulphur nanoparticles (SNPs) against two
phytopathogens, Fusarium solani (causing early blight and wilt diseases) and
Venturia inaequalis (causing apple scab disease). SNPs (35 nm) were found to be
more effective compared to the bigger-sized particles in preventing fungal growth.
Microscopic study confirmed that the fungicidal effect was mainly because of the
deposition of particles on the cell wall, resulting in subsequent damage. The NP
deposition caused an imbalance in the cell wall structure as supported by a Biuret
assay test. Hence, nano sulphur fungicides can effectively control the fungal diseases
of crops.
For the management of plant pathogens, copper in several formulations has been
used since ancient times. Copper sulphate is a compound which has antifungal and
antibacterial properties and is a key ingredient in most of the commercially available
fungicides for disease management in agricultural crops. Copper nanoparticles
(CuNPs) inhibit the invasion or colonization of plant pathogenic fungi viz.,
Alternaria alternata, Fusarium oxysporum, Curvularia lunata and Phoma
destructiva (Kanhed et al. 2014). Bramhanwade et al. (2016) also reported the
antifungal effect of CuNPs on the colonization of Fusarium culmorum,
F. graminearum and F. oxysporum under in vitro conditions. Treatment with
CuNPs caused suppression of colonization of F. culmorum, F. equiseti and
F. oxysporum. Giannousi et al. (2013) reported that foliar application of Cu
nanoparticles (11–25 nm) on tomato effectively inhibited the infection of
Phytophthora infestans at a concentration much lower than the commercial fungi-
cidal formulations. Similarly copper-chitosan complex nano-gels effectively
inhibited F. graminearum (Brunel et al. 2013). Salem et al. (2019) evaluated silver
nanoparticles (AgNPs) for their antifungal effects on the incidence and severity of
gray mold in tomato fruits caused by Botrytis cinerea. The results revealed that there
is no incidence of grey mold in case of tomato fruits with silver nanoparticles for
forty days. Finally the lowest incidence (0.3%) and severity (1.5%) of diseases were
after thirty days of cold storage in case tomato fruits were treated with Nano–
Silver + Inoculated. However, the highest incidence and severity (7.5%) of grey
mold was noticed after ten days of cold storage in case of treatment of tomato fruits
with Botrytis cinerea, in comparison with the incidence and severity (1%) of the
disease percentage after twenty days of cold storage in case of untreated tomato fruits
(control).
Lamsal et al. (2011a, b) evaluated silver nanoparticles (AgNPs) against powdery
mildew of cucumber and pumpkin under in vitro and in vivo conditions at various
concentrations to determine antifungal activities. In both fields and in vitro
evaluations, the application of 100 ppm AgNPs showed the highest inhibition of
Table 26.2 Evaluation of metal nanoparticles against major fungal and bacterial plant pathogens
602
Sl. Tested Effective concentrations/zone of

no. Nanoparticles Size (nm) Plant pathogens concentration inhibition/per cent inhibition References
1. Silica-silver Silica ¼ 20 Pythium ultimum, Magnaporthe 0.3, 3, 10 and P. ultimum, M. grisea, Park et al.
Silver ¼ 100 grisea, Colletotrichum 100 ppm C. gloeosporioides and B. cinerea, (2006)
gloeosporioides, Botrytis cinerea, R. solani ¼ 10 ppm
Rhizoctonia solani Bacillus subtilis. B. subtilis. A. chrococuum, R. tropici,
Azotobacter chrococuum, P. syringae and X. c.
Pseudomonas syringae and pv. vesicatoria ¼ 100 ppm
Xanthomonas compestris
pv. vesicatoria
2. Silica-silver Silica ¼ 20 Sphaerotheca fuliginea 0.3 ppm Squash powdery mildew ¼ 100% Park et al.
Silver ¼ 100 disease control (2006)
3. Sulphur 50 Aspergillus niger 125, 24.33 mm ¼ 2000 ppm Choudhurya
500, 1000 Control ¼ 20.33 mm et al. (2009)
and
2000 ppm
4. Silver 25 Bipolaris sorokiniana and 25, 50 and 50 and 100 ppm ¼ most effectively Jo et al.
Magnaporthe grisea 200 ppm inhibited the spore germination (2009)
5. Nano-silver 7–25 Sclerotium cepivorum 1, 3, 5, 7, 10, 90% inhibition of Jung et al.
liquid 25, 50 and S. cepivorum ¼ 7 ppm (2010)
100 ppm
6. Silver 4–8 Colletotrichum gloeosporioides 10, 30, 50 90% inhibition of fungal mycelium Lamsal et al.
and 100 ppm under in vitro ¼ 100 ppm lowest (2011a, b)
disease incidence (9.7%), over the
control (84.1%) at 50 ppm
7. Zinc oxide 30 & 50 Alternaria alternate, Fusarium 0.3, 0.2 and Spore germination inhibition Wani and
and oxysporum and Rhizopus stolonifer 0.1 ml A. alternata ¼ 44.94 to 9.80% Shah (2012)
magnesium F. oxysoprum ¼ 66.28 to 12.96%
oxide M. plumbeus ¼ 41.54 to 6.40%
26
8. Sulphur Erysiphe cichoracearum of okra 100 ppm E. cichoracearum ¼ least conidial Gogoi et al.
germination (4.56%) (2013)
9. Copper 3–10 Phoma destructiva (DBT-66), 20 μg/disc P. destructiva ¼ 22 1 mm Kanhed et al.
Curvularia lunata (MTCC no. 2030), C. lunata ¼ 21 0.5 mm (2014)
Alternaria alternate (MTCC A. alternate ¼ 18 1.1 mm
no. 6572) F. oxysporum ¼ 24 0.5 mm
Fusarium oxysporum (MTCC
no. 1755)
10. Copper 32 Exserohilum turcicum 1–2000 ppm 250 ppm ¼ 100% spores inhibited Chikkanna
et al. (2016a)
11. Copper 58–100 Exserohilum turcicum 1800 ppm No inhibition Nargund
et al. (2016c)
12. Silver 50–200 Curvularia lunata, Xanthomonas 169 ppm C. lunata ¼ 95% spores inhibited Nargund
axonopodis pv. punicae Xap ¼ 9.25 mm et al. (2016a)
13. Copper, 30–80 CuNPs ¼ Xap and Xac 1–2000 ppm CuNPs ¼ Xap & Xac ¼ no inhibition Nargund
sulphur <50 SNPs ¼ cucumber powdery mildew 500 ppm SNPs ¼ recorded only 20% PDI et al. (2016b)
14. Zinc oxide <100 Pectobacterium carotovorum subsp. 1–30 mg/ml Effective conc. ¼ 30 mg/ml Hafez et al.
wasabiae Zone of inhibition ¼ 32 mm (2014)
15 Iron oxide 17–42 Xanthomonas sp. 10–50 mg/ml Effective conc. ¼ 50 mg/ml Prabhu et al.
Proteus vulgaris Xanthomonas sp. ¼ 14 mm (2015)
P. vulgaris ¼ 22 mm
16 Zinc oxide <100 Xanthomonas axonopodis pv. citri 1–50 μg/ml Effective conc. ¼ 30 μg/ml Poovizhi and
Aspergillus sp. Xac ¼ 16 mm Krishnaveni
Penicillium sp. Spore germination and mycelial (2015)
inhibition in Aspergillus sp. and
Green Nanotechnology and Its Application in Plant Disease Management
Penicillium sp.@ 30 μg/ml

17. Zinc oxide <100 Aspergillus fumigatus 20, 40, 60 Mycelia inhibition of both fungi @ Navale et al.
Aspergillus flavus and 80 μg/ml 80 μg/ml ¼ 75% (2015)
(continued)
603
604
Sl. Tested Effective concentrations/zone of

no. Nanoparticles Size (nm) Plant pathogens concentration inhibition/per cent inhibition References
18. Zinc <100 Peronospora tabacina 1–10 mg/l Inhibited leaf infection @ 10 mg/l Wagner et al.
Reduction of germ tube elongation @ (2016)
5 mg/l ¼ >60%
19. Silver 50–200 Curvularia lunata 169 ppm C. lunata spore germination Nargund
Xanthomonas axonopodis pv. punicae inhibited ¼ 95% et al. (2016)
Xap ¼ 9.25 mm
20. Copper 32 Exserohilum turcicum 1–2000 ppm Inhibition of spore germination @ Chikkanna
250 ppm ¼ 100% et al. (2016b)
21. Iron oxide 35 Escherichia coli 10–50 mg/ml Effective conc. ¼ 50 mg/ml Manyasree
Staphylococus aureus E. coli ¼ 36 mm et al. (2016)
S. aureus ¼ 30 mm
disease in field and maximum inhibition of growth of fungal hyphae and conidial
germination in vitro. The foliar spray technique was used to apply silver
nanoparticles on pumpkin plants 3–4 weeks before the outbreak of the disease and
after disease occurrence, and distilled water was used as control. Gogoi et al. (2013)
evaluated the one Sulphur NPs ,and three commercial products viz., commercial
sulphur (Merck), commercial nano-sulphur (MK Impex, Canada) and Sulphur
80 WP (Corel Insecticide) were evaluated in vitro for their fungicidal efficacy at
1000 ppm against Erysiphe cichoracearum of okra. All the sulphur fungicides
significantly inhibited the germination of conidia of E. cichoracearum as compared
to control. The lowest conidial germination was noticed in synthesized nano-sulphur
treatment (4.56%) followed by Canadian nano-sulphur (14.17%), sulphur 80 WP
(15.97%) and control (23.09%). Cleistothecial appendages were also disrupted in
contact with nano-sulphur and the cleistothecia became sterile. Miguel et al. (2011)
evaluated the antifungal activity of AgNPs against Colletotrichum gloeosporioides,
which causes anthracnose of papaya fruits. Two different mean nanoparticle sizes
(5 and 24 nm) and various concentrations (13, 26 and 52 μg silver/mL PDA) of
AgNPs were tested against anthracnose of papaya in vitro. The inhibition of the
fungus was maximum (90%) at 52 μg/mL. The various nanoparticles that were tested
against different plant pathogens are presented in Table 26.2.
References
Acharyulu NPS, Dubey RS, Swaminadham V, Pratap Kollu RLK, Pammi SVN (2014) Green
synthesis of CuO nanoparticles using Phyllanthus amarus leaf extract and their antibacterial
activity against multidrug resistance bacteria. Int J Eng Res Tech 3:639–641
Al-Zubaidi S, Al-Ayafi A, Hayam A (2019) Biosynthesis, characterization and antifungal activity
of silver nanoparticles by Aspergillus niger isolate. J Nanotechnol Res 1:023–036
Angrasan JKV, Subbaiya R (2014) Biosynthesis of copper nanoparticles by Vitis vinifera leaf
aqueous extract and its antibacterial activity. Int J Curr Microbiol App Sci 3:768–774
Arciniegas-Grijalba PA, Patiño-Portela MC, Mosquera-Sánchez LP, Guerrero-Vargas JA,
Rodríguez-Páez JE (2017) ZnO nanoparticles (ZnO-NPs) and their antifungal activity against
coffee fungus Erythricium salmonicolor. Appl Nanosci 7:225–241
Awwad A, Salem NM, Abdeen AM (2014) Noval approach for synthesis of sulphur (S-NPs)
nanoparticles using Albizia julibrissin fruits extract. Adv Mater Let 10:99–102
Awwad AM, Salem NM, Abdeen AO (2015) Noval approach for synthesis sulfur (S-NPs)
nanoparticles using Albizia julibrissin fruits extract. JVBRI Press 6:432–443
Banerjee P, Satapathy M, Mukhopahayay A, Das P (2014) Leaf extract mediated green synthesis of
silver nanoparticles from widely available Indian plants: synthesis, characterization, antimicro-
bial property and toxicity analysis. J Biores Bioproc 10:233–238
Bramhanwade K, Shende S, Bonde S, Gade A, Rai M (2016) Fungicidal activity of Cu
nanoparticles against Fusarium causing crop diseases. Environ Chem Lett 14:229–235
Brunel F, El Gueddari NE, Moerschbacher BM (2013) Complexation of copper (II) with chitosan
nanogels: toward control of microbial growth. Carbohydr Polym 92:1348–1356
Chikkanna S, Nargund VB, Madhu SG, Hasansab AN, Hulagappa N, Puneeth R (2016a) Biosyn-
thesis of plant mediated copper and silver nanoparticles and their toxicity studies on crop plants.
Proc Int Conf on Emerging Trends in Synthesis of Nanoparticles in Agri Biotechnol Res and
Commercialization, 25–27, February, Loyola Academy Degree & PG Coll, Secunderabad
(India), pp. 86–87
Chikkanna S, Nargund VB, Byadgi AS, Patil RR, Savalgi VP (2016b) Green synthesis of copper
nanoparticles and their efficacy against Exserohilum turcicum causing leaf blight of in maize.
Proc Nation Symp Rec Adv Pl Health Manage Sust Prod. 15–16, December, UAS, Dharwad
(India), p. 139
Choudhurya SR, Nair KK, Kumar R, Gogoi R, Srivastavad C, Gopal G, Subhramanyam BS,
Devakumar C, Goswamia A (2009) Nanosulphur: a potent fungicide against food pathogen,
Aspergillus niger. Proc Int Conf Adv Nanomater Nanotechnol New Delhi (India), pp. 154–157
El-Hai KA, El-Metwally MA, El-Baz SM, Zeid AM (2009) The use of antioxidants and
microelements for controlling damping-off caused by Rhizoctonia solani and charcoal rot
caused by Macrophomina phaseolina on sunflower. Plant Pathol J 8:79–89
El-Kassas HY, Aly-Eldeen MA, Gharib SM (2017) Green synthesis of iron oxide (Fe3O4)
nanoparticles using two selected brown seaweeds: characterization and application for lead
bioremediation. Acta Oceanol Sin 35:89–98
Fernandez RG, Prats E, Jorrin-Novo JV (2010) Proteomics of plant pathogenic fungi. J Biomed
Giannousi K, Avramidis I, Dendrinou-Samara C (2013) Synthesis, characterization and evaluation
of copper based nanoparticles as agrochemicals against Phytophthora infestans. RSC Adv
3:21743–21752
Gogoi R, Singh PK, Kumar R, Nair KK, Alam I, Srivastava C, Yadav S, Gopal M, Choudhury SR,
Goswami A (2013) Suitability of nano-sulphur for biorational management of powdery mildew
of okra (Abelmoschus esculentus Moench) caused by Erysiphe cichoracearum. J Plant Pathol
Microb 4:225–229
Gopinath M, Subbaiya R, Selvam MM, Suresh D (2014) Synthesis of copper nanoparticles from
Nerium oleander leaf aqueous extract and its antibacterial activity. Int J Curr Microbiol App Sci
3:814–818
Hafez EE, Hassan HS, Elkady M, Salama E (2014) Assessment of antibacterial activity for
synthesized zinc oxide nanorods against plant pathogenic strains. Int J Sci Technol Res
3:318–324
Harikumar PS, Aravind A (2016) Antibacterial activity of copper nanoparticles and copper
nanocomposites against Escherichia coli bacteria. Int J Sci 2:84–90
Harishkumar K, Savalgi VP (2017) Microbial synthesis of zinc nanoparticles using fungus isolated
from rhizosphere soil. Int J Curr Microbiol App Sci 6:2359–2364
He L, Liu Y, Mustapha A, Lin M (2011) Antifungal activity of zinc oxide nanoparticles against
Botrytis cinerea and Penicillium expansum. Microbiol Res 166:207–215
Honary S, Barabadi H, Gharaeifathabad E, Naghibi F (2012) Green synthesis of copper oxide
nanoparticles using Penicillium aurantiogriseum, Penicillium citrinum and Penicillium
waksmanii. Digest J Nano Mat Biostr 7:999–1005
Hussain I, Singh NB, Singh A, Singh H, Singh SC (2016) Green synthesis of nanoparticles and its
potential application. Biotechnol Lett 38:545–560
Jalaluddin M, Ashraf, Ansari M, Haris M, Khan Mohammad A (2016) Green synthesis of silver
nanoparticles and characterization of their inhibitory effects on AGEs formation using biophys-
ical techniques. www.nature.com/scientificreports
Jo YK, Kim BH, Jung G (2009) Antifungal activity of silver ions and nanoparticles on phytopatho-
genic fungi. Pl Dis 93:1037–1043
Jores K, Mehnert W, Drecusler M, Bunyes H, Johan C, Mader K (2004) Investigation on the stricter
of solid lipid nanoparticles and oil-loaded solid nanoparticles by photon correlation spectros-
copy, field flow fractionasition and transmission electron microscopy. J Control Release
17:217–227
Jung JH, Kim SW, Min JS, Kim YJ, Lamsal K, Kim KS, Lee YS (2010) The effect of nano-silver
liquid against the white rot of green onion caused by Sclerotium cepivorum. Mycobiol 38:39–45
Jyoti K, Baunthiyal M, Singh A (2016) Characterization of silver nanoparticles synthesized using
Urticadioica Linn. leaves and their synergistic effects with antibiotics. J Radiat Res Appl 9 (3):
217–227
Kanhed P, Birla S, Gaikwad S, Gade A, Amedea BS, Rubilar O, Duran N, Rai M (2014) In vitro
antifungal efficacy of copper nanoparticles against selected crop pathogenic fungi. Mater Lett
115:13–17
Kasprowicz MJ, Kozioł M, Gorczyca A (2010) The effect of silver nanoparticles on phytopatho-
genic spores of Fusarium culmorum. Can J Microbiol 56:247–253
Kim SW, Jung JH, Lamsal K, Kim YS, Min JS, Lee YS (2012) Antifungal effects of silver
nanoparticles (AgNPs) against various plant pathogenic fungi. Mycobiology 40:53–58
Korbekandi H, Iravani S, Abbasi S (2009) Production of nanoparticles using organisms. Crit Rev
Krishnaraj C, Ramachandran R, Mohan K, Kalaichelvan PT (2012) Optimization for rapid synthe-
sis of silvervnanoparticles and its effect on phytopathogenic fungi. Spectrochim Acta A Mol
Biomol Spectrosc 93:95–99
Lamsal K, Sang-Woo K, Jung JH, Kim YS, Kim KU, Lee YS (2010) Inhibition effects of silver
nanoparticles against powdery mildews on cucumber and pumpkin. Microbiology 39:26–32
Lamsal K, Kim SK, Jung JH, Kim Y, Kim K, Lee Y (2011a) Application of silver nanoparticles for
the control of Colletotrichum species in vitro and pepper anthracnose disease in field. Mycobiol
39:194–199
Lamsal K, Kim SW, Jung JH, Kim YS, Kim KS, Lee YS (2011b) Inhibition effects of silver
nanoparticles against powdery mildews on cucumber and pumpkin. J Mycobiol 39:26–32
Manyasree D, Kiranmayi P, Ravikumar RN (2016) Synthesis, characterization and anti bacterial
activity of iron oxide nanoparticles. Indo Am J Pharm Res 6:5992–5997
Miguel A, Aguilar-Mendez M-MES, Ortega-Arroyo L, Cobian-Portillo G, Sanchez-Espindola E
(2011) Synthesis and characterization of silver nanoparticles: effect on phytopathogen
Colletotrichum. J Nanopart Res 13:2525–2532
Moghaddam AB, Namvar F, Moniri M, Tahir PM, Azizi S, Mohamad R (2015) Nanoparticles
biosynthesized by fungi and yeast: a review of their preparation, properties, and medical
applications. Molecules 20:16540–16565
Molpeceres J, Aberturas MR, Guzman M (2000) Biodegradable nanoparticles as a delivery system
for cyclosporine: Preparation and characterization. J Microencapsul 17:599–614
Muhlen AZ, Muhlen EZ, Niehus H, Mehnert W (1996) Atomic force microscopy studies of solid
lipid nanoparticles. Pharm Res 13:1411–1416
Nadaroglu H, Onem H, Gungor AA (2017) Green synthesis of Ce2O3 NPs and determination of its
antioxidant activity. IET Nanobiotechnol 11:411–419
Nargund VB, Chikkanna S, Madhu SG, Hulagappa RH (2016a) Green nanotechnology: synthesis,
characterization of silver and copper nanoparticles and their efficacy against major pathogens of
maize and pomegranate. Indian Phytopath 69:598–601
Nargund VB, Chikkanna S, Vinay JU, Nadaf A (2016b) Copper and sulphur green nanoparticles in
plant disease management. Proc. Nation. Symp. on Rec. Adv. Pl. Health Manage. Sust. Prod.,
15–16, December, UAS, Dharwad (India), p. 134
Nargund VB, Patil RR, Chikkanna S, Madhu S, Puneeth RM, Hulagappa, Naik I, Ramangouda H
(2016c) Phytomining based nanotechnology: a novel approach in plant protection. Nation.
Symp. Rec. Trends. Pl. Pathol. Res. Edu, UAS, Raichur (India), p. 82
Navale GR, Thripuranthaka M, Late DJ, Shinde SS (2015) Antimicrobial activity of ZnO
nanoparticles against pathogenic bacteria and fungi. JSM Nanotechnol Nanomed 21:115–205
Niederberger M, Garnweitner G (2006) Reduction of metal ions in polymer matrices as a conden-
sation method of nanocomposite synthesis. Eur J 12:7282
Park HJ, Kim SH, Kim HJ, Choi SH (2006) A new composition of nanosized silica-silver for
control of various plant diseases. Pl Pathol J 22:295–302
Park S, Sung HK, Kim Y (2016) Green synthesis of metal nanoparticles using sprout plants: pros
and cons. J Nanosci Nanotechnol 16:4444–4449
Pearce JM (2012) Make nanotechnology research open-source. Nature 491:519–521
Polakovic M, Gorner T, Gref R, Dellacherie E (1999) Lidocaine loaded biodegradable nanospheres:
modelling of drug release. J Control Release 60:169–177
Ponarulselvam S, Panneerselvam C, Murugan K, Aarthi N, Kalimuthu K, Thangamani S (2012)

Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their
antiplasmodial activities. Asian Pac J Trop Biomed 2:574–580
Poovizhi J, Krishnaveni B (2015) Synthesis, characterization and antimicrobial activity of zinc
oxide nanoparticles synthesized from Calotropis procera. Int J Pharm Sci Drug Res 7:425–431
Prabhu YT, Rao KV, Kumari BS, Kumar VSS, Pavani T (2015) Synthesis of Fe3O4 nanoparticles
and its antibacterial application. Int Nano Lett 5:85–92
Rai M, Yadav A, Gade A (2009) Silver nanoparticles as a new generation of antimicrobials.
Biotechnol Adv 27:76–83
Rao MD, Gautam P (2016) Synthesis and characterization of ZnO nanoflowers using
Chlamydomonas reinhardtii: a green approach. Environ Prog Sustain Energy 35:1020–1026
Rao K, Paria S (2013) Use of sulfur nanoparticles as a green pesticide on Fusarium solani and
Venturia inaequalis phytopathogens. RSC Adv 3:10471–10478
Safavi K, Mortazaeinezahad F, Esfahanizadeh M, Javad AM (2011) In vitro antibacterial activity of
nanomaterial for using in tobacco plants tissue culture. World Acad Sci Eng Technol
79:372–373
Salem NM, Albannaa LS, Awwad AM (2016a) Green synthesis of sulfur nanoparticles using
Punica granatum peels and the effects on the growth of tomato by foliar spray applications. J
Env Nano Monitoring Manage 6:83–87
Salem NM, Luma SA, Awwad AM, Ibrahim QM, Abdeen AO (2016b) Green synthesis of nano-
sized sulphur and its effect on plant growth. J Agric Sci 8:88–194
Salem E, Nawito S, El-Rahman A, Ahmed A (2019) Effect of silver nano-particles on gray mold of
tomato fruits. J Nanotechnol Res 1:108–118
Santhoshkumar J, Venkat Kumar S, Rajeshkumar S (2017) Synthesis of zinc oxide nanoparticles
using plant leaf extract against urinary tract infection pathogen. Res Eff Technol 3:459–465
Schneider S, Ullrich WR (1994) Differential induction of resistance and enhanced enzyme activities
in cucumber and tobacco caused by treatment with various abiotic and biotic inducers. Physiol
Mol Plant Pathol 45(291):304
Shah M, Fawcett D, Sharma S, Tripathy SK, Poinern GE (2015) Green synthesis of metallic
nanoparticles via biological entities. Materials 8:7278–7308
Singh D, Kumar A, Singh AK, Tripathi HS (2013) Induction of resistance in field pea against rust
disease through various chemicals/micronutrients and their impact on growth and yield. Plant
Pathol J 12:36–49
Singh P, Kim YJ, Zhang DB, Yang DC (2016) Biological synthesis of nanoparticles from plants
and microorganisms. Trends Biotechnol 34:588–599
Tran QH, Van Quy N, Le AT (2013) Silver nanoparticles: synthesis, properties, toxicology,
applications and perspectives. Adv Nat Sci 4:1–21
Vaseeharan B, Sivakamavalli J, Thaya R (2015) Synthesis and characterization of chitosan-ZnO
composite and its antibiofilm activity against aquatic bacteria. J Compos Mater 49:177–184
Vijayakumar S, Krishnakumar C, Arulmozhi P, Mahadevan S, Parameswari N (2018) Biosynthesis,
characterization and antimicrobial activities of zinc oxide nanoparticles from leaf extract of
Glycosmis pentaphylla (Retz.) DC. Microb Pathog 116:44–48
Vinay JU, Nargund VB, Jahagirdhar S, Hedge RV, Patil RR, Chikkannaswamy (2016) Synthesis
and characterization of chitosan based zinc nanoparticles. In: Nat. Symp. on Recent Advances in
Plant Health Management for Sustainable Productivity. Univ. Agric. Sci., Dharawad, December
15–16, p. 139
Vinay JU, Nargund VB, Patil RR, Jahagirdhar S, Hedge RV (2017) Biogenic mediated synthesis of
Zinc Oxide Nanioparticles (ZnONPs) using Pseudomonas fluorescens and their efficacy against
phytopathogens. In: Nat. Symp. Emerging Trends in Plant Health Management in Realtion to
Climate Change. College of Horticulture, Bengaluru. September 12–13, p. 45
Wagner G, Korenkov V, Judy JD, Bertsch PM (2016) Nanoparticles composed of Zn and ZnO
inhibit Peronospora tabacina spore germination in vitro and P. tabacina infectivity on tobacco
leaves. Nanomedicine 6:50–58
Wani A, Shah MA (2012) A unique and profound effect of MgO and ZnO nanoparticles on some
plant pathogenic fungi. J App Pharma Sci 2:40–44
Wei D, Sun W, Qian W, Ye Y, Ma X (2009) The synthesis of chitosan-based silver nanoparticles
and their antibacterial activity. J Carbohydr Res 344:2375–2382
Yu J, Dabing Z, Deok-Chun Y (2016) Biological synthesis of nanoparticles from plants and
microorganisms. Trends Biotechnol 34:7–18
Plant Disease Management in Organic
Farming System: Strategies and Challenges 27
Laxmi Rawat, T. S. Bisht, and Dinesh Chandra Naithani
Abstract
Organic farming can be stated as an ecologically, economically, and socially

dependable way of farming that may provide continuous supply of safe and
healthy food and fibres, with the least probable loss of nutrients and energy,
and the least negative impacts on the environment. Though the use of chemical
inputs in agriculture is inevitable to combat dreaded pests and meet the growing
demand for food in a populous nation like India, there are opportunities where
organic production can be encouraged to meet the domestic and export demand
for fresh fruits and vegetables. Organic agriculture can be seen as a pioneering
effort to create sustainable development based on different principles compared to
mainstream agriculture. Modification in cultural practices, mechanical destruc-
tion of the source of inoculum, clean cultivation, use of organic amendments,
developing pesticides of organic origin, encouraging biocontrol agents, use of
cover and trap crops and use of heat treatment, cold temperature, solar energy, etc.
can be conveniently used to manage disease incidence below the economic injury
level under organic farming. This chapter provides an overview of the potential
role and challenges of organic agriculture in this global perspective where organic
farming servs as an alternative example for the broader implementation of
ecological justice in agriculture and society.
L. Rawat (*) · D. C. Naithani

Plant Pathology Division, College of Forestry, VCSG Uttarakhand University of Horticulture and
Forestry, Ranichauri, Tehri Garhwal, Uttarakhand, India
VCSG Uttarakhand University of Horticulture and Forestry, Bharsar, Pauri Garhwal, Uttarakhand,
India
T. S. Bisht
Department of Horticulture, School of Agriculture and Allied Sciences, HNB Garhwal University
(A Central University), Srinagar (Garhwal), Uttarakhand, India

https://doi.org/10.1007/978-981-15-6275-4_27
612 L. Rawat et al.
Keywords
Agriculture · Organic farming · Biocontrol agents · Agro-chemicals and trap crop
27.1 Introduction
The human civilization from “hunting and gathering” to present day agriculture has
underwent several changes. The fast changes witnessed depletion of production
bases in many fertile grounds coupled with contamination of natural resources and
human health hazards, which warranted a change in the direction of agriculture for
sustainable development. Modern agriculture and food systems, including organic
agriculture, are undergoing a technological and structural modernization with grow-
ing globalization. Organic agriculture can be seen as a pioneering effort to create
sustainable development based on different principles compared to mainstream
agriculture (Kristensen 2005). Mainstream agriculture is conventional agriculture
where externally industry-produced inputs such as fertilizers and pesticides are used.
The indiscriminate use of fertilizers and plant protection chemicals to increase the
yield potential and save the crops from insect pests and diseases respectively, no
doubt, has doubled or tripled our total food production that has helped us to sustain
low cost along with more food supply, but has also created a number of health
hazards and deteriorated the agro-ecosystem badly. Their negative impacts far
outweigh their social benefits. In recent years, more ecological approaches are
now being researched and there has been a world wide swing to the use of
eco-friendly methods for protecting the crops from pest and disease. The challenge
today is how to achieve not only food security but also food safety (Rawat
2011). This situation has compelled us to switch over to organic agriculture to
cultivate valuable crops and safer foods for health and at the same time to safeguard
our environment.
27.2 Concepts and Definitions of Organic Agriculture
The concept of organic agriculture has been perceived differently by different

people. To most of them, organic agriculture is a way of agriculture that relies on
ecosystem management rather than external agriculture inputs. This approach
excludes the use of synthetic inputs, such as synthetic fertilizers and pesticides,
and genetically modified organisms and usually subscribes to the principles of
sustainable agriculture. Organic agriculture has been defined by Lampkin (1990)
as a production system which avoids or largely excludes the use of synthetic
compounded fertilizers, pesticides, growth regulators and livestock feed additives.
Palaniappan and Annadurai (1999) stated that the organic agriculture system
depends on different approaches and their integrations, which include crop rotation,
crop residues, animal manures, legumes, green manures, off-farming wastes and the
aspect of biological pest and disease control, to maintain soil productivity and tilth to
27 Plant Disease Management in Organic Farming System: Strategies and Challenges 613
supply plant nutrients, and to control insects, pests and weeds. Later on the Food and
Agriculture Organization (1999) defined organic agriculture as a holistic production
management system which promotes and enhances agro-ecosystem health, including
biodiversity, biological cycles and soil biological activity. This definition aims at
three strategies viz., (i) the use of management practices in preference, (ii) the use of
off-–farm inputs and (iii) taking into account the regional conditions that require
locally adapted systems. Therefore, the basic concept of these definitions is to
maintain the soil as a living system that develops the activities of beneficial micro-
organisms present in the soil.
Organic agriculture is not the simple replacement of chemical fertilizers,
herbicides, fungicides and insecticides with biologically active formulations and
other organic inputs; instead it adopts broad management practices to improve the
soil productivity by enhancing soil health, soil life and mineral particles. Soil air and
water exist in a stage of dynamic equilibrium and regulate the ecosystem processes
in mutual harmony by complementing and supplementing each other. In healthy soil,
the population of soil microfauna and microflora multiplies rapidly, which in turn
sustains the biochemical process of dissolution and synthesis at a high rate, and as a
result, the regeneration capacity of soil will enhance making it resilient to absorb the
effects of climate variability and occasional failures in agronomic management. The
principal elements to be considered while practicing organic agriculture are:
(i) maintaining a living soil, (ii) making all the essential nutrients available, (iii)
organic mulching for conservation and (iv) attaining a sustainable high yield
(Palaniappan and Annadurai 1999).
In traditional agriculture, generally, we use chemical inputs (synthetic pesticides
and water-soluble synthetically purified fertilizers) while in organic agriculture these
inputs are replaced with natural inputs (bio pesticides and bio fertilizers). Therefore,
organic agriculture integrates the scientific knowledge of ecology, developed mod-
ern technologies and conventional agricultural tactics based on natural biological
processes. The motive of organic farming is to raise the crops in such a way that it
keeps the soil alive and healthy by incorporating organic wastes and bio-fertilizers in
the soil and releases nutrients to the crops for sustainable production in an
eco-friendly pollution-free environmental condition. The basic rules of organic
agriculture are based on crop rotation, incorporation of green manures in soil,
biological control of diseases and insect pests along with mechanical cultivation of
crops. The enhancement of crop productivity depends on the integration of these
basic rules, because crop rotations renew the soil and confuse pests, incorporation of
legume crops in rotation fix the atmosphere-free nitrogen in the soil, while mulches
in the crops are used to conserve soil moisture and control diseases and weeds. Fi
(2006) argued that some of the methods developed for organic agriculture have been
borrowed by more conventional agriculture such as integrated pest management,
which is a multifaceted strategy that uses various organic methods of pest control
whenever possible, but could include synthetic pesticides only as a last resort.
614 L. Rawat et al.
27.2.1 Why Organic Agriculture?
• To produce food of high nutritional quality in sufficient quantity

• To work with natural systems rather than seeking to dominate them
• To encourage and enhance the biological cycles within the farming system
involving microorganisms, soil flora and fauna and plants and animals
• To maintain and increase the long-term fertility of soil
• To use, as far as possible, renewable resources in a locally organized agriculture
system
• To give better conditions of life to all livestock that allow them to perform all
aspects of their innate behaviour
• To avoid all forms of pollution that may result from agricultural techniques
• To maintain the genetic diversity of the agricultural system and its surroundings,
including protection of both plant and wildlife habitats
• To consider the wider social and ecological impact of the farming system
27.3 Components of Organic Agriculture
The ideal organic agriculture approach is that where the conservation of biological
potential of soil and other natural resources is maintained by adopting the integrated
crop models (Fig. 27.1). These models are devoid of chemical inputs, by which the
interaction among beneficial soil microorganisms is stimulated and sustained and the
soil life and health are also improved. Crop production and health in organic farming
systems are attained through a combination of structural factors and tactical man-
agement components to ensure products of sufficient quality and quantity for human
and livestock consumption. Following are the important components of organic
agriculture:
Fig. 27.1 Components of

organic agriculture
1. Diverse crop
rotation
5. Synthetic
pesticides Components of 2. Soil fertility
control organic management
agriculture
4. Natural pest
and disease 3. Weeds
control management
27.3.1 Diverse Crop Rotation
Diversification of crops helps to maintain soil health and enhances its efficiency to
produce more food by reducing disease and pest incidence enhancing the soil
rhizosphere system and soil fertility and renewing the root zone soil. Divers crop
rotation systems include many cropping systems such as rotational cropping,
sequential cropping, intercropping, multistoried cropping system, etc. These crop-
ping systems can be adopted in organic agriculture to reduce soil erosion and soil-
borne diseases and improve weed control and soil water holding capacity. The
success of crop rotation depends on the choice of crops and their varieties and
spatial and temporal design. According to Stockdale et al. (2001), development and
implementation of well-designed crop rotations is central to the success of organic
agriculture. So crop rotation can be designed in such a way as to improve soil health
and minimize the spread of weeds, diseases and pests as stated by Altieri (1995).
Herridge et al. (2008) observed that the inclusion of pulse crops in the farming
system can enhance the availability of nitrogen in the soil. Enhancement of soil
nitrogen level is due to the ability of many pulse crops to fix atmospheric nitrogen
through symbiosis with Rhizobium, as stated by Peoples et al. (1995). Pulse crops in
the rotation not only enhance the crop yield and nitrogen use efficiency but also
decrease inputs of inorganic fertilizers, as argued by Gan et al. (2009). Kirkegaard
and Ryan (2014) reported that dry pea and lentil crops use 15–35% less water than
cereal and oilseed crops, thereby enhancing water-use efficiency in the semiarid
northern regions.
27.3.2 Soil Fertility Management
Management of soil fertility in organic agriculture is a core component, because

efficient management of nutrients and soil structure ensure good yield of crops,
while poor management can result in poor yield, poor animal health and increased
environmental pollution. In organic agriculture, soil fertility can be improved by
inclusion of organic manures, crop residues, dung and urine from domesticated
animals, wastes from slaughter houses, human excreta and sewage, biomass of
weeds and organic wastes from fruit and vegetable production and processing
units. For the good physical conditions of soil viz., soil structure, aeration and
water holding capacity, it is essential to have high amounts of organic matter.
Organic farming uses a variety of methods to improve soil fertility, including crop
rotation, cover cropping, reduced tillage and application of compost. Due to zero
tillage or less tillage, soil is not much disturbed, less exposed to air and the loss of
soil carbon to the atmosphere is minimum, resulting in higher organic carbon
depositions into the soil, which can reduce the effect of greenhouse gases and help
fight changing environmental conditions. The other tactics to enhance soil fertility
through biological nitrogen fixation can be achieved by using Azolla, Blue green
algae for rice, Rhizobium for pulses and Azatobactor and Azospirillum for other
crops. Different crops can fix different amounts of nitrogen into the soil. For
616 L. Rawat et al.
example, white clover can fix up to 250 kg N ha 1 year 1, as reported by Kristensen

et al. (1995), red clover up to 240 kg N ha 1 year 1 (Schmidt et al. 1999) and lucerne
fix up to 500 kg N ha 1 year 1, as recorded by Spiertz and Sibma (1986), while Van
Kessel and Hartley (2000) estimated up to 200 kg N ha 1 year 1 fixed by field
beans.
Plants require a number of nutrients in varying quantities for growth and devel-
opment. Supplying enough nitrogen, and particularly synchronization, so that plants
can get enough nitrogen at the time when they need it most is a challenging task for
organic farmers, as stated by Watson et al. (2002). They also observed that crop
rotation, green manure and intercropping fix nitrogen into the soil from the atmo-
sphere through symbiosis with rhizobial bacteria, the crop residues can be ploughed
back into the soil, and different plants leave different amounts of nitrogen, poten-
tially aiding synchronization, but the competition between legumes and crops can be
problematic and thus wider spacing between crop rows is required. Organic farmers
also use animal manure, certain processed fertilizers such as seed meal and various
mineral powders such as rock phosphate and green sand, a naturally occurring form
of potash that provides potassium. Gillman (2008) stated that in some cases pH may
need to be amended through natural pH agents, i.e. lime and sulfur, but in the United
States some compounds such as iron sulfate, aluminum sulfate, magnesium sulfate
and soluble boron products are allowed in organic farming. Biological research of
soil and soil organisms has proven beneficial to organic farming because these soil
microorganisms (bacteria and fungi) break down chemicals, plant matter and animal
waste into productive soil nutrients, resulting in healthier yield and more productive
soil for future crops, as argued by Ingram (2007), while fields with less or no manure
display significantly lower yield due to decreased soil microbe community. Simi-
larly, Fliebbach (2006) observed that increased manure improves biological activity,
providing a healthier, more arable soil system and higher yields. Soil microbial
biomass gets benefitted by the introduction of organic amendments because
microorganisms use the available C more efficiently and also contribute to nutrient
mineralization (Fliebbach and Mader 2000).
27.3.3 Weed Management
Weeds are identified as a major problem in organic farming, so an appropriate

integrated strategy is necessary to control weeds in the organic agriculture system.
Experimentally it has been proved that deep summer ploughing, inter-cultivation
tactics, field operation time, timely sowing, line sowing, mulching and soil solariza-
tion suppress weed germination and favour crop growth and development. Kathleen
and Robert (2003) stated that organic weed management promotes weed suppres-
sion, rather than weed elimination, by enhancing crop competition and phytotoxic
effects on weeds of cover crops and crops with dissimilar life cycles to discourage
weeds associated with a particular crop. They also suggested other cultural practices
to enhance crop competitiveness resulting in reduced weed pressure viz., selection of
competitive crop varieties, high-density planting, tight row spacing and late planting
into warm soil to encourage rapid crop germination. Szykitka (2004) classified the
mechanical and physical weed control practices into different groups such as:
tillage – turning the soil between crops to incorporate crop residues and soil
amendments; removing existing weed growth and preparing a seedbed for planting;
turning soil after seeding to kill weeds, including cultivation of row crops; mowing
and cutting – removing top growth of weeds; flame weeding and thermal weeding –
using heat to kill weeds; and mulching– blocking weed emergence with organic
materials, plastic films, or landscape fabric. However, still researches are required to
develop organic methods that suppress the growth or germination of common weeds
through promoting the growth of natural microorganisms.
27.3.4 Natural Pest and Disease Control
Pest and disease management without the use of synthetic plant protection chemicals
is an important feature of organic agriculture. Plant protection in organic agriculture
is based on the maintenance of on-farm diversity, improvement of soil and plant
health through crop diversification and the use of bio-agents and plant-based
biopesticides. Organic agriculture, which is free from the indiscriminate and irrele-
vant use of synthetic chemicals, results in an increase in the population of naturally
occurring beneficial insects and micro-organisms that are known as bio-control
agents, having potential to control insects and diseases in organic farming. In
India, organic farmers predominantly use neem oil, fermented butter milk, cow
urine, panchgavya and baking soda for the control of pests as well as diseases in
organic farming. Butter milk and panchgavya have potential to control foliage
diseases in plants. Organic farmers also use baking soda for the control of mildew
and rust diseases on plants, while they use butter milk against blight, mildew, mosaic
virus and other fungal and viral diseases. Amrit paani, which is a fermented product
of cow dung and urine, is used by Indian farmers to enhance crop growth and disease
management. Such fermented solutions are known to have high bacterial population
of cellulose degraders, nitrogen fixers, P-solubilizers, plant growth promoters and
antagonists of disease-causing fungi, as reported by Venkateswarlu et al. (2008).
The use of essential oils extracted from aromatic plants as insecticides has
increased considerably owing to their popularity with organic growers and environ-
mentally conscious consumers, as stated by Hikal et al. (2017). According to Shelton
et al. (2002), these plant-based products have repellent, insecticidal, antifeedant,
growth inhibitory, oviposition inhibitory, ovicidal and growth-reducing effects on a
variety of insects and pathogens. Organic farmers are allowed to use naturally
occurring insecticides such as Bacillus thuringiensis (a bacterial toxin), pyrethrum
(a chrysanthemum extract), spinosad (a bacterial metabolite), neem (a tree extract)
and rotenone (a legume root extract) in organic farming. But only 10% organic
farmers use these naturally derived insecticides regularly, according to the survey
carried by Lotter (2003). These pesticides are not always safe or environmentally
friendly than synthetic pesticides and can cause harm, as stated by Gillman (2008).
Marking and Bills (1976) said that rotenone and pyrethrum are particularly
618 L. Rawat et al.
controversial because they work by attacking the nervous system, like most conven-
tional insecticides, while rotenone is extremely toxic to fish and can induce
symptoms resembling Parkinson’s disease in mammals, as argued by Panov et al.
(2005). Pyrethrum (natural pyrethrins) is more effective against insects when used
with piperonyl butoxide (which retards degradation of the pyrethrins), as reported by
Jones (1998). Scheuerell and Mahaffee (2004) said that the compost tea contains a
mix of beneficial microbes, which may attack or out-compete certain plant
pathogens but variability among formulations and preparation methods may contrib-
ute to inconsistent results or even dangerous growth of toxic microbes in compost
teas, as stated by Brinton et al. (2004). They also said that some naturally derived
pesticides like nicotine sulfate, arsenic and strychnine are not allowed for use in
organic farms.
27.3.5 Synthetic Pesticide Control
The oldest synthetic fungicide is Bordeaux mixture which is prepared by reaction of

copper sulphate and calcium hydroxide in water to manage a number of diseases in
agricultural and horticultural crops. Gayon and Sauvageau (1903) stated that the first
copper-based antimicrobial compound used in agriculture was Bordeaux mixture,
which was accidentally discovered in 1885 by a French scientist, Pierre-Marie
Alexis Millardet. Copper acts in two ways, (i) as a co-factor for several enzymes
involved in respiration and electron transport proteins in all living organisms
including plants (Sommer 1931) and (ii) as a broad-spectrum fungicide at higher
concentrations due to its interaction with nucleic acids, disruption of enzyme active
sites, interference with the energy transport system and finally the disruption of the
integrity of cell membranes, as argued by Fleming and Trevors (1989). A wide range
of copper-based antimicrobial compounds are formulated for the use of foliar disease
management in annual and perennial crops, especially in organic agriculture since
the application of conventional fungicides is prohibited in this system.
Diseases caused by oomycetes viz., downy mildew of grapevine and late blight of
potato are mostly managed by using copper-based antimicrobial compounds in
organic farming (Tamm et al. 2015; Finckh et al. 2015a, b). However, apple scab
and various coffee diseases which are difficult to manage without fungicides also
benefit from the use of copper-based antimicrobial compounds, as reported by Holb
et al. (2003) and Souza et al. (2015). The other diseases, i.e. tomato spot (Roberts
et al. 2008), citrus canker (Behlau et al. 2017), fire blight of pome fruits (Elkins et al.
2015), walnut blight (Ninot et al. 2002), stone fruit canker (Sayler and Kirkpatrick
2003), mango apical necrosis (Cazorla et al. 2006) and olive knot (Teviotdale and
Krueger 2004) are controlled by using copper-based antimicrobial compounds in
organic farming. However, copper sulfate and Bordeaux mixture (copper sulfate plus
lime) approved for organic use in various jurisdictions can be more environmentally
problematic than some synthetic fungicides disallowed in organic farming, as stated
by Edwards-Jones and Howells (2001). Application of copper-based formulations
like copper sulfate and copper nitrate in agricultural soil either in too high dose or too
frequently may cause plant stress and reduce soil fertility, having adverse effects on
the crop yield and quality (Dumestre et al. 1993), because these formulations release
Cu ions when they are dissolved in water and thus an excessive uptake of Cu ions by
plants at any time may lead to damage, also known as phytotoxicity. Lamichhane
et al. (2018) observed that the prolonged application of copper-based antimicrobial
compounds for over a century has resulted in accumulation of this heavy metal in the
soil particularly at upper 15 cm top soil. However, the potential toxicity of Cu varies
from one soil to another independently of the concentration of Cu accumulated in the
soil, for example, alkaline soils with increased calcium availability ameliorate the
effects of Cu phytotoxicity, and downward movement of copper through the soil
profile is greater in sandy soils than soils rich in clay or organic matter, as reported by
Alva et al. (1993 and 1995). Furthermore, copper availability and toxicity in the soil
is greatly increased as the soil pH decreases below 5.5, as observed by Fan et al.
(2011). Therefore, research is also needed to seek alternatives for replacement of
copper-based fungicides in organic agriculture.
27.4 Principles of Organic Agriculture
Organic farming has set out to be an alternative to conventional agriculture, and

Luttikholt (2007) stated that it is based on principles and values. The four basic
principles which are the roots from which organic agriculture develops are described
below:
1. Health
The first principle of organic agriculture is health, which is sustained and enhances
the health of soil, plant, animal and human as one and indivisible, as suggested by
IFOAM (2006). According to this principle, there is a relationship between
healthy soil and human and animal health, because healthy crop produced from
healthy soil fosters human and animal health. Balfour (1943) argued that the
living soil is a necessary condition for healthy plant growth and human being. The
key characteristics of health in organic farming are immunity, resilience, regen-
eration and maintenance of social, ecological, mental and physical well-being.
Therefore, the goal of organic agriculture is to produce healthy and quality food
by avoiding chemicals like fertilizers and pesticides that contributes to preventive
health care of humans, animals and ecosystem.
2. Ecology
Organic agriculture should be based on living ecological systems and cycles, work
with them, emulate them and help sustain them, as suggested by IFOAM (2006).
According to this principle, the living ecosystem is the heart of organic agricul-
ture because nutritious food can be achieved through ecology of specific produc-
tion environment, which is based on ecological process and recycling. Darnhofer
et al. (2010) elaborated the meaning of ecology in organic management, to build
up resilience of the agro-ecological system, attain ecological balance through
620 L. Rawat et al.
cropping systems, inputs reduced by reuse, recycling, efficient management and

must be adapted to local conditions, ecology, culture and scale.
3. Care
Organic agriculture should be managed in a precautionary and responsible manner to
protect the health and well-being of current and future generations and the
environment as suggested by IFOAM (2006). Kirchmann et al. (2008) argued
that the caring for the environment is a basic principle necessary for its
sustainability in order to provide humans with well-being, food and other
essentials. Scientific knowledge alone is not sufficient, practical experience,
accumulated wisdom and traditional and indigenous knowledge offer valid
solutions, tested by time as reported by Kirchmann et al. (2008). Therefore, the
key concern in organic management, development and choices of technology to
organic agriculture should be precaution and responsibility.
4. Fairness
Organic agriculture should be built on relationships that ensure fairness with regard
to the common environment and life opportunities, as suggested by IFOAM
(2006). The principle of fairness adds new aims to organic agriculture, not
explicitly addressed by the pioneers, such as equity, respect, justice, eradication
of poverty, animal welfare, equitable systems for distribution and trade as well as
social costs as stated by Kirchmann et al. (2008) This principle insists that organic
agriculture should prevent significant risks by adopting appropriate technologies
and rejecting unpredictable ones. Therefore, fairness requires systems of produc-
tion, distribution and trade that are open and equitable and account for real
environmental and social costs (IFOAM, 2006).
27.5 Strategies for Plant Disease Management in Organic

Farming System
Occurrence of a disease depends on the balanced interaction among host, pathogen

and environment. Disease management practices (Fig. 27.2) under organic agricul-
ture aim to disrupt this balance and disallow the pathogen to cause disease beyond
the economic injury level. Pathogens need suitable environmental conditions like
humidity, temperature, moisture, host exudates, etc. to germinate, survive and infect.
In the absence of these, pathogens cannot survive and perish. Most of the strategies
under agronomic, physical, biological and genetic methods described below inter-
fere with the micro-environmental conditions to make them uncongenial for patho-
gen propagation, multiplication and initiating infection. Further, a majority of these
strategies are specific to a particular disease in a crop and hence a combination of
strategies based on the crop growth stages and disease cycle need to be integrated as
a module for a crop in a particular agro-climatic region. The following methods are
used for plant disease management under organic agriculture:
Fig. 27.2 Methods of plant

disease management in
organic farming system Agronomic
Methods
Plant Disease
Management
Genetic Physical
Methods Under Methods
Organic Farming
System
Biological
Methods
27.5.1 Agronomic Methods
The crop productivity potential depends on its production environment and farmers
expertise to identify and overcome the factors that minimize the production poten-
tial. Modification in crop management tactics to disallow disease development is one
of the oldest and mostly accepted methods in plant disease management. The
following agronomic practices can be helpful for management of plant diseases
under the organic agriculture system.
(i) Sanitation
The aim of field sanitation is to completely or partially destroy the source of infection
present in the soil. The periodic clean off of diseased plants from a population is a
basic sanitary precaution of organic agriculture. For the viral disease control, field
sanitation is one of the effective recommendations. It includes removal of dis-
eased plants, pruning of infected parts of plants, removing or effectively treating
plant material, burning infected crop stubble, etc. and preventing the inoculum
from finding suitable infection courts, by preventing wounding, establishing
barriers and defoliation (Zentmyer and Bald 1977). Sanitation particularly is
applicable to pathogens that do not spread from plant to plant in the field and
that require a large amount of inoculum to develop an epiphytotic condition when
crops are grown in the same field for several years. As an example, a sanitation
practice for late blight of potato is to eliminate the refuse piles where infected
tubers give an early start for the disease (Finckh et al. 2006). In vineyards and
orchards, diseased branches are pruned away and plant residues are removed from
greenhouses (Finckh et al. 2015a, b).
(ii) Crop Residue Management
Crop residues are non-economic plant parts that are left in the field after harvesting.
The organic carbon in the soil can be enhanced through decomposition of crop
residues that foster soil health and disease control. Generally, the growth of
622 L. Rawat et al.
pathogen spores, their sporulation and survival depend on the crop residue
amount and quality that is left in the field after harvesting of host and non-host
plants. After decomposition of the crop residues through the release of fungicidal
and fungistatic compounds, it provides food to facultative pathogens for feed
on. The factors that determine the rate of decomposition are depth of placement,
type of crop, quantity of residues, allelopathic interactions among existing soil
biota and time as stated by Bailey and Lazarovits (2003). They also reported that
the partial disease control can be attained using residue management methods,
such as tillage and crop rotation that lower the pathogen’s inoculum density in the
soil, reduce its ability to survive, deprive the pathogen of its host, and create
conditions that favour the growth of other microorganisms at the expense of the
pathogen. Palaniappan and Annadurai (1999) suggested two principle methods of
residue decompositions, (i) thermo-chemical including direct combustion, pyrol-
ysis, liquefaction and gasification with air or oxygen and (ii) biological including
anaerobic digestion and hydrolysis followed by fermentation.
(iii) Tillage
The basic objective of tillage is to prepare seed-bed for seeding by sizeable distur-
bance of the soil, while zero tillage or single tillage involves minimum amount of
soil disturbance. Lupwayi et al. (1999) stated that the organic matter accumula-
tion through sequestering carbon in the soil can be achieved by minimizing
tillage, and as a result the rate of decomposition increases by microflora and
microfauna, as reported by Kennedy and Smith (1995). Soil having higher
organic matter showed their potential to prevent spore germination of Cannabis
sativa and the total population of soil microbes enhances with increasing soil
organic matter as observed by Chinn (1967). Plough practices directly or indi-
rectly displace the spore of pathogens through the crop residue placement in the
soil, which fosters the activity and competition among soil microbes (Cook
1990). Therefore, reduce tillage practices play an important role for disease
management, because they minimize the potential of disease-causing agents by
removing the primary source of inoculums.
(iv) Crop Rotation
Crop rotation is a long established practice to reduce the activity, pathogenesis and
survival of soil-borne fungi, nematodes or other pathogens (Baker and Cook
1974). Typical examples of soil invaders that can be controlled in 3–4 year
rotation with non-host crops include the organisms causing cabbage black rot,
bacterial blight of bean, cabbage blackleg and bean anthracnose. Sequeira (1958)
stated that the bacterial wilt of banana can be controlled by disking the surface
24 cm of soil during the dry season followed by 9 months fallow taking advantage
of the sensitivity of the causal organism to desiccation. Long periods of rotation
are often necessary particularly for soil inhabitants and pathogens that survive by
means of sclerotia or thick-walled resting spores. The development and imple-
mentation of well-designed crop rotations is central to the success of organic
production systems (Stockdale et al. 2001). However, crop rotation can be
ineffective if the pathogen is long-lived in the soil with a wide host range. For
the formation and maintenance of healthy soil through organic crop rotation, it
should comprise multi-year grass ley or grass-legume ley or an alfalfa crop, as
stated by Van Bruggen and Termorshuizen (2003). The effectiveness of crop
rotation in disease management depends on (i) wide host range, (ii) mechanisms
of pathogen survival, (iii) amount of inoculums, (iv) crop susceptibility, (v) types
of crops that stimulate the formation of resting structure, (vi) poor crop residue
management, (vii) residue management techniques, (viii) frequency of soil infes-
tation with pathogens from external sources and (ix) type of soil conducive to
diseases.
(v) Soil Disinfestations
Soil disinfestations play an important role for reducing the initial inoculum. The
following methods can be used for soil disinfestations under organic agriculture.
(a) Flooding
This is a pre-planting practice which can be regarded as soil disinfestation
treatment. The harmful effect of flooding on soil-borne pathogens may be
related to lack of oxygen, increased CO2 or various microbial interactions,
e.g. production of substances that are toxic to the pathogen upon anaerobic
processes (Bruehl 1987). A classic case of management on a large scale was
demonstrated with the panama wilt disease of banana caused by Fusarium
oxysporum f.sp. cubense (Stover 1962). The soil is flooded for 3–4 months or
more with a minimum of 30 cm of water. Flooding is not effective when large
populations of the pathogen are present, or in soil which contain unknown
factors which may favour the pathogen. However, flooding can only be used
as a cultural practice for disease management only in countries where large
resources of water are available (Patil 1981).
(b) Soil Solarization
In soil solarization, moist soil is covered with transparent, UV-resistant
plastic and exposed to sunlight for a few weeks (Gamliel and Katan 2012).
Most plant-pathogenic fungi, bacteria and nematodes, except for some heat-
tolerant fungi and viruses, are quite sensitive to increased temperatures,
i.e. 45–55 C (Klein et al. 2011). The solarization effect can be enhanced
by incorporation of isothiocyanate producing residues from brassica crops
into soil before covering with plastic (Klein et al. 2012). Along with the
direct heat effects on pathogens, soil solarization can also enhance plant
growth by increasing the availability of mineral nutrients and improving
soil tilth (Gamliel and Katan 2012). Fungal diseases such as damping-off,
root rots, stem rots, fruit rots, wilts and blights caused by Pythium spp.,
Phytophthora spp., Fusarium spp., Sclerotium rolfsii, Rhizoctonia solani,
Sclerotinia sclerotiorum, and Verticillium spp. can easily be managed by soil
solarization. Similarly, a number of bacterial diseases like bacterial canker of
tomato and nematode diseases such as Ditylenchus dipsaci, Globodera
rostochiensis, Heterodera spp., and Meloidogyne spp. have been success-
fully managed in fields by soil solarization (Akhtar et al. 2008).
624 L. Rawat et al.
(c) Anaerobic Soil Disinfestations

In anaerobic soil disinfestations, fresh organic material is incorporated into
soil, and the soil is moistened and covered by airtight plastic for 3–6 weeks
(Momma 2008). Proliferating bacteria deplete the available oxygen until
anaerobic bacteria continue to decompose the carbon source. Toxic products,
including alcohols, aldehydes, organic acids and other volatile compounds
accumulate and soil pH is reduced, affecting the survival of soil-borne
pathogens (Huang et al. 2015). Anaerobic bacteria such as Bacillus and
Clostridium spp. may also contribute to pathogen inactivation. Anaerobic
soil disinfestation results in the control of many soil-borne plant-pathogenic
fungi, bacteria, and nematodes, viz., Rhizoctonia, Fusarium, Verticillium,
Sclerotinia, Phytophthora, Ralstonia, Meloidogyne and Globodera spp., as
well as most weeds (Butler et al. 2012). The changes in microbial
communities’ characteristics for anaerobic soil disinfestation often result in
general disease suppression that can remain active for several years (Goud
et al. 2004).
(d) Biofumigation
It involves the addition of organic amendments to soil, generating biologi-
cally derived volatile compounds that are toxic to soil microorganisms.
Green manure crops that contain glucosinolates, mainly Brassica spp., are
most commonly used (Cohen et al. 2005). After tissue decomposition,
hydrolysis results in the release of various toxic compounds, such as organic
cyanides, nitriles, and thiocyanates, which have fungistatic or biocidal
properties (Finckh et al. 2015a, b). Application of animal-derived residues
that are high in nitrogen, such as manure or compost, can result in the
production of ammonia gas, which is toxic to a wide range of pathogens
and nematode pests (Lazarovits et al. 2001).
(vi) Application of Organic Amendments
Soils with low microbial diversity promote establishment of plant pathogenic
organisms. Healthy soil is the mainstay of organic agriculture. Improved soil
biological activity is known to play a key role in suppressing weeds, pests and
diseases (IFOAM 1998). Improving soil health through use of cover crops, green
manures and animal manures to fertilize the soil not only helps in restricting soil-
borne pathogens but also maximizes biological activity and maintains long-term
soil health. Application of composts and organic amendments tends to increase
the quantity and diversity of soil microbial diversity and consecutive disease
suppressiveness. Organic amendments are biodegradable and are generally avail-
able on the farmer’s fields. Neem cake used for soil amendment @ 0.25 to 0.5 t/ha
contributes significantly in control of nematodes and soil-borne pathogens. Soils
rich in organic matter are high in soil biodiversity with abundance of beneficial
soil microorganisms (Singh 2003).
(vii) Cultural Control
Cultural control is more like habit of good agricultural practices, which promote
healthy soils and healthy plants. From choosing the date of planting to field
sanitation and weed management, the specific cultural measures reduce the initial
load of inoculum and favourable conditions for growth of pathogens. Litterick

et al. (2002) opined that pest control strategies in organic farming systems are
mainly preventive rather than curative. The management of cropped and
un-cropped areas, crop species and variety choice and the temporal and spatial
pattern of the crop rotations are actually aimed to reduce interaction between
susceptible host and virulent pathogen, while maintaining a diverse population of
beneficial organisms in the field. Ensuring good drainage is essential for disease
management. Poor drainage in the fields not only reduces general health of the
plant but also allows the pathogen to multiply rapidly. Many pathogens can
survive on debris and weeds. Tilling and cleaning of plant residue at the end of
the season allows the break down of the organic matter, leaving potential
pathogens without a host. Moderate fertilization induces steady growth and
makes a plant less vulnerable to infection.
(viii) Management of Environment
The changing climatic conditions pose a potential threat to agricultural production
and productivity throughout the world and this might affect the crop yields,
incidence of weeds, pests and plant diseases and the economic costs of agricul-
tural production. This changing climatic condition has a twin effect in front of
agricultural production. First, the frequent outbursts of diseases are obviously due
to break of resistance chain of the respective crops and fast acclimatization of
disease causing agent, i.e. causal organism under changing climate conditions
(IPCC 2007). Modification of the environment may play an important role in
resistance of plant to disease. Walker (1969) stated that the polygenic resistance is
less stable than monogenic resistance in relation to temperature change. Mono-
genic resistance to fusarium wilt of cabbage is stable up to 26 C while polygenic
resistance breaks down at 24 C temperature. Therefore, the microclimate can be
modified by using the following methods for management of diseases under the
organic agriculture system.
(a) Date of Seeding
Some diseases are very destructive when susceptible age of the host and
optimum soil and atmospheric conditions for aggressiveness of the pathogen
coincide. Alteration of the date of sowing in such a way that the susceptible
stage of the plant growth does not coincide with the favourable environment
for the pathogens helps in reducing losses from such diseases. Delayed
emergence of seedlings above the soil surface gives the pathogen more
time for causing infection. Regulation of the depth of sowing in such cases
helps young plants escape infection. Dickson (1923) reported that the wheat,
a low-temperature crop, grows well and escapes invasion by Fusarium
roseum f. sp. cerealis in early spring when the soil temperature is in the
range of 8–12 C. Planting times can be adjusted to avoid heavy aphid flights
or periods when other diseases will surge by planting crops at the proper time
of the year or by ensuring enough crop growth before the onset of an
epidemic (Finckh et al. 2015a, b).
626 L. Rawat et al.
(b) Spacing of Crop

Growth habit and density of planting can be significant factors in increasing
microclimate near the leaf surface and thus increasing some foliage diseases.
Therefore, spacing between plants can help reduce disease incidence because
of better ventilation, sunlight and lesser humidity in the crop and those
organisms that flourish in high humidity such as downy mildews are
discouraged. Steadman et al. (1973) found that the wider spacing provided
satisfactory control of white mold disease in bean. Less disease inci-
dence by Cercospora apii on celery due to wider space was reported by
Berger (1975) who stated that the less disease incidence was due to modified
micro-climatological factors rather than less inoculum of Cercospora apii.
(c) Mixed Cropping System
Mixed cropping system is defined as the cultivation of a mixture of two crops
together in the same field. Mixed cropping system can be characterized
according to the degree to which roots of different crop species interact,
which is determined not only by the mixed cropping system but also by the
root architecture of each of the crops in the mixture (De Kroon 2007).
Pigeonpea mixture with sorghum reduces the wilt disease incidence probably
by increasing the distance between host plants and between infected and
healthy roots by creating root barriers between the roots of diseased and
healthy plants, and through toxic root exudates (Singh, 2007). The following
mechanisms are reported to reduce diseases under mixed cropping system:
(i) Host Dilution
Host dilution plays an important role in reduction of soil-borne diseases
or pathogens in mixed cropping systems. Mundt (2002) found host
dilution to be a dominant factor for disease-reducing mechanism for
air-borne pathogens in mixed cropping systems. While, the effect of
host dilution likely is a reduction in disease incidence rather than
disease severity on infected plants, as stated by Burdon and Chilvers
(1982). Host dilution might have a direct effect on the pathogen itself as
well as indirect effects on other factors than the pathogen on disease
suppression in mixed crops. Otten et al. (2005) reported the reduced
incidence of Rhizoctonia damping-off in radish–mustard mixtures
because of increasing densities of the non-host mustard plants and
spread halted at host densities below a threshold density. The intensity
of root intermingling in mixed cropping may be an important determi-
nant for the interference processes (Kroon 2007) and the level of disease
suppression may therefore be determined by the crops or cultivars
grown and their root architectures. The non-host crop simply acts as a
physical barrier, thus reducing disease spread (Vilich-Meller 1992). The
barrier function can reduce the impact of raindrops thus reducing
dispersal, and it can intercept splashing spores that would reach a host
plant under conditions of monoculture (Soleimani et al. 1996).
(ii) Allelopathy
Allelopathy is defined as any biochemical interaction among plants,
including those mediated by microorganisms, resulting in either detri-
mental or beneficial effects on the interacting plants (Wu et al. 2001).
When watermelon was intercropped with rice, allelopathic substances
from rice roots reduced production and germination of conidia of
Fusarium oxysporum f.sp. melonis, leading to a 67% reduction in wilt
(Ren et al. 2007). The allelopathic exudates only reduced Fusarium
conidial density in the rhizosphere and not in bulk soil indicating a
limited diffusion. Natarajan et al. (1985) found delayed germination of
spores of F. udum, causing wilt in pigeonpea because of allelopathic
substances exuding from sorghum roots. To be effective in inhibiting
rhizosphere-inhabiting pathogens, allelopathic substances should be
present at sufficiently high concentrations in the micro sites where the
pathogen is located and roots of mixed crops should be in close prox-
imity. Roots of non-hosts can sometimes stimulate the germination of
the survival propagules of the pathogen leading to a decline in the
inoculum density (Mol and Van Riessen 1995). In relay mixed crops,
this premature germination might have a disease-suppressive effect,
especially in combination with inoculum burial and enhanced microbial
antagonism.
(iii) Microbial Antagonists
Enhanced antagonistic populations are main mechanisms for disease
reduction in mixed-cropping. In mixed crops, increased plant diversity
leads to more diverse root exudates and consequently to a more diverse
rhizosphere-inhabiting microbial community (Kowalchuk et al. 2002).
Rhizospheres of mixed crops support different bacterial and fungal
microbial communities compared to the corresponding single-crop
rhizospheres (Song et al. 2007). Wheat root infection by G. graminis
var. tritici was reduced by 25% in wheat-trefoil mixed cropping system
(Lennartsson, 1988) while 75% reduction in Fusarium wilt was reached
when bottle gourd was mixed with Chinese chive because of stimulation
of Pseudomonas gladioli populations on the Chinese chive roots (Arie
et al. 1987). Also, increased occupation of available niches by
non-pathogenic fusaria was held responsible for increased disease sup-
pression in oil-palm–legume mixed cropping (Abadie et al. 1998).
Rhizosphere microbial communities, including pathogens, antagonists
and plant-growth-promoting bacteria are crop- and cultivar-specific
(Germida and Siciliano 2001). Cultivar-specific resistance against
races of pathogens is widely known and often applied in mixed crops.
Mazzola (2004) used wheat to stimulate the natural antagonistic
populations of fluorescent pseudomonads, which led to control of
apple replant disease.
628 L. Rawat et al.
27.5.2 Physical Methods
Heat therapy of seeds is commonly used to control certain seed-borne pathogens

while leaving the host tissue viable. Many media have been used for heat treatment
including water, steam, air and microwave radiation.
27.5.2.1 Concept of Heat Therapy

The concept of heat therapy is that microorganisms are killed or viruses are
destroyed at temperatures not injurious to seed (Baker 1962). However, it is univer-
sally accepted that heat cause inactivation and immobilization of pathogens. There
are two schools of thoughts regarding inactivation of pathogens (viruses) by heat.
One holds the opinion that the heat treatment stimulates enzymes that cause the
degradation of virus, though according to Benda (1972) this has not been
established. The others pursue the idea that heat causes loosening of bonds both in
nucleic acid and the protein components of the virus. In the nucleic acid, when the
bonds are disrupted the linear arrangement of nucleotides is disrupted and thus the
virus loses infectivity. In proteins, the bonds holding the chains of amino acids may
be destroyed. Disruption of bonds causes denaturing of protein molecules, which
become less soluble in water, and finally leads to coagulation. The rate at which the
pathogen is inactivated is determined by temperature and the duration of treatment
(Table 27.1). Seeds with low moisture content are ideal for heat therapy, while seeds
with high moisture content are killed during heat therapy because of denaturation of
protein, lipid liberation, hormone destruction, tissue asphyxiation, depletion of food
reserves and metabolic injury with or without accumulation of toxic intermediates,
as stated by Baker (1962).
Table 27.1 Details of temperature and exposure time for control of plant pathogens by heat
therapy methods
Crop Disease Temperature Duration
Brassica spp. Black rot 50 C 20–30 min.
Cluster bean Bacterial blight 50 C 10 min
Cucumber Seeding blight 50 C and 75% 3 days
relative humidity
Lettuce Leaf spot 70 C 1–4 days
Groundnut Testa nematode 60 C 5 min after soaking for
15 min in cool water
Pearl millet Downy mildew 55 C 10 min
Potato Potato phyllody 50 C 10 min
Rice White tip 51 C to 53 C 15 min after soaking for
1 day in cool water
Safflower Leaf spots 50 C 30 min
Tobacco Hollow stalk 50 C 12 min
Tomato Bacterial black speck 52 C 1 hours
Source: Chaube and Singh (1990)
(i) Hot Water

Hot water is used widely to control the pathogens especially bacteria and viruses.
The following steps are included in hot water heat therapy:
(a) Selection of Seeds
Seeds that can withstand hot water therapy are preferred. Hot water therapy
is recommended for seeds with deep-seated infection.
(b) Pre-soaking the Seeds
Pre-soaking is done to replace the air between the embryo and seed coat with
water, which is a better heat conductor. The water soak may stimulate
pathogen growth, by which the pathogen becomes more heat susceptible.
(c) Pre-heating
Pre-heating is done to counter the cooling effects of seeds soaked in cool
water. In pre-heating, seeds are heated for 1–2 min at 9–10 C below the
temperature of the final treatment.
(d) Hot Water Soak
The temperature and time required varies with the pathogen and crop. Time
must be precise otherwise seed viability is lost. A large volume of water
helps maintain a constant temperature. Seeds should be packed loosely in
porous bags, screen boxes or frames with sufficient provision for an ample
water flow.
(e) Cooling
Treated seeds are spread out for cooling and drying immediately after
treatment.
(f) Drying
Seeds are dried quickly to prevent sprouting.
Hot water therapy is an eco-friendly technique for controlling the pathogens and
economically viable, but because the seed temperature must be raised quickly, only
small quantities of seeds can be treated at one time and germination particularly of
older seeds may be reduced.
(ii) Hot Air Therapy

Hot air treatment is less injurious to seed and easy to operate but also less
effective than hot water treatment. It has been used against several diseases of
sugarcane. Red rot of sugarcane is completely controlled by hot air treatment of
54 C for 8 h (Singh 1973). Similarly, grassy shoot disease of sugarcane has
been controlled by hot air at 54 C for 8 h (Singh 1968). Drying tomato seeds in
an oven for 6 h at 29.5–37.5 C eliminated Phytophthora infestans from
discoloured seeds of infected fruits (Vartanian and Endo 1985). Dry heat
treatment of tomato seeds for 2 days at 78 C temperature reduced tomato
mosaic virus without an effect on germination. Zeigler and Alvarez (1988)
found dry heat treatment effective for controlling the Pseudomonas avenae and
Pseudomonas glumae in rice. Similarly, dry heat treatment of capsicum seeds
for 7 days at 70 C gave control of capsicum mosaic virus (Stijger and Rast
1988).
630 L. Rawat et al.
(iii) Aerated Steam Treatment

The use of aerated steam is safer than hot water and more effective than hot air
in controlling seed-borne infections. The heating capacity of water vapour is
about half that of water and 2.5 times that of temperature control and no damage
to seed coat of legumes (Agarwal and Sinclair 1996). Most frequent application
of steam and aerated steam has been in greenhouses where steam also provides
heat during cold seasons. As gas, it moves readily through soil, in contrast to
the slow, inefficient movement of water. Aerated steam provides an opportu-
nity to treat soil at temperatures lower than those possible with pure steam.
Navaratnam et al. (1980) obtained complete eradication of Septoria apiicola
from celery seeds after treatment for 30 min at 56 C and X. campestris
pv. campestris in cabbage seeds after treatment for 30 min at 54 C. ØThe
use of aerated steam to disinfect seeds is an easy-to-handle, inexpensive method
that was already shown to be effective in eliminating seed-borne plant-patho-
genic bacteria and fungi (Heller and Zoller 2010).
(iv) Moist Hot Air Therapy
The moist hot air therapy has been proposed by Singh (1973) to eliminate the
grassy shoot disease of sugarcane. Under this technique, the setts of sugarcane
are initially exposed to hot air for 8 h at 54 C, after that these setts are exposed
to aerated steam at 50 C for 1 h and finally to moist hot air at 54 C for 2 h.
(v) Solar Heat Therapy
Solar heat therapy is one of the simplest and oldest techniques used in India for
elimination of seed-borne pathogen of loose smut disease. This technique
devised by Landen (1939) to eliminate the deep-seated infection of Ustilago
nuda. Previously, the hot water treatment was followed to eliminate loose smut.
In this technique, seeds are soaked in cold water for 4 h in the forenoon on a
bright summer day followed by spreading and drying the seeds in hot sun for
4 h in the afternoon. But extensive care is necessary of the embryo to avoid
injuries from thermal death point because it is very close to pathogen and
embryo.
27.5.3 Biological Methods
The use of bio-agents puts forward a practical and economical alternative for plant
disease management in organic agriculture, in which the ecological community of
available microorganisms is maintained that enhance plant immunity by suppressing
the pathogens. Disease suppression by using bio-agents is a result of interaction
between plant, pathogen, bio-agent and microbial community on and surrounding
the plant and their physical environment. There are two basic strategies of biological
control (a) enhanced biological control via endemic natural enemies, including
competitors, antagonists, predators or parasites, by means of habitat management
and (b) inundative biological control via the release of specific competitors,
antagonists, predators or parasites. Srinivas and Ramakrishna (2005) stated that
the microbial bio-control agents isolated from native environments are relatively
safe, host specific and do not disturb other biotic systems. Commercial
bio-fungicides contain beneficial living organisms and used for disease management
in organic agriculture system. These are available in different forms viz., as powders
for seed treatments, as granulars for soil application and as suspensions for soil
drenches and foliar sprays.
27.5.3.1 Biocontrol Agents in Plant Disease Management

Biological control agents like Trichoderma spp., Pseudomonas spp. and Bacillus
spp. have proven their worth in managing a range of plant diseases. Handelsman and
Stabb (1996) observed that bacteria like Bacillus, Pseudomonas, Serratia and
Arthrobacter have shown efficacy to manage numerous fungal diseases. Bacterium
such as Pseudomonas fluorescens, which produces the antimicrobial polyketides,
has the ability to control several fungal pathogens, including Phythium spp.
(Girlanda et al. 2001), while Bacillus sp. accelerates plant growth by suppressing
soil-borne diseases (Utkhede and Smith 1992). Deka Boruah and Dileep kumar,
(2003) reported that Rhizoctonia solani, Fusarium solani, Fusarium semitectum and
other fungal diseases have been controlled by Bacillus sp., when it is used as a seed
coating agent. A combination of Pseudomonas strains is effective in siderophore-
mediated competition for iron and induction of systemic plant resistance to improve
control of Fusarium wilt of radish (De Boer et al. 2003). Seed treatment with
Trichoderma harzianum or Pseudomonas fluorescens @ 6 g/kg coupled with two
sprays of Pseudomonas fluorescens @ 0.3% at the time of flowering and second after
10 days can control leaf, neck and finger blasts very effectively in small millets
(Patro et al. 2008), while Nagaraja et al. (2012) stated that the blast can be managed
by seed dressing using bioformulation of Pseudomonas fluorescens.
27.5.3.2 Biocontrol Agents as a Major Component of Integrated Disease

Management System
An integrated pest management (IPM) system entails simultaneous or sequential use
of several methods of control. Biological control is of particular interest as a
component of, and can best be exploited within the framework of an IPM
system (Barzman et al. 2015). Biocontrol agents (BCAs) have distinct advantages
in being compatible with most of the agricultural practices and hence can be
successfully utilized as a part of total crop management practices or broadly, as a
component of the agro-ecosystem management (Rawat et al. 2012). Different
biocontrol agents have been integrated with cultural practices, soil solarization,
fungicides and disease-resistant varieties for managing different crop diseases.
Combination of the seed/root application of Trichoderma harzianum or Pseudomo-
nas fluorescens with soil solarization was very effective in management of seed and
seedling diseases of tomato, brinjal and capsicum in nursery at farmers field (Singh
2003). Wilt and root-rot complex of chickpea, lentil and pigeon pea were success-
fully managed by integration of Trichoderma harzianum or Trichoderma virens with
carboxin (Mukhopadhyay and Mukherjee 1996). Integration of fertilizers or
herbicides with biocontrol agents to control plant diseases has also been attempted.
Bacterial biocontrol agents are compatible with most of the fungicides. Even fungal
632 L. Rawat et al.
biocontrol agents like Trichoderma spp. are insensitive to fungicides like carboxins,
oxycarboxins, metalaxyl, tricyclazoles, carpropamid host defense inducers
(e.g. benzothiadiazole), etc. Therefore, they could be integrated easily. Integrating
biocontrol agents with reduced doses of fungicides seems to be an effective way of
controlling pathogens with less interference with biological equilibrium (Patibanda
and Ranganathswamy 2018). This would not only reduce the use of fungicides but
also improve the efficacy of a biocontrol system with reduced cost and lessen the
chances of development of fungicide-resistant strains of the pathogen. Integration of
biocontrol agents with compatible fungicides for seed treatment is very effective
even against high population of fast-growing pathogens like Rhizoctonia solani in
soil. Under such conditions, a biocontrol agent alone may not be very effective as by
the time it gets activated in soil, the pathogen is able to penetrate the host. Integration
of fungicide with biocontrol agents helps in early protection by fungicide and the
later protection by introduced biocontrol agents. A fungicide also provides a conge-
nial environment to compatible biocontrol agents to multiply and colonize
spermosphere and rhizosphere by suppressing other microbes sensitive to the fungi-
cide (Singh et al. 2003; Rawat et al. 2013).
27.5.3.3 Mechanisms of Biological Disease Control

The mechanisms underlying successful biological control vary from niche competi-
tion and antagonism to parasitism and predation. Van Bruggen and Termorshuizen
(2003) stated that the biocontrol mechanisms for soil-borne diseases are much
effective than aerial diseases because aerial diseases are influenced by micro-
climatic conditions.
In all cases, pathogens are antagonized by the presence and activities of other
organisms that they encounter. The different mechanisms of antagonism occur
across a spectrum of directionality related to the amount of interspecies contact
and specificity of the interactions (Table 27.2). Direct antagonism results from
physical contact and/or a high-degree of selectivity for the pathogen by the mecha-
nism(s) expressed by the biocontrol agent(s). In such a scheme, hyperparasitism by
obligate parasites of a plant pathogen would be considered the most direct type of
antagonism because the activities of no other organism would be required to exert a
suppressive effect. In contrast, indirect antagonisms result from activities that do not
involve sensing or targeting a pathogen by the BCA(s). Stimulation of plant host
defense pathways by non-pathogenic BCAs is the most indirect form of antagonism.
However, when the microbial products are applied on seeds or soil, they may also
induce systemic resistance to foliar diseases as stated by Vallad and Goodman
(2004). The mechanisms reveal by BCAs are a result of direct antagonism from
physical contact and/or a high-degree of selectivity for the pathogen. In such a
scheme, hyperparasitism by obligate parasites of a plant pathogen would be consid-
ered the most direct type of antagonism because the activities of no other organism
would be required to exert a suppressive effect. In contrast, indirect antagonisms
result from activities that do not involve sensing or targeting a pathogen by the
biological control agents. Stimulation of plant host defense pathways by
non-pathogenic biological control agents is the most indirect form of antagonism.
Table 27.2 Types of interspecies antagonisms resulting in biological control of plant pathogens
Type Mechanism Examples
Direct Hyperparasitism/ Lytic/some non-lytic mycoviruses Ampelomyces
antagonism predation quisqualis, Lysobacter enzymogenes, Pasteuria
penetrans, Trichoderma virens
Mixed-path Antibiotics 2,4-diacetylphloroglucinol, Phenazines, Cyclic
antagonism lipopeptides
Lytic enzymes Chitinases, Glucanases, Proteases
Unregulated Ammonia, Carbon dioxide, Hydrogen cyanide
waste products
Physical/ Blockage of soil pores, Germination signal
chemical consumption, Molecular cross-talk confused
interference
Indirect Competition Exudates/leachate consumption, Siderophore
antagonism scavenging, Physical niche occupation
Induction of host Contact with fungal cell walls, Detection of pathogen-
resistance associated, molecular patterns, Phytohormone-mediated
induction
Source: Pal, K. K. and B. McSpadden Gardener (2006)
For instance, Pseudomonads known to produce the antibiotic

2, 4-diacetylphloroglucinol (DAPG) may also induce host defenses, as reported by
Iavicoli et al. (2003). Additionally, DAPG-producers can aggressively colonize
roots, a trait that might further contribute to their ability to suppress pathogen activity
in the rhizosphere of wheat through competition for organic nutrients, as stated by
Raaijmakers and Weller (2001). Chandrashekara et al. (2012) reported direct-
(hyperparasitsm, predation), indirect- (induce host resistance and competition) and
mixed (antibiotic, lytic enzymes production, interference)-type antagonism.
In some instances, antibiotics produced by microorganisms have been shown to
be particularly effective at suppressing plant pathogens and the diseases they cause.
Some examples of antibiotics reported to be involved in plant pathogen suppression
are listed in Table 27.3.
27.5.3.4 Future Prospects for Large-Scale Commercialization

Microbial BCAs are ideal for both short- and long-term pest suppression and are also
compatible with most other control methods. Their mechanisms of action include
competition, antagonism, antibiosis, enhanced nutrient uptake, induction of host
resistance, plant growth promotion (Kloepper et al. 1997; Singh et al. 2003; Rawat
et al. 2011), etc. and unlike chemical pesticides, they are harmless to humans and
other non-target organisms, they do not leave chemical residues on crops, are easy
and safe to dispose of and do not contaminate water systems. A number of commer-
cial formulations of bio-control agents are available in the market for use as seed
treatment, soil application and foliar sprays. If a BCA is having high antagonistic
634 L. Rawat et al.
Table 27.3 List of some of the antibiotics produced by biocontrol agents

Antibiotic Source Target pathogen Disease
2, 4-diacetyl- Pseudomonas Pythium spp. Damping off
phloroglucinol fluorescens F113
Agrocin 84 Agrobacterium Agrobacterium tumefaciens Crown gall
radiobacter
Bacillomycin D Bacillus subtilis Aspergillus flavus Aflatoxin
AU195 contamination
Bacillomycin, Bacillus Fusarium oxysporum Wilt
fengycin amyloliquefaciens
FZB42
Xanthobaccin A Lysobacter sp. strain Aphanomyces cochlioides Damping off
SB-K88
Gliotoxin Trichoderma virens Rhizoctonia solani Root rots
Herbicolin Pantoea Erwinia amylovora Fire blight
agglomerans C9–1
Iturin A B. subtilis QST713 Botrytis cinerea and R. solani Damping off
Mycosubtilin B. subtilis BBG100 Pythium aphanidermatum Damping off
Phenazines P. fluorescens 2–79 Gaeumannomyces graminis Take-all
and 30–84 var. tritici
Pyoluteorin, P. fluorescens Pf-5 Pythium ultimum and R. solani Damping off
pyrrolnitrin
Pyrrolnitrin, Burkholderia R. solani and Pyricularia Damping off
pseudane cepacia oryzae and rice blast
Zwittermicin A Bacillus cereus Phytophthora medicaginis and Damping off
UW85 P. aphanidermatum
Source: Pal, K. K. and B. McSpadden Gardener (2006)
activity, competent saprophytic ability and besides this, if it is having plant growth
promotion activity, induce resistance in host, longer shelf life, tolerance to biotic and
abiotic stress and broad host range and is successfully commercialized, the specific
BCA will be a boon to the industry associated with its commercialization. The
broad-action spectrum of a BCA is a guarantee of its wide acceptability in the
market.
However, the following are the associated limitations of the BCAs:
• Inconsistence field performance

• Too specific or slow acting
• Poor shelf life of formulations
• Subject to environmental influences, i.e. effect of temperature, pH, salinity and
water stress.
These problems can be overcome by isolating or developing broad-action spec-

trum BCAs, which could be used under heterogeneous adverse environmental
conditions and these BCAs can be further improved for better stress tolerance by
using advanced techniques like mutagenesis, genetic transformation and protoplast
fusion. But the problems associated with these techniques are high cost, sophisti-
cated and required broader setup. Therefore, it is more feasible to isolate and screen
the BCAs for stress tolerance by testing them in vitro and natural field conditions,
where such adverse conditions prevail. Strains obtained by conventional methods
can be registered without any protest from environmental protection agencies. In this
regard, the first requirement of biological control is the identification and deploy-
ment of highly effective strains controlling several biotic stresses and at the same
time, it is important to have information about the effects of various environmental
factors on the biocontrol efficiency of BCAs in designing effective and safe biocon-
trol strategies (Rawat 2011). Biocontrol agents may be used in rotation or in
integration with compatible pesticides and or synthetic substances. There are some
synthetic substances such as copper hydroxide, copper oxychloride, copper oxide,
copper sulphate, hydrated lime, potassium bicarbonate and lime sulphur that are
accepted in organic farming provided that their use should be judicious and the
number of applications should be moderated.
There is a need for investigations on compatibility of BCAs with other agents,
cultural practices and agrochemicals. This will help in the development of more
effective and efficient formulations of BCAs. Industries should work closely with
researchers and extension service workers in resolving these matters before the
products reach the market.
27.5.4 Genetic Methods
Resistance of diseases and pests is a naturally occurring phenomenon in plants. That

is how they have been facing the attack of enemies probably since their evaluation.
Nature and pathogens have been eliminating the weak and susceptible ones from the
population while the farmers selected the best yielders from the survivors. These
surviving populations carried different sets of major or minor genes for resistance.
By using these surviving populations, numerous varieties or cultivars have been
developed for the cultivation. There are two approaches under genetic method for
managing diseases in the organic farming system.
27.5.4.1 Use of Disease-Resistant Varieties

The use of a resistant variety is an important low external input alternative to
chemical pesticides in organic agriculture. Exploiting the diversity and variability
in the host genetic constitution for resistance against a pathogen in a crop is the best
strategy for disease management without application of hazardous pesticides. It can
individually restrict the incidence of a particular disease in a crop. Successful disease
establishment depends on the compatible gene for gene interaction between a host
and a pathogen. Resistant varieties tend to remain disease free for a long period of
time owing to morphological manifestation of their genetic constitution in the form
of leaf and stem toughness, time of maturity, nutrient content, plant architecture and
growth habit which can deter growth of pathogen, their reproduction and host
preference. Care should be taken to include more than one resistant variety in a
636 L. Rawat et al.
region to dissipate selection pressure on the pathogen. However, this strategy is very
specific and tends to tackle only one or two diseases at a time owing to its resistance
(Srinivas 2017).
27.5.4.2 Use of Multiline Varieties

Multiline varieties and variety mixtures can also provide functional diversity that
limits pathogen and pest expansion cropping system (Finckh et al. 2000). These
approaches also reduce the risk of resistance breakdown, which are due to a range of
mechanisms including barrier and frequency effects as well as induced resistance.
Also, differential adaptation, i.e. adaptation within races to specific host genotypic
backgrounds, may prevent the rapid evolution of complex patho-types in mixtures
(Finckh et al. 2000). Therefore, yield stability is commonly greater in mixtures than
in pure stands (Finckh 2008). The wider application of variety mixtures in organic
farming is constrained by the concern of farmers and processors about
the anticipated negative effect on the homogeneity of the quality. However, if the
mixture components are carefully designed and selected for desired traits in the
breeding progress, product quality may be equal to or higher than that obtained in
pure stands (Finckh 2008). Nevertheless, there is still a risk that due to genotype
environment interaction, unacceptable heterogeneity may occur under different
environments.
References
Abadie C, Edel-Hermann V, Alabouvette C (1998) Soil suppressiveness to Fusarium wilt: influence
of a cover plant on density and diversity of Fusarium population. Soil Biol Biochem
30:643–649
Agarwal VK, Sinclair JB (1996) Principles of seed pathology. Lewis Publishers, New York, p 539
Akhtar Y, Yeoung YR, Isman MB (2008) Comparative bioactivity of selected extracts from
Meliaceae and some commercial botanical insecticides against two noctuid caterpillars,
Trichoplusia ni and Pseudaletia unipuncta. Phytochem Rev 7(1):77–88
Altieri MA (1995) Agroecology: the science of sustainable agriculture. Intermediate Technology
Publications, London
Alva AK, Graham JH, Tucker DPH (1993) Role of calcium in amelioration of copper phytotoxicity
for citrus. Soil Sci 155:211–218
Alva AK, Graham JH, Anderson CA (1995) Soil pH and copper effects on young “Hamlin” orange
trees. Soil Sci Soc Am J 59:481–487
Arie T, Namba S, Yamashita S, Doi Y, Kijima T (1987) Pseudomonas gladioli. Annu Rev
Phytopathol Soc Jpn 53:531–539
Bailey KL, Lazarovits G (2003) Suppressing soil-borne diseases with residue management and
organic amendments. Soil Tillage Res 72:169–180
Barzman M, Bàrberi P, Birch ANE, Boonekamp P, Dachbrodt-Saaydeh S, Graf B, Hommel B,
Jensen JE, Kiss J, Kudsk P, Lamichhane JR, Messéan A, Moonen AC, Ratnadass A, Ricci P,
Sarah JL, Sattin M (2015) Eight principles of integrated pest management. Agron Sustain Dev
35(4):1199–1215
Baker KF (1962) Thermotherapy of planting material. Phytopathology 53:1244
Baker KF, Cook RJ (1974) Biological control of plant pathogens. W. H. Freeman and Co San
Francisco, California, p 433
Balfour EA (1943) The living soil. Faber & Faber Ltd, London, p 276
Behlau F, Scandelai LHM, Da Silva Junior GJ, Lanza FE (2017) Soluble and insoluble copper
formulations and metallic copper rate for control of citrus canker on sweet orange trees. Crop
Prot 94:185–191
Benda GTA (1972) Hot water treatment for mosaic and ratoon stunting disease control. Sugar J
34:32–39
Berger RD (1975) Disease incidence and infection rates of Cercospora apii in plant spacing plots.
Brinton et al (2004) Compost teas: microbial hygiene and quality in relation to method of prepara-
tion (PDF). Biodynamics 249:36–45
Bruehl GW (1987) Soil borne plant pathogens. MacMillan Publishing Company, New York
Burdon JJ, Chilvers GA (1982) Host density as a factor in disease ecology. Annu Rev Phytopathol
20:143–166
Butler DM, Rosskopf EN, Kokalis-Burelle N, Albano JP, Muramota J, Shennan C (2012) Exploring
warm season cover crops as carbon sources for anaerobic soil disinfestations. Plant and Soil
355:149–165
Cazorla FM, Arrebola E, Olea F (2006) Field evaluation of treatments for the control of the bacterial
apical necrosis of mango (Mangifera indica) caused by Pseudomonas syringae pv. syringae.
Chandrashekare KN, Manivannan S, Chandrashekare C, Chakravarthi M (2012) Biological control
of plant diseases. In: Singh VK, Singh Y, Singh P (eds) Eco-friendly innovative approaches in
plant disease management. International Book Distributors and Publisher, New Delhi, pp
147–166
Chaube HS, Singh US (1990) Plant disease management: principles and practices. C.R.C, Press,
Boca Raton, p 319
Chinn SHF (1967) Differences in fungistasis in some Saskatchewan soils with special reference to
Cochliobolus sativus. Phytopathology 57:224–226
Cohen MR, Yamasaki H, Mazzola M (2005) Brassica napus seed meal soil amendment modifies
microbial community structure, nitric oxide production and incidence of Rhizoctonia root rot.
Soil Biol Biochem 37:1215–1227
Cook RJ (1990) Twenty-five years of progress towards biological control. In: Hornby D
(ed) Biological control of soilborne pathogens. CAB International, Wallingford, pp 1–14
Darnhofer I, Lindenthal T, Bartel-Kratochvil R, Zollitsch W (2010) Conventionalization of organic
farming practices: from structural criteria towards an assessment based on organic principles. A
review. Agron Sustain Dev 30:67–81
De Boer M, Bom P, Kindt F, Keurentjes JJB, Van der Sluis I, Van Loon LC, Bakker PAHM (2003)
Control of Fusarium wilt of radish by combining Pseudomonas putidastrains that have different
disease suppressive mechanisms. Phytopathology 93:626–632
De Kroon H (2007) How do roots interact? Science 318:1562–1563
Deka Boruah HP, Dileep Kumar BS (2003) Rhizoctonia wilt suppression of brinjal and plant
growth activity by Bacillus BS2. Indian J Exp Biol 41:627–631
Dickson JG (1923) Influence of soil temperature and moisture on the development of the seedling
blight of wheat and corn. J Agric Res 23:837–869
Dumestre A, Sauve S, McBride M (1993) Copper speciation and microbial activity in long-term
contaminated soils. Arch Environ Contam Toxicol 36:124–131
Edwards-Jones G, Howells O (2001) The origin and hazard of inputs to crop protection in organic
farming systems: are they sustainable. Agr Syst 67:31
Elkins RB, Temple TN, Shaffer CA (2015) Evaluation of dormant stage inoculum sanitation as a
component of a fire blight management program for fresh-market Bartlett pear. Plant Dis
99:1147–1152
Fan J, He Z, Ma LQ, Stoffella PJ (2011) Accumulation and availability of copper in citrus grove
soils as affected by fungicide application. J Soil Sediment 11:639–648
FAO (1999) Organic agriculture. Food and agriculture organization of the United Nations, Rome
Accessed 26 Feb 1999
638 L. Rawat et al.
Fi BL (2006) Use of potassium bicarbonate as a fungicide in organic farming Archived 11 January

2014 at the Wayback Machine
Finckh MR (2008) Integration of breeding and technology into diversification strategies for disease
control in modern agriculture. Eur J Plant Pathol 121:399–409
Finckh MR, Gacek ES, Goyeau HC, Lannou U, Merz CC, Mundt L, Munk J, Nadziak AC,
Newton C, Vallavieille-Pope D, Wolfe MS (2000) Cereal variety and species mixtures in
practice, with emphasis on disease resistance. Agronomie 20:813–837
Finckh MR, Schulte-Geldermann E, Bruns C (2006) Challenges to organic potato farming: disease
and nutrient management. Potato Res 49:27–42
Finckh MR, Van Bruggen AHC, Tamm L (2015a) Plant diseases and their management in organic
agriculture. APS Press, St. Paul
Finckh MR, Tamm L, Bruns C (2015b) Organic potato disease management. In: Finckh MR, van
Bruggen AHC, Tamm L (eds) Plant Diseases and their Management in Organic Agriculture.
APS Press, St Paul, pp 239–257
Fleming CA, Trevors JT (1989) Copper toxicity and chemistry in the environment: a review. Water
Air Soil Pollut 44:143–158
Fliebbach A, Mader P (2000) Microbial biomass and size-density fractions differ between soils of
organic and conventional agricultural systems. Soil Biol Biochem 32(6):757–768
Fliebbach A, Oberholzer H, Gunst L, Mäder P (2006) Soil organic matter and biological soil quality
indicators after 21 years of organic and conventional farming. Agric Ecosyst Environ 118
(1–4):273–284
Gamliel A, Katan J (2012) Soil solarization: theory and practice. APS Press, St. Paul, p 266
Gan Y, Campbell CA, Liu L, Basnyat P, McDonald CL (2009) Water use and distribution profile
under pulse and oilseed crops in semiarid northern high latitude areas. Agric Water Manag
96:337–348
Gayon U, Sauvageau C (1903) Notice sur la vie et les travaux de A. Millardet Mem la Soc des Sci
Phys Nat Bordeaux 6:9–47
Germida JJ, Siciliano SD (2001) Taxonomic diversity of bacteria associated with the roots of
modern, recent and ancient wheat cultivars. Biol Fertil Soils 33:410–415
Girlanda M, Perotto S, Moenne-Loccoz Y, Bergero R, Lazarri A, Defago G, Bonfante P, Luppi AM
(2001) Impact of biocontrol Pseudomonas fluorescens CHA and a genetically modified deriva-
tive on the diversity of culturable fungi in the cucumber rhizosphere. Appl Environ Microbiol
67:1851–1864
Gillman J (2008) The truth about organic gardening: benefits, drawnbacks, and the bottom line.
Timber press
Goud JKC, Termorshuizen AJ, Blok WJ, Van Bruggen AHC (2004) Long-term effect of biological
soil disinfestation on Verticillium wilt. Plant Dis 88:688–694
Handelsman J, Stabb EV (1996) Biocontrol of plant pathogens. Plant Cell 8:1855–1869
Herridge DF, Peoples MB, Boddey RM (2008) Global inputs of biological nitrogen fixation in
agricultural systems. Plant and Soil 311:1–18
Heller WE, Zoller C (2010) Pflanzenbau Desinfektion von Basilikum-Saatgut ist eine
Herausforderung. J Pediatr Matern Fam Health-Chiropr 1(5):190–193
Hikal WM, Baeshen RS, Said-Al Ahl HAH (2017) Botanical insecticide as simple extractives for
pest control. Cogent Biol 3:1404274
Holb IJ, DeJong PF, Heijne B (2003) Efficacy, phytotoxicity of lime sulphur in organic apple
production. Ann Appl Biol 142:225–233
Huang X, Wen T, Zhang J, Meng L, Zhu T (2015) Toxic organic acids produced in biological soil
disinfestation mainly caused the suppression of Fusarium oxysporum f. sp. cubense. BioControl
60:113–124
Iavicoli A, Boutet E, Buchala A, Metraux JP (2003) Induced systemic resistance in Arabidopsis
thaliana in response to root inoculation with Pseudomonas fluorescens CHA0. Mol Plant
IFOAM (1998) IFOAM basic standards for organic production and processing. IFOAM
Publications, Tholey-Tholey
IFOAM (2006) The four principles of organic farming. The International Federation of Organic
Agriculture Movements, www.IFOAM.org, Bonn, Germany, assessed 5/6 2006
Ingram M (2007) Biology and beyond: the science of back to nature farming in the United States.
Ann Am Assoc Geogr 97:298–312
IPCC (2007) In: Metz B, Davidson OR, RPR B, Meyer Dave LA (eds) Climate change: mitigation.
Contribution of Working Group III to the fourth assessment report of the Intergovernmental
Panel on Climate Change. Cambridge University Press, Cambridge/New York
Jones D (1998) Piperonyl butoxide: the insecticide synergist. Academic Press, London, p 323
Kathleen D, Robert H (2003) Weed management for organic farmers. Iowa State University
Extension Bulletin 1883
Kennedy AC, Smith KL (1995) Soil microbial diversity and the sustainability of agricultural soils.
Plant and Soil 170:75–86
Kirchmann H, Thorvaldsson G, Bergstrom L, Gerzabek M, Andren O, Eriksson L, Winninge M
(2008) Fundamentals of organic agriculture – past and present. In: Kirchmann H, Bergstrom L
(eds) Organic crop production – ambitions and limitations. Springer, Dordrecht, pp 13–38
Kirkegaard JA, Ryan MH (2014) Magnitude and mechanisms of persistent crop sequence effects on
wheat. Field Crop Res 164:154–165
Klein E, Katan J, Gamliel A (2011) Soil suppressiveness to Fusarium disease following organic
amendments and solarization. Plant Dis 95:1116–1123
Klein E, Katan J, Gamliel A (2012) Soil suppressiveness to Meloidogyne javanica as induced by
organic amendments and solarisation in greenhouse crops. Crop Prot 39:26–32
Kloepper JW, Tuzun S, Zehnder GW, Wei G (1997) Multiple disease protection by rhizobacteria
that induce systemic resistance – historic precedence. Phytopathology 87(2):136–137
Kowalchuk GA, Buma DS, De Boer W, Klinkhamer PGL, Van Veen JA (2002) Effect of above
ground plant species composition and diversity on the diversity of soil borne microorganisms.
Antonie Van Leeuwenhoek 81:509–520
Kristensen ES (2005) Organic farming in a global perspective, globalization, sustainable develop-
ment and ecological justice. Danish Research Centre for Organic Food and Farming, Tjele
Kristensen ES, Hogh-Jensen H, Kristensen IS (1995) A simple-model for estimation of
atmospherically-derived nitrogen in grass-clover systems. Bio Agric Hortic 12:263–276
Lampkin N (1990) Organic farming. Farming Press Books. Ipswich, UK. Plant Patho., Kyoto. 411
Landen EW (1939) Abstract in Rev Appl Mycol 19:84
Lazarovits G, Tenuta M, Conn KL (2001) Organic amendments as a disease control strategy for
soilborne diseases of high-value agricultural crops. Australas Plant Pathol 30:111–117
Lamichhane JR, Osdaghi E, Behlau F, Köhl J, Jones JB, Aubertot JN (2018) Thirteen decades of
antimicrobial copper compounds applied in agriculture. a review. Agron Sustain Dev 38(3):28
Lennartsson M (1988) Effects of organic soil amendments and mixed species cropping on take-all
disease of wheat. In: Allen P, Van Dusen D. (eds) Global perspectives on agroecology and
sustainable agricultural systems: proceedings of the sixth international scientific conference of
the international federation of organic agriculture movements, Santa Cruz, August 18–20,
p 575–580
Litterick AM, Watson CA, Atkinson D (2002) Crop protection in organic agriculture-a simple
matter? In: Proceedings of the UK organic research 2002 conference. Institute of Rural Studies,
University of Wales Aberystwyth, Organic Centre Wales, pp 203–206
Lotter D (2003) Organic agriculture. J Sustain Agri 21:59
Lupwayi NZ, Rice WA, Clayton GW (1999) Soil microbial biomass and carbon dioxide flux under
wheat as influenced by tillage and crop rotation. Can J Soil Sci 79:273–280
Luttikholt LW (2007) Principles of organic agriculture as formulated by the international federation
of organic agriculture movements. NJAS-Wageningen J Life Sci 54:347–360
Marking LL, Bills TD (1976) Toxicity of rotenone to fish in standardized laboratory tests. Dept. of
the Interior, Fish and Wildlife Service, Washington. No. 72, p 11
640 L. Rawat et al.
Mazzola M (2004) Assessment and management of soil microbial community structure for disease
suppression. Annu Rev Phytopathol 42:35–59
Mol L, Van Riesen HW (1995) Effect of plant roots on the germination of microsclerotia of
Verticillium dahliae. European J Pl Pathol 101(6):673–678
Momma N (2008) Biological soil disinfestation (BSD) of soilborne pathogens and its possible
mechanisms. Jpn Agric Res Q 42:7–12
Mukhopadhyay AN, Mukherjee PK (1996) Fungi as fungicides. Int J Trop Plant Dis 14:1–17
Mundt CC (2002) Use of multiline cultivars and cultivar mixture for diseases management. Annu
Nagaraja A, Kumar A, Raguchander T, Hota AK, Patro TSSK, Devaraje GP, Savita E, Gowda
MVC (2012) Impact of management practices of finger millet blast and grain yield. Indian
Natarajan M, Kannaiyan J, Willey R, Nene YL (1985) Studies on effects of cropping system on
Fusarium wilt of pigeonpea. Field Crop Res 10:333–346
Navaratnam SJ, Shuttleworth D, Wallace D (1980) The effect of aerated steam on six seed borne
pathogen. Australian J Exp Agril Ani Husban 20:97–101
Ninot A, Aleta N, Moragrega C, Montesinos E (2002) Evaluation of a reduced copper spraying
program to control bacterial blight of walnut. Plant Dis 86:583–587
Otten W, Filipe JAN, Gilligan CA (2005) Damping off epidemics, contact structure and disease
transmission in mixed species populations. Ecology 86:85–92
Pal KK, McSpadden GB (2006) Biological control of plant pathogens. The Plant Health Instructor.
https://doi.org/10.1094/PHI-A-2006-1117-02
Palaniappan SP, Annadurai K (1999) Organic farming: theory and practice. Scientific Publishers,
Jodhpur, p 257
Panov A, Dikalov S, Shalbuyeva N, Taylor G, Sherer T, Greenamyre JT (2005) Rotenone model of
Parkinson disease: multiple brain mitochondria dysfunctions after short term systemic rotenone
intoxication. J Biol Chem 280:42026–42035
Patil J (1981) Cultural practices and infection crop diseases. Springer-Verlog, Barlin, p 243
Patro TSSK, Rani C, Kumar GV (2008) Pseudomonas fluorescens, a potential bioagent for the
management of blast in Eleusine coracana. J Mycol Pl Pathol 38:298–300
Patibanda AK, Ranganathswamy M (2018) Effect of agrichemicals on biocontrol agents of plant
disease control. In: Microorganisms for green revolution, pp 1–21
Peoples MB, Herridge DF, Ladha JK (1995) Biological nitrogen fixation: an efficient source of
nitrogen for sustainable agricultural production? Plant and Soil 174:3–28
Raaijmakers JM, Weller DM (2001) Exploiting the genetic diversity of Pseudomonas spp. charac-
terization of superior colonizing P. fluorescens strain Q8r1-96. Appl Environ Microbiol
67:2545–2554
Rawat L, Singh Y, Shukla N, Kumar J (2011) Alleviation of the adverse effect of salinity stress in
wheat by seed biopriming with salinity tolerant isolates of Trichoderma harzianum. Plant and
Soil 347:387–400
Rawat L (2011) Characterization of native Trichoderma spp. from Indian Himalayas and their
evaluation against salinity stress. Ph.D. Thesis, GB Pant University of Agriculture and Tech-
nology, Pantnagar, Uttarakhand, India
Rawat L, Singh Y, Kumar J (2012) Seed biopriming with salinity tolerant isolates of Trichoderma
harzianum alleviates salinity stress in rice: growth, Physiological and Biochemical
characteristics. J Plant Pathol 94:353–365
Rawat L, Singh Y, Shukla N, Kumar J (2013) Salinity tolerant Trichoderma harzianum reinforces
NaCl tolerance and reduces population dynamics of Fusarium oxysporum f.sp. ciceri in
chickpea (Cicer arietinum L.) under salt stress conditions. Arch Phytopathol Plant Protect
46:1442–1467
Ren LX, Su SM, Yang XM, Xu YC, Huang QW, Shen QR (2007) Intercropping with aerobic rice
suppressed Fusarium wilt in watermelon. Soil Biol Biochem 40:834–844
Roberts PD, Momol MT, Ritchie L (2008) Evaluation of spray programs containing famoxadone
plus cymoxanil, acibenzolar-Smethyl, and Bacillus subtilis compared to copper sprays for
management of bacterial spot on tomato. Crop Prot 27:1519–1526
Sayler RJ, Kirkpatrick BC (2003) The effect of copper sprays and fertilization on bacterial canker in
French prune. Plant Dis 25:406–410
Scheuerell SJ, Mahaffee WF (2004) Compost tea as a container medium drench for suppressing
seedling damping-off caused by Pythium. Phytopathology 94:1156–1163
Schmidt H, Philipps L, Welsh JP, Von Fragstein P (1999) Legume breaks in stockless organic
farming rotations: nitrogen accumulation and influence on the following crops. Bio Agric Hortic
17:159–170
Sequeira L (1958) Bacterial wilt of banana: dissemination of the pathogen and control of disease.
Shelton AM, Zhao JZ, Roush RT (2002) Economic, ecological, food safety and social
consequences of the deployment of B-transgenic plants. Annu Rev Entomol 47:845–881
Singh K (1968) Hot air therapy against grassy shoot disease of sugarcane. Curr Sci 37:592
Singh K (1973) Hot air therapy against red rot of sugarcane. Plant Dis Rep 57:220
Singh RS (2003) Diseases of vegetable crops. Oxford & IBH Publishing Co. Pvt. Ltd, New Delhi
Singh RS (2007) Plant diseases. Oxford and IBH Publishing Co. PVT. Ltd., New Delhi, p 720
Singh US, Zaidi NW, Joshi D, Varshney S, Khan T (2003) Current status of Trichoderma as a
biocontrol agent. In: Ramanujam B, Rabindra RJ (eds) Current status of biological control of
plant diseases using antagonistic organisms in India. Project Directorate of Biological Control,
Bangalore, pp 13–48
Soleimani MJ, Deadman ML, Mc Cartney HA (1996) Splash dispersal on Pseudocercosporella
herpotrichoides spores in wheat-clover crop canopies from simulated rain. Plant Pathol
45:1065–1070
Sommer AL (1931) Copper as an essential for plant growth. Plant Physiol 6:339–345
Song YN, Zhang FS, Marschner P, Fan FL, Gao HM, Bao XG, Sun JH, Li L (2007) Effect of
intercropping on crop yield and chemical and microbiological properties in rhizosphere of
wheat, maize, and faba bean. Biol Fertil Soils 43:565–574
Souza AGC, Maffia LA, Silva EF (2015) A time series analysis of brown eye spot progress in
conventional and organic coffee production systems. Plant Pathol 64:157–166
Spiertz JHJ, Sibma L (1986) Dry-matter production and nitrogen utilization in cropping systems
with grass, lucerne and maize. 2. Nitrogen yield and utilization. Neth J Agric Sci 34:37–47
Srinivas P (2017) Disease management in organic farming. International conference on organic
farming for sustainable agriculture. Organized by Orissa University of Agriculture and Tech-
nology and Centre for Environment and Economic Development from 2–3 June, 2017 at
University of Agriculture and Technology, Bhubaneswar
Srinivas P, Ramakrishna G (2005) Biological management of rice seed borne pathogens by native
biocontrol agents. Ann Plant Protec Sci 13:422–426
Steadman JR, Coyne DP, Cook GE (1973) Reduction of severity of white mold disease on great
northern beans by wider row spacing and determinate growth habit. Pl Dis Rep 12:1070–1071
Stijger CCMM, Rast ATB (1988) Prospects of dry heat treatment at 0 C disinfection of pepper seed
infected with capsicum mosaic virus. Meded Facul 53:473
Stockdale EA, Lampkin NH, Hovi M, Keatinge R, Lennartsson EKM, MacDonald DW, Padel S,
Tattersall FH, Wolfe MS, Watson CA (2001) Agronomic and environmental implications of
organic farming Systems. Adv Agron 70:261–327
Stover RH (1962) Studies on Fusarium wilt of banana. VIII differentiation clones by cultural
interaction and volatile substances. Can J Bot 40:1467–1471
Szykitka W (2004) The big book of self-reliant living: advice and information on just about
everything you need to know to live on planet earth. Globe-Pequot, p 343
Tamm L, Willer H, van Bruggen AHC (2015) Organic perennial crop production. In: Finckh MR,
van AHC B, Tamm L (eds) Plant diseases and their management in organic agriculture. APS
Press, St Paul, MN, pp 33–41
642 L. Rawat et al.
Teviotdale BL, Krueger WH (2004) Effects of timing of copper sprays, defoliation, rainfall, and
inoculum concentration on incidence of olive knot disease. Plant Dis 88:131–135
Utkhede RS, Smith EM (1992) Promotion of apple tree growth and fruit production by the EBW-4
strain of Bacillus subtilis in apple replant disease soil. Can J Microbiol 38:1270–1273
Vallad GE, Goodman RM (2004) Systemic acquired resistance and induced systemic resistance in
conventional agriculture. Crop Sci 44:1920–1934
Van Kessel C, Hartley C (2000) Agricultural management of grain legumes: has it led to an increase
in nitrogen fixation? Field Crop Res 65:165–181
Van Bruggen AHC, Termorshuizen AJ (2003) Integrated approaches to root disease management in
organic farming systems. Australas Plant Pathol 32:141–156
Vartanian VG, Endo RJ (1985) Survival of Phytophthora infestans in seeds extracted from tomato
fruits. Phytopathology 75:375
Venkateswarlu B, Balloli SS, Ramakrishna YS (2008) Organic farming in rainfed agriculture:
opportunities and constraints. Central Research Institute for Dryland Agriculture, Hyderabad,
p 185
Vilich-Meller V (1992) Pseudocercosporella herpotrichoides, Fusarium spp and Rhizoctonia
cerealis stem rot in pure stands and interspecific mixtures of cereals. Crop Prot 11(1):45–50
Walker JL (1969) Diffusion of innovations among American states. Am Polit Sci Rev 63:880–899
Watson CA, Atkinson D, Gosling P, Jackson LR, Rayns FW (2002) Managing soil fertility in
organic farming systems. Soil Use Manage 18:239–247
Wu FZ, Meng LJ, Wen JZ (2001) Effect of root exudates of cucumber on mycelium growth of
Fusarium wilt. China Veg 5:26–27
Zentmyer GA, Bald JG (1977) Management of the environment. In: Plant disease: an advanced
treatise, vol 1. Academic Press Inc, New York, pp 121–144
Zeigler RS, Alvarez E (1988) Pseudomonas spp. causing grain and sheath rot of rice in Latin
America. Proc. 5th international congress of plant pathology, Kyoto, p 411
Organic Agriculture for Plant Disease
Management 28
D. K. Shahi, Sweta Kachhap, Arvind Kumar, and B. K. Agarwal
Abstract
Green Revolution ushered the nation into a millennium of self-sufficiency, but we

have again been presented with the threat of diminishing agricultural production
due to deterioration in soil health and, ultimately, risking the sustenance of the
human race. Indiscriminate and reckless use of chemicals has fuelled the concerns
of environmental pollution, pesticide toxicity and soil health even further. Amidst
this, organic agriculture has come out to be a holistic paradigm for sustaining life
as we know it. Though diseases are not a threat in organically managed farms,
still in order to prevent disease incidence from reaching economically damaging
levels, organic agriculture uses the natural process and fundamental components
of the ecosystem, such as nutrient cycling and microorganism population as soil
management tools. Disease control through soil management can be accom-
plished either through modifying nutrient availability or by modifying its uptake.
Organic agriculture follows composting, manuring, crop rotation and reduced
tillage systems for suppression of pathogens and control of disease.
Keywords
Organic farming · Holistic approach · Soil management · Disease management ·
Soil fertility · Biodiversity · Crop rotation · Compost · Organic matter
D. K. Shahi (*) · S. Kachhap · A. Kumar · B. K. Agarwal

Department of Soil Science and Agricultural Chemistry, Birsa Agricultural University, Ranchi,
Jharkhand, India

https://doi.org/10.1007/978-981-15-6275-4_28
644 D. K. Shahi et al.
28.1 Introduction
Agriculture has changed a lot since the evolution of mankind, but the world
witnessed a dramatic change in the agriculture system especially since the end of
World War II. With the challenge to meet the growing demands of the burgeoning
population and the “Green Revolution” in full flight, the government policies
favoured unchecked use of mechanization, fertilizers and pesticides. And, though
the food and fibre production soared and food surplus could be attained as Green
Revolution ushered the nation into a millennium of self-sufficiency, sadly, today,
even with high inputs, agricultural production has realized a plateau which is
sustained with diminishing returns of falling dividends. Moreover, the reckless and
indiscriminate use of chemicals has contributed to concerns of environmental pollu-
tion, pesticide toxicity and soil health. And the world has again been presented with
the threat of diminishing agricultural production due to deterioration in soil health
and, ultimately, risking the sustenance of the human race.
So, embracing a holistic notion that the health of any agriculture-based nation is
dependent on the long-term vitality of its soil, and motivated by a desire to reverse
these agricultural problems the concept of organic agriculture was born in the early
twentieth century. But plant diseases continue to create challenging problems and
pose real economic threats in agricultural ecosystems. Despite a wide use of chemi-
cal pesticides for crop produce, it has been manifested that the losses due to diseases
are significant, apart from causing toxicity to the soil and environment and entering
the food chain through contaminated food.
While there’s still a debate if organic agriculture can sustain the growing demands
of the ever-growing population, a more compelling question is whether organic
agriculture has feasible options for disease management. Our objective here is to
describe the potential of soil management practices in organic agriculture for disease
management as an alternative way for conventional agricultural practices, which can
lead to a sustainable resource utilization and contribute in mitigating global soil
health problems. This chapter makes an attempt to highlight the strategies of soil
management for crop protection in organic farming to suppress disease-causing
pathogens and decrease losses due to crop failure.
28.2 Organic Agriculture – Concept, Principles and Components
28.2.1 Definitions of Organic Agriculture
Organic agriculture is one of the several approaches to sustainable agriculture. It has

many definitions and has been described differently by different people, but
Lampkin (1990) provides the most apt and comprehensive definition of organic
agriculture. He defines organic agriculture as a production system, which exclusively
avoids the use of synthetic compounded chemical fertilizers, growth regulators,
livestock feed additives and pesticides, and instead uses only organic-based
28 Organic Agriculture for Plant Disease Management 645
fertilizers, such as compost, farmyard manure, biofertilizers, green manures,

vermicompost and natural pesticides and biocontrol agents.
The Food and Agriculture Organization (FAO) suggests organic agriculture to be
“a unique production management system which promotes and enhances agro-
ecosystem health, including biodiversity, biological cycles and soil biological activ-
ity which is accomplished by excluding all synthetic off-farm inputs and instead
making use of on-farm agronomic, biological and mechanical methods”
(FAO/WHO Codex Alimentarius Commission 1999).
Over the years, organic agriculture has emerged as an ecologically, economically
and socially dependable way of production which provides a continuing supply of
healthy and safe food and fibre, with minimal probable nutrients and energy losses,
and the least negative forces on the environment, as regulated by certification
agencies which scientists are looking at as a viable alternative way of restoring the
lost production and soil health equilibrium (Finckh et al. 2015).
28.2.2 Principles of Organic Agriculture
The International Federation of Organic Agriculture Movements (IFOAM) goes

further beyond biophysical aspects to define organic agriculture as “a production
system that sustains the health of soils, ecosystems and people. It relies on ecological
processes, biodiversity and cycles adapted to local conditions, rather than the use of
inputs with adverse effects. Organic Agriculture combines tradition, innovation and
science to benefit the shared environment and promote fair relationships and a good
quality of life for all involved.” IFOAM also gives four principles which are the roots
from which organic agriculture grows and develops (IFOAM 2020).
Organic agriculture, according to IFOAM, is guided by four principles that
include how people tend plants, animals, water and soil for production of food and
its distribution, and can be applied to agriculture in the broadest sense
(Fig. 28.1). These principles which concern the manner in which people relate to
each other interact with living landscapes and influence the future generations to
come and are as follows:
Fig. 28.1 Principles of organic agriculture

1. The Principle of Health – Health means the integrity and wholeness of living
systems. The principle of health points out that organic agriculture should be able
to sustain as well as enhance not only human and animal health but also the health
of soil and plant including the planet as one and indivisible. It not only means
avoiding illness but also to maintain physical, social, mental and ecological well-
being. In the same context, it in turn means largely excluding the use of fertilizers,
pesticides, food additives and animal drugs that can cause adverse health effects.
2. The Principle of Ecology – It points out that the basis of production in organic
agriculture should be ecological processes including efficient management of
materials and recycling, working with them, emulating them and helping in
sustenance and should be aimed at achieving ecological balance, improving the
environmental quality and conserving resources.
3. The Principle of Fairness – Fairness encompasses equity, justice, respect and
stewardship, and this principle emphasizes that organic agriculture should be
directed towards building a good quality of life by ensuring fairness at all levels of
food production and consumption in a manner that is socially just and environ-
mentally viable too.
4. The Principle of Care – Organic agriculture should be directed towards respon-
sibly managing the supply to the demands and conditions (both external and
internal) in a manner that not only ensures health, safety and ecological soundness
of the present generation but also discards risks that jeopardize the health and
well-being of the future generations. Organic agriculture should make use of
science and technology to increase productivity and enhance efficiency, but with
care and precaution so as to prevent significant risks.
28.2.3 Components of Organic Agriculture
Organic agriculture has come out to be a holistic paradigm for sustaining life as we
know it on earth and hence is so much more than just a way to naturally treat soil,
plants and animals. Organic agriculture involves:
1. Reducing soil erosion and improving and maintaining soil fertility, soil structure
and soil biodiversity.
2. Reducing risks of exposure of toxic materials to humans, animals and environ-
ment alike.
3. Fine-tuning farming practices to meet production and distribution and attaining
sustainable yield.
For these, organic agriculture must employ components (Fig. 28.2) that envisage
a comprehensive management approach to enhance and maintain soil productivity
without compromising the soil fertility and soil health and regulate the ecological
processes working together in synergy and maintain a stage of dynamic equilibrium.
Fig. 28.2 Components of organic agriculture
28.2.4 Organic Agriculture Vs. Conventional Agriculture
Organic agriculture does not merely imply a production system that works on simple
replacement of synthetic fertilizers and other chemical pesticides, but there’s much
more to it besides restricting and eliminating the entry of chemicals into the
environment and there are a lot of fundamental differences between organic agricul-
ture and conventional agriculture (Table 28.1). When we compare organic agricul-
ture to conventional farming, organic agriculture production systems by and large:
(a) have higher plant diversity, both in space and time; (b) have enhanced soil
organic matter content by means of cover crops and crop rotation; (c) have a higher
diversity of soil flora and fauna; (d) have enhanced water-holding capacity with
enhanced water-use efficiency and (e) have increased cycling, improved cation
exchange capacity (CEC) and reduced loss of nutrients (Aparna et al. 2014;
Gomiero et al. 2011; Tuck et al. 2014).
Table 28.1 Fundamental differences between organic and conventional farming

Organic agriculture Conventional agriculture
1 Synthetic fertilizers and synthetic pesticides Synthetic fertilizers and synthetic
are not permitted pesticides are allowed
2 Genetically modified organisms (GMOs) are GMOs can be used
not allowed
3 Soils have higher water holding capacity Soils have lower water holding capacity
4 Has larger floral and faunal biodiversity Has smaller biodiversity (simple crop
(complex crop pattern) pattern)
5 The agricultural landscape is characterized The agricultural landscape is characterized
by heterogeneity (multicultural system) by homogeneity (monocultural system)
6 Minimizing the use of non-renewable Depends largely on non-renewable
resources by recycling plant and animal resources (off-farm inputs)
waste into the soils (on-farm inputs)
7 More sustainable Less sustainable
8 Strictly regulated by international and Not strictly regulated
national institutional bodies such as Codex
Alimentarius and IFOAM
9 Crop protection depends mainly on natural Crop protection relies mainly on human
processes such as soil fertility, crop cycle, intervention with synthetic chemicals
and biodiversity (more preventive) (more curative)
10 Optimum input: output ratio Low input: output ratio
11 No pollution Considerable pollution
12 Stability due to diversification Market based programme
13 Ecological orientation and holistic approach Economical orientation and economical
approach
28.3 Disease Incidence and Disease Management in Organic

Agriculture
There are, basically, three conditions for a plant disease to happen. Plant diseases
occur only when a disease-causing pathogen comes in contact with a susceptible host
in a conducive environment (Fig. 28.3). And if any of these three conditions are not
satisfied, disease incidence will fail to happen. Hence, the disease management
strategies in organic agriculture, which are mainly of preventive nature rather than
being curative, are aimed at creating the environments less favourable and the hosts
less susceptible for disease invasion.
Successful disease management in organic agriculture demands an exhaustive
understanding of life cycles of the crop and the disease-causing pathogens and their
interactions with themselves, soil, climate and other factors of the production
system. To achieve this, organic agriculture relies heavily on the concept that within
an agro-ecosystem, all natural processes are reciprocally dependent on one another,
and its management should be aimed at supporting and maintaining self-regulation
through these natural processes (Birkhofer et al. 2008). Therefore, to replenish
nutrients taken from the soil, organic agriculture largely depends on the natural
Fig. 28.3 A schematic representation of the interacting components involved in plant disease
(Huber and Haneklaus 2007)
breakdown of organic matter by means of agricultural practices like composting and

green manuring, which are driven by beneficial soil microorganisms and allow
natural cycling of the nutrients in the soil.
28.4 Soil – A Resource of Infinite Possibilities
Soil, a complex mix of organic and inorganic matter, is the fundamental medium for
crop growth in all production systems where plant roots along with living
microorganisms bind organic matter and mineral particles into a dynamic structure
that governs air, water and nutrient flow. A healthy and fertile soil is the basis of a
productive, profitable and environmentally sound agro-ecosystem. A healthy soil
allows for so many functions that support crop growth by means of nutrient cycling,
regulation of water and air supply and also by way of biological control of plant
pests. And in context to the agricultural production system, soil health pertains to the
ability of the soil to sustain crop productivity simultaneously protecting the
environment.
Soil being a critical resource can improve or degrade the quality of that resource
owing to the ways in which it is managed. And, the success of this dynamic living
resource, to a great extent, depends on characteristics which affect the rooting of
crops, such as structural characteristics and nutrient supply. However, soil condition
is one of the factors that can influence plant growth and development via occurrence
and severity of disease incidence. And so by understanding the physical, chemical

and biological components of a healthy soil and by understanding how the soil
processes work and how they support plant growth, it is possible to design a soil
management system that maintains and improves soil health.
28.4.1 Disease-Suppressive Soils
Disease-suppressive soils are defined as the kind of soils in which either the
pathogen is unable to establish or persist, the pathogen though gets established but
is unable to cause damage or though the pathogen is able to cause some damage, the
disease fails to progress in severity despite the pathogen persisting in the soil (Cook
and Baker 1983). In other words, a soil is referred to as disease-suppressive when,
despite a conducive environment for disease incidence, a disease-causing organism
does not establish; it gets established but is unable to produce disease, or even if it
gets established and produce disease it is only for a short time after which it declines
(Schneider 1982).
The concept of disease-suppressive soils is not new and has been getting
acknowledged since several decades now and involves two types of disease sup-
pression strategies which are as follows:
(1) General Suppression – of a disease-causing organism is a consequence of high

microbial biodiversity and microbial activity in the soil or the plant at a vital time
in the life cycle of the pathogen that create an unfavourable environment for the
disease to develop. General suppression operates against most pathogens and is
mostly non-specific.
(2) Specific Suppression – of a pathogen is a result of one organism targeting and
operating against a certain type of known pathogen to suppress it and reduce
disease incidence. Specific suppressiveness has been described for Phytophthora
spp., Pythium spp., Rhizoctonia solani, Fusarium wilts, Gaeumannomyces
graminis var. tritici and Thielaviopsis basicola (Table 28.2).
Table 28.2 Examples of different plant pathogens controlled by disease-suppressive soils

(Chandrashekara et al. 2012)
Pathogen involved Reference
1 Phytophthora cinnamomi Broadbent and Baker (1974)
2 Pythium spp. Hancock (1977)
3 Rhizoctonia solani Henis et al. (1978, 1979)
4 Fusarium oxysporum Stotzky and Martin (1963) and Scher and Baker
(1980)
5 Gaeumannomyces graminis var. Cook and Rovira (1976)
tritici
6 Plasmodiophora brassicae Murakami et al. (2000)
7 Streptomyces scabies Menzies (1959)
8 Cyst nematode Heterodera spp. Kerry (1988) and Westphal and Becker (1999)
While the degree of suppresiveness is associated with the physical, chemical and
biological attributes of the soil including soil pH, fertility level, the type and
population of soil organisms, nature of the soil and soil management (Sullivan
2001), the mechanisms employed in disease suppression include predation, parasit-
ism, antibiosis, induced resistance and nutrient competition by beneficial soil
organisms. But, although abiotic properties and characteristics of soil can help in
disease suppression, the level of disease suppression is typically a function of
microbial activity and microbial metabolites in a soil. So, the higher the microbial
mass and microbial activity in the soil, the higher is its capacity to utilize carbon,
nutrients and energy to suppress pathogens.
28.5 Soil Management for Disease Management
The soil is home to many living organisms since it is the ultimate source of their
mineral nutrients. A soil takes a period of time to develop suppression to pathogens,
and while natural suppressiveness is often related to the natural properties of the soil
independent of the crop history, we are more interested in induced suppressiveness,
which is completely dependent on soil management and agricultural practices.
Hence, soil management is important to crop productivity, human and animal health
and environmental sustainability, both directly and indirectly. Good soil manage-
ment ensures that appropriate mineral elements enter the food chain and that mineral
elements do not become deficient or toxic to plants and humans. All these functions
are governed by the physical, chemical and biological properties of soil, which in
turn are influenced by the soil management practices. In order to prevent disease
incidence from reaching economically damaging levels, organic agriculture uses the
natural process and fundamental components of the ecosystem such as nutrient
cycling and microorganism population as soil management tools, both directly and
indirectly. Some key soil management strategies and tools that are employed under
organic agriculture for successful disease control have been discussed below under
different subheads.
28.5.1 Soil Fertility
A fertile soil is the one which is abundant in essential and beneficial plant nutrients
and rich in organic matter. Soil fertility is referred to as the inherent capacity of the
soil to provide plants with essential nutrients in adequate quantities and proportions
at the optimum time for desirable plant growth. Soil fertility is a major factor in
disease management since nutrients are not only crucial for growth and development
of plants and microorganisms but their availability in the soil also plays a key role in
disease control. The nutrient status of the soil and the use of a specific fertilizer and
amendment may have substantial impacts on the environment of the disease-causing
pathogen (Agrios 2005).
28.5.1.1 Soil Nutrients

The availability of a particular nutrient in the soil can affect the severity of a disease
both positively as well as negatively by way of decreasing or increasing disease
incidence, respectively, under different environmental conditions (Marschner 1995;
Graham and Webb 1991; Huber 1980). Adequate crop nutrition makes plants more
tolerant of or resistant to a disease-causing organism since nutrients can govern
disease control through influencing disease tolerance or resistance. While disease
tolerance is the ability of the plant to maintain its growth and development despite
the disease infection, disease resistance refers to the ability of the plant to confine the
penetration, establishment and reproduction of the disease-causing pathogen
(Graham and Webb 1991). And though the genetic make-up of the plant species
controls tolerance and resistance to pathogens, nutrient deficiency and toxicity in the
soil and soil environment can affect them to a large extent and hence correct nutrient
management can help in disease control for achieving a higher yield (Agrios 2005;
Marschner 1995; Krauss 1999; Huber and Graham 1999; Graham and Webb 1991).
When a pathogen infects a susceptible host plant, it impairs its physiology
including the uptake, assimilation, translocation and utilization of a nutrient. Some
pathogens are also capable of immobilizing the nutrients in the infected regions or in
the rhizosphere and interfering with their translocation or utilization causing nutrient
deficiency or toxicity. Plant pathogens may also compete with the plants for nutrients
utilizing a significant amount of nutrients for their growth causing nutrient defi-
ciency and further increasing plant susceptibility to diseases (Marschner 1995;
Huber and Graham 1999; Timonin 1965). The level of nutrients available in
the soil has been also known to affect disease development either by affecting the
pathogen, by affecting plant physiology or by impacting both by influencing the
microclimate and hence infection and sporulation of the disease-causing pathogen
(Marschner 1995). Hence, it becomes crucial to manage nutrient availability in soil
in order to impact nutrient availability, and help in plant disease management.
Nitrogen (N) is the most important nutrient for plant growth and extensive
literature is available regarding the effect of N on diseases because its function in
disease resistance has been frequently demonstrated, but in spite of the fact that N is
one of the most important nutrients for plant growth and disease development, there
are several factors which impact the effect of N disease development such as the type
of pathogen – obligate vs. facultative parasite, the form of N nutrition available to the
host and the crop growth stage of N application (Table 28.3). For example, there is
an increase in the severity of disease caused by obligate parasites such as Puccinia
graminis and Erysiphe graminis at high N supply, whereas disease severity by
facultative parasites such as Xanthomonas vesicatoria and Alternaria solani is
greatly suppressed at high N availability. Similarly, the severity of infection of
Pythium spp., Rhizoctonia solani and Fusarium oxysporum has been found to
decrease at high nitrate (NO3 ) availability while high ammonium (NH4+) caused
decreased disease infection in Pyricularia, Sclerotium rolfsii and Gibberella zeae.
Phosphorus (P) is considered as the second most important nutrient for successful
crop growth and development and it has been observed to play a significant part in
influencing disease build-up. P application at the right crop growth stage has been
Table 28.3 Effect of N level on disease severity of several diseases (Dordas 2008)
Pathogen or disease Low N High N References
Obligate Puccinia graminis Decrease Increase Howard et al. (1994)
parasite Erysiphe graminis Decrease Increase Büschbell and Hoffmann
(1992)
Oidium lycopersicum Decrease Increase Hoffland et al. (2000)
Plasmodiophora brassicae Decrease Increase Kiraly (1976)
Tobacco mosaic virus Decrease Increase Singh (1970)
Pseudomonas syringae Decrease increase Hoffland et al. (2000)
Facultative Xanthomonas vesicatoria Increase Decrease Chase (1989)
parasite Alternaria solani Increase Decrease Blachinski et al. (1996)
Fusarium oxysporum Increase Decrease Woltz and Engelhar (1973)
Table 28.4 Effect of K level on disease severity of several diseases (Dordas 2008)
Pathogen or disease Low K High K References
Puccinia graminae Increase Decrease Lam and Lewis (1982)
Xanthomonas oryzae Increase Decrease Chase (1989)
Tobacco mosaic virus Increase Decrease Ohashi and Matsuoka (1987)
Alternaria solani Increase Decrease Blachinski et al. (1996)
Fusarium oxysporum Increase Decrease Srihuttagum and Sivasithamparam
(1991)
Pyrenophora tritici-repentis Increase Decrease Sharma et al. (2005)
Erysiphe graminis Increase Decrease Menzies et al. (1992)
found to reduce blast disease and bacterial leaf blight in rice, Pythium root rot in
wheat, root rot and soil smut in maize, pod and stem blight in soybean, downy
mildew and leaf curl virus disease in tobacco and brown stripe disease in sugarcane.
However, in some studies it has been shown that P application resulted in increased
disease severity of flag smut in wheat and increased disease infection by Bremia in
lettuce and Sclerotinia in several garden plants.
The role of potassium (K) in disease suppression is by way of decreasing disease
susceptibility of the host to various obligate and facultative parasites through
promoting the synthesis of high molecular-weight compounds (proteins, starch and
cellulose) and development of thicker outer walls in epidermal cells to help in
preventing disease attack. K application was found helpful in reducing disease
incidence of black rust in wheat, bacterial leaf blight, sheath blight and stem rot in
rice, bacterial leaf blight in cotton, tikka leaf spot in groundnut, sugary disease in
sorghum, Cercospora leaf spot in mungbean and seedling rot by Rhizoctonia solani
(Table 28.4) (Dordas 2008).
Micronutrients do not have a direct role in disease resistance but can affect
disease suppression indirectly. Deficiency of micronutrients not only impairs the
defence mechanism of the host but it also makes the host more prone to diseases by
causing several metabolites like reducing sugars and amino acids to leak outside of
Table 28.5 Role of micronutrient deficiency on soil-borne diseases

Micronutrient deficiency Disease Causal organism
1 Boron (Bo) Tomato Wilt Verticillium albo-atrum
Beans Root rot Fusarium solani
2 Zinc (Zn) Take all of wheat G. graminis var. tritici
Rhizoctonia Root rot Rhizoctonia solani
3 Manganese (Mn) Take all of wheat G. graminis var. tritici
4 Copper (Cu) Take all of wheat G. graminis var. tritici
Powdery mildew of wheat Blumeria graminis
Take all of wheat G. graminis var. tritici
Sunflower Alternaria
the plant cell, making it a more suitable feeding substrate. Micronutrients play a vital
part in controlling the permeability of cell membranes and maintaining the structural
integrity, deficiency of which causes the membranes to become unstable and leaky.
The deficiency of micronutrients in soil and plants can also reduce the production
of fungus-inhibiting natural antifungal compounds, which, in turn, can increase
the susceptibility of the host plants to diseases (Table 28.5) (Dordas 2008;
Chandrashekara et al. 2012).
Nutrient supply can impact the development of plant disease under field
conditions either directly through the nutritional status of the plant or indirectly by
affecting the environment, which can influence disease development such as crop
population and density, difference in light interception and humidity within the crop
stand. Disease control through nutrient management can be accomplished either
through modifying the nutrient availability or by modifying its uptake. It is very
crucial to supply the plants with balanced nutrition and at the optimum time when the
nutrient can be most effective not just for achieving higher yields but also for disease
control. On the other hand, nutrient uptake can be altered to suit our needs of disease
suppression and higher yields by changing root absorption, translocation and meta-
bolic efficiency (Dordas 2008).
28.5.1.2 Soil Organic Matter

Soil organic matter (SOM) is referred to as the index of soil fertility and
sustainability of agricultural systems. Soil organic matter improves the physical,
chemical and biological properties of soil, protects the soil surface from erosion and
acts as a reservoir of plant nutrients. The quantity as well as quality of soil organic
matter can influence not just the soil nutrient status but also several other soil
functions associated with soil health, including nutrient availability through micro-
bial activity, infiltration and water retention. Hence, soil organic matter can affect
disease incidence indirectly, if not directly, by means of promoting plant growth by
increasing plant resistance and escaping disease by altering the pathogen’s environ-
ment. Stone et al. (2004) has proposed several organic matter-mediated mechanisms
for disease suppresiveness which are listed below:
1. Microbiostasis
2. Microbial colonization of propagules of the disease-causing organism
3. Destruction of propagules
4. Antibiosis and antagonism
5. Competition for energy and nutrient sources and for substrate colonization
6. Competition for root infection sites
7. Induced systemic resistance or systemic acquired resistance
28.5.2 Soil Microbial Biomass
The importance of microorganisms in organic agriculture is irrefutable not only

because abundance of beneficial microorganisms in the soil renders it healthy but
also because a number of soil-borne diseases result from a reduction of ample
microbial populations. Whereas conventional agriculture banks on synthetic chemi-
cal fertilizers and pesticides for crop production, organic agriculture utilizes the
natural capital of the soil and its microbial population for agricultural production and
sustainability. These processes involve different soil microorganisms and reinstating
the soil with those beneficial microorganisms that tend to repel, attack or antagonize
disease-causing organisms make a soil disease suppressive. And plants which are
grown in disease-suppressive soils are more likely to resist disease attack better than
those which are grown in soil with lower microbial flora and fauna.
Pathogens flourish in soil environments with abundance of free nutrients. The
food for microorganisms comes from nutrients; therefore, putting a limit to available
nutrients can play a crucial role in disease suppression. Beneficial microorganisms
make the soil environment unfavourable for the disease-causing pathogen by com-
peting for food and energy paired with secretion of antibiotics. And for the very
reason that they secrete antibiotics and antifungal metabolites microorganisms are
presently also being used as a substitute for synthetic pesticides and fertilizers for
many different crops.
28.5.3 Soil pH
Soil pH has an important role to play in disease management, both directly and
indirectly. Soil pH directly affects disease incidence by influencing pathogen popu-
lation and its environment and indirectly by influencing the availability of nutrients
to the pathogen as well as the host. Pathogens require a favourable environment with
an optimum pH to reproduce, complete their lifecycle, flourish and cause disease. If
the soil pH is not suitable for the plant pathogen it hinders with its activity and hence
help in disease suppression by helping the host plant escape disease. Indirectly, soil
pH affects the release of nutrients from soil making nutrients unavailable for
utilization by the pathogens. Beneficial microorganisms thrive in slightly low pH,
but if the pH is too high it affects the soil bacteria that are involved in the breakdown
of organic matter, resulting in the nutrient getting locked up in the undecomposed
organic matter making it unavailable for the pathogens. Hence, plant diseases can be
managed by altering soil pH to suit the plant and beneficial microorganisms and to
hinder the growth of disease-causing organisms.
28.5.4 Soil Structure and Texture
Soil structure and texture do not have a direct impact on disease suppression but may
have an indirect effect on disease development as soil structure and texture influence
the nutrient status, water holding capacity and gas exchange. Beneficial organisms
thrive in soils with good aeration but soils with poor aeration resulting from a poor
soil structure and water-logged conditions make an environment conducive for the
development of Pythium spp. which causes cavity spot in carrot. Increased bulk
density resulting from soil compaction has been shown to affect fresh weights of pea
due to infection from root rot incidence (Chang 1994). Thus, the soil structure and
texture can be managed to play an important role in disease control.
28.5.5 Soil Moisture and Temperature
As discussed earlier, a pathogen requires a favourable environment to flourish, and a

favourable environment would mean abundant nutrients, optimum pH and an opti-
mal amount of soil moisture content with a favouring temperature. Well-drained soil
seldom have disease attacks, but soils with poor water drainage leads to water
logging and lowered temperature, resulting in an environment conducive for the
growth of several soil-borne pathogens such as Pythium spp. and Phytophthora spp.,
which are responsible for causing damping off and seed decay in many different
crops. Raised soil moisture due to poor drainage leads to reduced oxygen availabil-
ity, hindering the ability of the host plant to defend itself and contrastingly favouring
soil-borne pathogens to get established and multiply. The severity of cavity spot
disease of carrot by Pythium violae and Pythium sulcatum and root rot complex of
pea by Fusarium spp. have been found to be directly proportional to the amount of
soil moisture (Agrios 1997).
28.5.6 Cropping System, Cultural Practices and Residue

Management
28.5.6.1 Soil Amendment with Organic Fertilizers

Since synthetic chemical fertilizers are not permitted in organic agriculture, nutrients
applied to organic farms are generally through organic fertilizers which include
compost, farmyard manure and green manure. Owing to the multiple benefits that
can be derived from organic wastes in agricultural practices, they provide us with
myriad opportunities including a remarking potential in disease management. But
these organic amendments take time to decompose, which results in gradual nutrient
supply to crops and hence need to be planned well beforehand.
Compost
Compost has been used traditionally for the countless benefits it provides, which
include enhancing soil organic matter, enriching physico-chemical properties of the
soil and encouraging microbial population and microbial activity. While, some
studies have shown a decline in disease incidence of Sclerotinia minor in lettuce
and Pythium arrhenomanes in sugarcane, others have recorded a significant reduc-
tion in the inoculum of Rhizoctonia solani, Fusarium oxysporum f. sp. asparagi, and
Verticillium dahlia with continuous application of composts (Lumsden et al. 1983;
Dissanayake and Hoy 1999; Blok et al. 2000).
Compost has been proven to be effective in disease management and suppression
because it helps in nurturing a diverse soil environment with diverse life forms
buzzing with activity. Compost serves as a food source for beneficial
microorganisms which antagonize and parasitize plant pathogens and produce
antibiotics. Successful suppression of Pythium spp. and Phytophthora spp. causing
root rots in crops has been achieved from high diversity and activity of beneficial
microorganisms present in compost (Harrison and Frank 1999).
There exists a direct correlation between the level of decomposition in compost
and its ability to suppress a disease-causing pathogen. Fresh undecomposed organic
matter offers a conducive environment for highly competitive pathogens like
Pythium spp. and Rhizoctonia spp. to grow and colonize because immature composts
have good supply of readily available carbon compounds which gradually decrease
as the compost matures. So the level of decomposition plays a central part in disease
suppression owing to the direct relationship among the level of decomposition,
microbial population, diversity and activity and the degree of disease suppression.
Farmyard Manure
As discussed in the previous section, fresh organic matter is deleterious to crops
since it promotes growth of soil-borne pathogens such as Streptomyces spp. How-
ever, studies reveal that application of liquid swine manure has a positive role in
suppressing pathogens such as Streptomyces spp. and Verticillium dahliae and
reducing plant pathogenic nematode population (Conn and Lazarovits 1999,
2000). Besides, liquid swine manure was also found to promote Trichoderma
populations further enabling the management of soil-borne pathogens.
28.5.6.2 Tillage
Tillage is a necessary cultural practice for preparing a field for cultivation. Tillage
practices have been shown to play a great role in displacing pathogens directly as
well as indirectly – directly by the placement of the residues in the soil and indirectly
by promoting the soil microbial activity (Cook 1990). Soil microorganisms are
concentrated mainly within the top 15 cm of the soil and more tillage practice
would pose a greater risk of soil erosion and causing disturbances to the top layer
of the soil which is home to beneficial soil biota. Conventional agriculture involves
Fig. 28.4 Schematic diagram showing how agricultural practices recycle organic residues by the
process of decomposition for utilization by the pathogen and beneficial microorganisms
intensive tillage practices, which result in considerable disturbance to the soil

leading to low organic matter in the soil and therefore low microbial population
and microbial activity in the soil (Lupwayi et al. 1999; Kennedy and Smith 1995).
But the choice of tillage practice holds tremendous potential in disease suppression
owing to its role in altering the ability of the pathogen to survive and its inoculum
density.
Organic agriculture follows reduced tillage, minimum tillage or zero tillage
systems which involve reduced mixing of the soil and thus retaining and maintaining
healthy soil biota. These systems accumulate soil organic matter through carbon
sequestration and stubble retention and encourage soil microfloral and microfaunal
populations and activity (Fig. 28.4).
We have already discussed how soils with higher levels of organic matter have
improved the soil structure, water infiltration, water retention and water drainage.
Farming systems with reduced tillage practices have been shown to have lesser
inoculum density of Cochliobolus sativus causing common root rot in wheat and
barley compared to conventional tillage due to increased microbial activity, which
created an antagonistic environment for the pathogen. Reduced tillage also led to
increased populations of mycophagous amoebae as well as fungi which feed on C.
sativus and creating a more hostile environment for its dormant spores. Reduced
tillage systems also influence the availability of various nutrients in the soil and soil
moisture impacting survival and infection of Pythium and Phytophthora spp. that
cause root rots and damping-off (Tinline and Spurr 1991; Duczek 1983, 1986;
Duczek and White 1986; Workneh et al. 1996).
28.5.6.3 Crop Rotation

Several diseases build-up in the soil only because the same type of crops are sown in
the same field year after year. Crop rotation serves as the perfect strategy for
managing those pathogens that survive in the soil or on the crop residue and for
avoiding disease build-up by breaking this cycle. Since disease-causing organisms
differ in the lengths of time for which they persist in the soil, crop rotation needs to
be planned accordingly. Since pathogens usually attack plants belonging to the same
family, it becomes helpful to group the susceptible host plant, related plants and
alternate host pants together and keep them out during the rotation period (Fig. 28.4).
Crop rotation helps in disease suppression by either starving the pathogen or by
killing it with toxic root exudates and for it to be most effective, crop rotation cycles
should be 3–7 years between susceptible crops. A three-year crop rotation has been
reported as the standard recommendation for Fusarium oxysporum causing stem rot,
Ceratocystis fimbriata causing black rot, and Monilochaetes infuscans causing scurf
in sweet potato. Three-year rotations also work best for several soil-borne pathogens
such as Fusarium spp., Verticillium spp. and Ralstonia spp. that cause wilts in plants
(Chandra and Baiswar 2007). However, crop rotation doesn’t work effectively for
those pathogens that survive for long periods in the soil without a host plant.
28.5.6.4 Organic Amendments and Organic Pesticides

Organic amendments prove to be an effective mechanism for disease suppression
in organic agricultural systems because they enhance beneficial edaphic
microorganisms along with antagonists which compete with the pathogens for
available nutrients and suitable ecological niches. Organic amendments utilize
natural products obtained from plants and animals for disease management also
improving the soil structure, texture and water-holding capacity.
Neem seed and cake have been shown to effectively suppress many plant
diseases, such as bacterial blight of rice and rhizome rot of ginger, and effectively
control several disease pathogens such as Rhizoctonia solani, Fusarium solani,
Macrophomina phaseolina and Phytophthora capsici. While cotton cake has been
found to be effective in reducing disease incidence of seedling blight in eucalyptus,
and several neem derivatives such as Nimbicidine, Neemark Neemoil have been
reported to be effective against yellow mosaic virus in chickpea (Lazarovits et al.
2001; Bailey and Lazarovits 2003).
28.6 Conclusion
Crop protection in organic agriculture is not a simple matter. It heavily depends on a

thorough knowledge of the crops grown and their likely pathogens. Successful
organic crop protection strategies also rely on an understanding of the influence
the abiotic factors such as soil, weather, climate and topography are likely to have on
the crop performance as well as the disease-causing pathogens. Organic agriculture
is rapidly expanding in its scope and area to include different food and fibre items for
large-scale production, giving a strong competition to conventional farming systems.
Different innovative strategies to avoid, suppress and control persistent disease

problems have already been established and several are being devised rapidly. We
do need to optimize these strategies and look for further new avenues to tackle the
disease problem in organic farms for which research needs to be done.
References
Agrios GN (1997) Plant pathology. Academic Press, San Diego
Agrios GN (2005) Plant pathology, 5th edn. Elsevier Academic Press, London
Aparna K, Pasha MA, Rao DLN, Krishnaraj PU (2014) Organic amendments as ecosystem
engineers: microbial, biochemical and genomic evidence of soil health improvement in a
tropical arid zone field site. Ecol Eng 13:268–277
Bailey KL, Lazarovits G (2003) Suppressing soilborne diseases with residue management and
organic amendments. Soil Tillage Res 72:169–180
Birkhofer K, Bezemer TM, Bloem J, Bonkowski M, Christenen S, Dubois D, Ekelund F,
Fliebbach A, Gunst L, Hedlund K, Mader P, Mikola J, Robin C, Setala H, Tatin-Froux F, van
der Putten WH, Scheu S (2008) Long-term organic farming fosters below and aboveground
biota: implications for soil quality, biological control and productivity. Soil Biol Biochem
40:2297–2308
Blachinski D, Shtienberg D, Dinoor A, Kafkafi U, Sujkowski LS, Zitter TA, Fry WE (1996)
Influence of foliar application of nitrogen and potassium on Alternaria diseases in potato,
tomato and cotton. Phytoparasitica 24:281–292
Blok WJ, Lamers JG, Termorshuizen AJ, Bollen G (2000) Control of soilborne plant pathogens by
incorporating fresh organic amendments followed by tarping. Phytopathology 90:253–259
Broadbent P, Baker KF (1974) Behavior of Phytophthora cinnamomi in soils suppressive and
conducive to root rot. Aust J Agric Res 25:121–137
Büschbell T, Hoffmann GM (1992) The effects of different nitrogen regimes on the epidemiologic
development of pathogens on winter wheat and their control. J Plant Dis Protect 99:381–403
Chandra S, Baiswar P (2007) Disease management in organic agriculture. In: Munda GC, Ghosh
PK, Das A, Ngachan SV, Bujarbaruah KM (eds) Advances in organic farming Technology in
India. ICAR-Research Complex for NEH Region, Imiam, Meghalaya, pp 429–438
Chandrashekara C, Bhatt JC, Kumar R, Chandrashekara KN (2012) Supressive soils in plant
disease management. In: Singh VK, Singh Y, Singh A (eds) Eco-friendly innovative approaches
in plant disease management, vol 14. International Book Distributers, Dehradun, pp 241–256
Chang TJ (1994) Effects of soil compaction, temperature, and moisture on the development of the
Fusarium root rot complex of pea in south western Ontario. Phytoprotection 75:125–131
Chase AR (1989) Effect of nitrogen and potassium fertilizer rates on severity of Xanthomonas
blight of syngoniumpodophyllum. Plant Dis 73:972–975
Conn KL, Lazarovits G (1999) Impact of animal manures on Verticillium wilt, potato scab, and soil
microbial populations. Can J Plant Pathol 21:81–92
Conn KL, Lazarovits G (2000) Soil factors influencing the efficacy of liquid swine manure added to
soil to kill Verticillium dahliae. Can J Plant Pathol 22:400–406
Cook RJ (1990) Diseases caused by root-infecting pathogens in dryland agriculture. Adv Soil Sci
13:215–239
Cook RJ, Baker KF (1983) The nature and practice of biological control of plant pathogens. APS
Press, St. Paul
Cook RJ, Rovira AD (1976) The role of bacteria in the biological control of Gaeumannomyces
graminis by suppressive soils. Soil Biol Biochem 8:267–273
Dissanayake N, Hoy JW (1999) Organic material soil amendment effects on root rot and sugarcane
growth and characterization of materials. Plant Dis 83:1039–1046
Dordas C (2008) Role of nutrients in controlling plant diseases in sustainable agriculture. A review.
Agronomy for Sustainable Development, Springer Verlag/EDP Sciences/INRA. 28 (1): 33–46.
hal-00886444
Duczek LJ (1983) Populations of mycophagous amoebae in Saskatchewan soil. Plant Dis
67:606–608
Duczek LJ (1986) Populations in Saskatchewan soils of spore-perforating amoebae and an amoeba
(Thecamoeba granifera s.sp. minor) which feeds on hypha of Cochliobolus sativus. Plant Soil
92:295–298
Duczek LJ, White GP (1986) Chalara heteroderae a fungal antagonist that parasitizes hyphae of
Bipolaris sorokiniana. Soil Biol Biochem 18:655–659
Finckh MR, van Bruggen AHC, Tamm L (eds) (2015) Plant diseases and their Management in
Organic Agriculture. APS PRESS, St. Paul, p 424
Food and Agriculture Organization of the United Nations and World Health Organization (1999)
(GL 32 – 1999, Rev. 1 – 2001) Guidelines for the Production, Processing, Labelling and
Marketing of Organically Produced Foods FAO/WHO Codex Alimentarius Commission
Gomiero T, Pimentel D, Paoletti MG (2011) Environmental impact of different agricultural
management practices: conventional vs. organic agriculture. Crit Rev Plant Sci 30:95–124
Graham DR, Webb MJ (1991) Micronutrients and disease resistance and tolerance in plants. In:
Mortvedt JJ, Cox FR, Shuman LM, Welch RM (eds) Micronutrients in agriculture, 2nd edn. Soil
Science Society of America, Inc, Madison, pp 329–370
Hancock JG (1977) Factors affecting soil populations of Pythium ultimum in the San Joaquin Valley
of California. Hilgardia 45:107–122
Harrison UJ, Frank JL (1999) Disease management through suppressive soils. Department of plant
pathology. North Carolina State University, Raleigh. (draft document), 23 Sept. 14
Henis Y, Ghaffar A, Baker R (1978) Integrated control of Rhizoctonia solani damping-off of radish:
effect of successive plantings, PCNB and Trichoderma harzianum on pathogen and disease.
Henis Y, Ghaffar A, Baker R (1979) Factors affecting suppressiveness to Rhizoctonia solani in soil.
Hoffland E, Jegger MJ, van Beusichem ML (2000) Effect of nitrogen supply rate on disease
resistance in tomato depends on the pathogen. Plant Soil 218:239–247
Howard DD, Chambers AY, Logan J (1994) Nitrogen and fungicide effects on yield components
and disease severity in wheat. J Prod Agric 7:448–454
Huber DM (1980) The role of mineral nutrition in defense. In: Horsfall JG, Cowling EB (eds) Plant
disease, an advanced treatise, volume 5, how plants defend themselves. Academic Press,
Huber DM, Graham RD (1999) The role of nutrition in crop resistance and tolerance to disease. In:
Rengel Z (ed) Mineral nutrition of crops fundamental mechanisms and implications. Food
Product Press, New York, pp 205–226
Huber DM, Haneklaus S (2007) Managing nutrition to control plant diesease. Landbauforschung
Völkenrode 57(4):313–322
IFOAM (International Movement of Organic Agriculture Movements) (2020) Definition of organic
agriculture. Available from: https://www.ifoam.bio/en/organic-landmarks/definition-organic-
agriculture Accessed:03-02-2020
Kennedy AC, Smith KL (1995) Soil microbial diversity and the sustainability of agricultural soils.
Kerry BR (1988) Fungal parasites of cyst nematodes. Agric Ecosyst Environ 24:293–305
Kiraly Z (1976) Plant disease resistance as influenced by biochemical effects of nutrients and
fertilizers. In: Fertilizer use and plant health, proceedings of colloquium 12. International Potash
Institute, Atlanta, GA, pp 33–46
Krauss A (1999) Balanced nutrition and biotic stress. IFA agricultural conference on managing
plant nutrition, 29 June–2 July 1999. Barcelona, Spain
Lam A, Lewis GC (1982) Effects of nitrogen and potassium fertilizer application on Drechslera spp.
and Puccinia-coronata on perennial ryegrass (Lolium perenne) foliage. Plant Pathol 31:123–131
Lampkin N (1990) Organic farming. Farming Press, Ipswich

Lazarovits G, Tenuta M, Conn KL (2001) Organic amendments as a disease control strategy for
soilborne diseases of high-value agricultural crops. Australas Plant Pathol 30:111–117
Lumsden RD, Lewis JA, Millner PD (1983) Effects of composted sewage sludge on several soil
borne pathogens and diseases. Phytopathology 73:1543
Lupwayi NZ, Rice WA, Clayton GW (1999) Soil microbial biomass and carbon dioxide flux under
wheat as influenced by tillage and crop rotation. Can J Soil Sci 79:273–280
Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic Press, London, p 889
Menzies JD (1959) Occurrence and transfer of a biological factor in soil that suppresses potato scab.
Menzies J, Bowen P, Ehret D, Glass ADM (1992) Foliar applications of potassium silicate reduce
severity of powdery mildew on cucumber, muskmelon and zucchini squash. J Am Soc Hortic
Sci 117:902–905
Murakami H, Tsushima S, Shishido Y (2000) Soil suppressiveness to clubroot disease of Chinese
cabbage caused by Plasmodiophora brassicae. Soil Biol Biochem 32:1637–1642
Ohashi Y, Matsuoka M (1987) Localization of pathogenesis-related proteins in the epidermis and
intercellular spaces of tobacco-leaves after their induction by potassium salicylate or tobacco
masaic-virus infection. Plant Cell Physiol 28:1227–1235
Scher FM, Baker R (1980) Mechanism of biological control in a Fusarium-suppressive soil.
Schneider RW (ed) (1982) Suppressive soils and plant disease. The American Phytopathological
Society, St. Paul
Sharma S, Duveiller E, Basnet R, Karki CB, Sharma RC (2005) Effect of potash fertilization on
helminthosporium leaf blight severity in wheat, and associated increases in grain yield and
kernel weight. Field Crop Res 93:142–150
Singh R (1970) Influence of nitrogen supply on host susceptibility to tobacco mosaic virus
infection. Phyton-Annales Rei Botanicae 14:37–42
Srihuttagum M, Sivasithamparam K (1991) The influence of fertilizers on root-rot of field peas
caused by Fusarium oxysporum, Pythium vexans and Rhizoctonia solani inoculated singly or in
combination. Plant Soil 132:21–27
Stotzky G, Martin RT (1963) Soil mineralogy in relation to the spread of Fusarium wilt of banana in
Central America. Plant Soil 18:317–337
Stone AG, Scheuerell SJ, Darby HM (2004) Suppression of soil-borne diseases in field agricultural
systems: organic matter management, cover cropping and other cultural practices. In:
Magdoff F, Weil RR (eds) Soil organic matter in sustainable agriculture. CRC Press, Boca
Raton, pp 131–177
Sullivan P (2001) Sustainable management of soil-borne plant diseases. ATTRA, USDA’s Rural
Business Cooperative Service, https://www.attra.org
Timonin ME (1965) In: Gilmore CM, Allen ON (eds) Interaction of higher plants and soil
microorganisms. In microbiology and soil fertility. Oregon State University Press, Corvallis,
pp 135–138
Tinline RD, Spurr DT (1991) Agronomic practices and common root rot in spring wheat: effect of
tillage on disease and inoculum density of Cochliobolus sativus in soil. Can J Plant Pathol
13:258–266
Tuck SL, Winqvist C, Mota F, Ahnström J, Turnbull LA, Bengtsson J (2014) Land-use intensity
and the effects of organic farming on biodiversity: a hierarchical meta-analysis. J Appl Ecol 51
(3):746–755
Westphal A, Becker JO (1999) Biological suppression and natural population decline of Heterodera
schachtii in a California field. Phytopathology 89:434–440
Woltz SS, Engelhar AW (1973) Fusarium wilt of chrysanthemum effect of nitrogen source and lime
on disease development. Phytopathology 63:155–157
Workneh F, Wegulo SN, Yang XB (1996) Effects of cropping practices on Sclerotinia stem rot of
soybean in Iowa. Phytopathology 86:S43. Abstr
Pest Risk Analysis and Plant Quarantine
Regulations 29
V. Celia Chalam, Kavita Gupta, Ruchi Sharma, Vaishali Dutt Sharma,
and A. K. Maurya
Abstract
International trade of agri-horticultural commodities and exchange of germplasm

plays an important role in the long-distance dissemination of insect pests and
pathogens, which may pose potential risk to the agriculture of the importing
country. The National Plant Protection Organizations have the responsibility of
protecting their countries from the unwanted entry of new insect pests and
pathogens. The exclusion can be achieved by a combination of regulatory and
technical approaches that can ensure biosecurity for a country/region. The Inter-
national Plant Protection Convention (IPPC) of the Food and Agriculture Orga-
nization in the United Nations develops the International Standards for
Phytosanitary Measures (ISPMs) which provide guidelines on pest prevention,
detection and eradication. To date, 43 ISPMs have been developed and adopted.
In India, the Directorate of Plant Protection, Quarantine and Storage under the
Ministry of Agriculture and Farmers Welfare is responsible for enforcing quar-
antine regulations and for quarantine inspection and disinfestation of agricultural
commodities meant for commercial purpose. The imported germplasm materials
including transgenics are subject to quarantine processing at the ICAR-National
Bureau of Plant Genetic Resources, New Delhi. The strategies for biosecurity for
insect pests and pathogens include pest risk analysis and stringent quarantine
regulations for the imported material, domestic quarantine and use of certified
disease-free seed and other planting materials within the country. Adopting a
workable strategy such as post-entry quarantine (PEQ) growing in PEQ
greenhouses/containment facility, electron microscopy, conventional, serological
and molecular diagnostics, several pests including 45 viruses have so far been
intercepted, which include 19 viruses not yet reported from India. Adopting the
right strategy and appropriate technique for pest detection would go a long way in
V. C. Chalam (*) · K. Gupta · R. Sharma · V. D. Sharma · A. K. Maurya

Division of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources, New Delhi, India
e-mail: celia.chalam@icar.gov.in

https://doi.org/10.1007/978-981-15-6275-4_29
664 V. C. Chalam et al.
ensuring the biosecurity of Indian agriculture from transboundary introduction of

plant viruses.
Keywords
Pest risk analysis · Quarantine · Pathogens · Viruses · Diagnostics · Germplasm ·

Commercial material · India
29.1 Introduction
Plant diseases substantially reduce crop production every year, resulting in serious
economic losses throughout the world. Trade and exchange of germplasm at the
international level play a key role in the long distance dissemination of a destructive
pest or its virulent pathotype/race/strain along with agri-horticultural produce. Due
to liberalization under WTO, the recent years have seen a significant growth in trade
and exchange of agri-horticultural crops. The global movement of seed and other
planting materials has the potential of introducing new pathogens and insect pests,
which may pose potential risk to the agriculture of the importing country.
The devastating effects resulting from pathogens introduced, along with interna-
tional movement of seed and other planting materials, are well documented. The
Irish famine of 1845, which forced people to migrate en masse from Europe, was the
result of almost total failure of potato crop due to attack of late blight pathogen
(Phytophthora infestans) introduced from Central America. Coffee rust (Hemileia
vastatrix) appeared in Sri Lanka in 1875 and reduced the coffee production by
>90% in 1889. The disease entered India in 1876 from Sri Lanka and within a
decade, the coffee industry of South India was badly affected. Bulk import of seeds
and other planting materials without proper phytosanitary measures, indiscriminate
exchange of germplasm and the distribution of seed and other planting materials by
international agencies have increased the possibility of dissemination of pathogens
in areas previously considered pathogen-free (Khetarpal et al. 2006). Further, the
threat may become severe, if more virulent strains or races of the pathogen are
introduced into previously disease-free areas. Even a low seed transmission rate of a
pathogen, especially viruses may lead to an epiphytotic proportion of the disease in
field, if other conditions of field spread and climate are favourable. The worldwide
distribution of many economically important viruses such as Bean common mosaic
virus, Soybean mosaic virus, Pea seed-borne mosaic virus, Wheat streak mosaic
virus, Peanut mottle virus, etc. is attributed to the unrestricted exchange of seed lots.
Like in other countries, a number of exotic pathogens and insect pests got
introduced in India along with imported planting material causing serious crop losses
from time to time. These included potato late blight (Phytophthora infestans in
1883), coffee rust (Hemileia vastatrix in 1879), potato tuber moth (Pthorimaea
operculella in 1906), flag smut of wheat (Urocystis tritici in 1906), San Jose scale
(Quadraspidiotus perniciosus in 1910), fluted scale (Icerya purchasi in 1912),
codling moth (Cydia pomonella in 1919) and the weed, Lantana camara (in the
29 Pest Risk Analysis and Plant Quarantine Regulations 665
early part of nineteenth century). These introductions highlighted the fact that
increased pace of international travel and trade had exposed countries to the danger
of infiltration of exotic pathogens and insect pests harmful to the agriculture.
The most fundamental approach to the management of a disease is to ensure that
it is not present through exclusion (quarantine) or eradication. National Plant
Protection Organizations (NPPOs) assume responsibility for protecting their
countries from the unwanted entry of new pests by applying appropriate measures.
Such measures must either be based on international standards or based on risk
assessment for the country. Therefore, plant quarantine measures should be based on
pest risk analysis (PRA). The present day definition of a pest is any species, strain or
biotype of plant, animal or pathogenic agent injurious to plants or plant products.
The PRA is calculating, speculating or extrapolating the “risk (known or perceived)
involved” if an organism will enter, colonize and/or become established in an area
where it is not known to occur. Most NPPOs determine the entry status of imported
agricultural commodities based on perceived risk and economics involved in miti-
gation measures, in case the pest establishes. The PRA consists of risk assessment
(scientific estimation of likelihood and magnitude of establishment of a given pest
risk) and impact assessment (estimation of the consequences of the establishment of
pest). It is a process of evaluating biological or other scientific and economic
evidence to determine whether a pest should be regulated and the strength of any
phytosanitary measures to be taken against it. Risk analysis is an important element
of biosecurity and forms the basis of prevention and initial control of problems
arising from damaging new areas or introduction of new pests.
Theoretically for achieving zero risk, a country should not allow any imports of
planting material/other commodities and be equally strict to not permit the move-
ment of human beings and animals/birds from one part of the country to other. It is
not easy to achieve this; therefore, if the overall risk is perceived to be high, the entry
status of the imported material should be conservative, and conversely if the risk is
perceived to be low the entry status should be liberal. Further to ensure that the best
option available has been chosen out of all the available ones, PRA needs to be
conducted for all. A PRA should be sufficiently documented so that when a dispute
arises, the PRA will clearly state the source of information and the rationale used in
reaching a management decision regarding phytosanitary measures taken or to be
taken.
The risk analysis is carried out on the basis of information about the pest status in
both importing and exporting countries. The justification for regulating pests
requires that the pest should qualify as a quarantine pest or as Regulated
Non-Quarantine Pest (RNQP), which is based on the PRA. The procedures to be
followed for risk analysis of pests are given in the ISPM-2 (framework for pest risk
analysis), ISPM-11 (pest risk analysis for quarantine pests) and ISPM-21 (pest risk
analysis for regulated non-quarantine pests).
All the member countries of WTO are required to prepare and update lists of
regulated pests to facilitate safe trade. In India, the Plant Quarantine (Regulation of
Import into India) Order 2003 notified to the WTO Secretariat gives a list of >1200
regulated pests on ~700 host commodities. The Schedules V and VI pertain to the
specific requirements for regulated pests, but listing of RNQPs is yet to be done
specifically. All the crops not listed under any of the present schedules can only be
introduced in India only after carrying out detailed PRA. The requirements under
these existing schedules become a part of the additional declarations sought from the
importing country in the import permit. The same pest-free status and treatments
given are followed by the exporting country and confirmed on the phytosanitary
certificate.
29.2 Pest Risk Analysis
The PRA is evaluation of several factors through transparently collected data,

quantitative or qualitative grading for the pest status, pathway, biology of the host,
climatic conditions, economic impact and official control. Overall risk is then
calculated and mitigation measures are discussed to reach to an objective of the
minimum possible risk while carrying out trade under the prevailing situations. The
preparation of PRA is an elaborate process requiring scientific input on various
aspects of the pest and the associated risk involved in its inadvertent introduction. As
per the IPPC, the term plant pest refers to all organisms harmful to plants or plant
products including other plants, bacteria, fungi, insects and other animals, mites,
molluscs, nematodes and viruses. Pests can be either regulated or not, and the IPPC
recognizes and defines two categories of regulated pests of plants viz., quarantine
pests and regulated non-quarantine pests. PRA assists with determining whether a
pest fits either of these two categories.
A quarantine pest is defined by the IPPC as “a pest of potential economic
importance to the area endangered thereby and not yet present there, or present but
not widely distributed and being officially controlled” FAO (2019). In other words, it
is any organism that is injurious or potentially injurious, directly or indirectly, to
plants or plant products or by-products of plants and includes bacteria, fungi, insects,
mites, molluscs, nematodes, other plants and viruses, not present in a specified area
at risk or, if present, being controlled by an NPPO.
A regulated non-quarantine pest (RNQP) is defined by the IPPC as “a
non-quarantine pest whose presence in plants for planting affects the intended use
of those plants with an economically unacceptable impact and which is therefore
regulated within the territory of the importing contracting party” (FAO 2019).
RNQPs are generally those that are established in the importing country, may be
widely distributed and regulated on specified hosts to keep them below a level, at
which they would cause an unacceptable economic impact.
The requirement for a PRA consists of three stages viz., initiation, risk assessment
and risk management whose compilation should be fully documented in the event a
review or whenever a dispute arises. The PRA should clearly document information
sources and the rationale used in reaching a management decision regarding mitiga-
tion measures that have been taken or are to be taken. PRA is a process to answer the
questions such as Is the organism a pest? What is the likelihood of introduction,
establishment and spread? How much economic (including environmental and
social) damage (unacceptable impacts) does it cause? and What can be done to
mitigate unacceptable impacts? Consequently, any phytosanitary measures
implemented by a country to mitigate against a particular pest should be technically
justified by the PRA.
29.2.1 Pest-Initiated vs. Pathway-Initiated PRAs
There are two widely adopted approaches to conducting a PRA, one focused on a
pathway, the other focused on a particular pest associated with one or more
pathways. A commodity PRA is one type of pathway PRA. In the case of a pathway
PRA, it is necessary to conduct PRAs on those pests associated with the pathway that
are identified as potential quarantine pests. In the case of a pest PRA, consideration
needs to be given to all possible pathways or commodities with which the pest may
be associated.
29.2.2 Stages in PRA
Pest risk analysis (PRA) consists of risk assessment (scientific estimation of likeli-
hood and magnitude of establishment of a given pest risk) and impact assessment
(estimation of the consequences of the establishment of pest). Therefore, to ensure
that imported commodity presents a minimal pest or no pest risk to our agriculture
and forestry, PRA must be conducted.
Initiating the process involves identification of pests or pathways for which the
PRA is needed. The list of pests is generated by any combination of databases,
literature sources or expert consultation. Once the list of pests has been established, it
is preferable to prioritize it by using expert judgement before the next step.
According to the results obtained, it may or may not be necessary to conduct a
risk assessment on all pests on the list.
Pest risk assessment determines whether each pest identified as such, or
associated with a pathway, is a quarantine pest, characterized in terms of likelihood
of entry, establishment, spread and economic importance. In doing so, the PRA
considers all aspects of each pest and in particular actual information about its
geographical distribution, biology and economic importance. Expert judgement is
then used to assess the establishment, spread and economic importance potential in
the PRA area. Finally the potential for introduction into the PRA area is
characterized.
Pest risk management involves developing, evaluating, comparing and selecting
options for reducing the risk. A list of options for reducing risks to an acceptable
level should be assembled. These options will primarily concern pathways and in
particular the conditions for permitting entry of commodities. The pest risk manage-
ment to protect the endangered areas should be proportional to the risk identified in
the pest risk assessment.
A PRA should be sufficiently documented so that when a review or a dispute

arises, the PRA will clearly state the sources of information and the rationales used in
reaching a management decision regarding phytosanitary measures taken or to be
taken. ISPMs specifically related to pest risk analysis.
29.2.3 International Standards for Phytosanitary Measures
The International Standards for Phytosanitary Measures (ISPMs) that are most
relevant to pest risk analysis (PRA) rare given below:
29.2.3.1 ISPM No. 2 Framework for Pest Risk Analysis (Adopted in 2007
and Published in 2019)
This standard provides a framework that describes the pest risk analysis (PRA)
process within the scope of the IPPC. It introduces the three stages of pest risk
analysis – initiation, pest risk assessment and pest risk management. The standard
focuses on the initiation stage. Generic issues of information gathering, documenta-
tion, risk communication, uncertainty and consistency are addressed.
29.2.3.2 ISPM No. 3 Guidelines for the Export, Shipment, Import

and Release of Biological Control Agents and Other Beneficial
Organisms (Adopted in 2005 and Published in 2017)
This standard provides guidelines for risk management related to the export, ship-
ment, import and release of biological control agents and other beneficial organisms.
The standard addresses biological control agents capable of self-replication (includ-
ing parasitoids, predators, parasites, nematodes, phytophagous organisms and
pathogens such as fungi, bacteria and viruses), as well as sterile insects and other
beneficial organisms (such as mycorrhizae and pollinators), and includes those
packaged or formulated as commercial products. Provisions are also included for
import for research in quarantine stations of non-indigenous biological control
agents and other beneficial organisms. The scope of this standard does not include
living modified organisms, issues related to registration of biopesticides or microbial
agents intended for vertebrate pest control.
29.2.3.3 ISPM No. 11, Pest Risk Analysis for Quarantine Pests (Adopted
in 2013 and Published in 2019)
This standard provides details for the conduct of PRA to determine if pests are
quarantine pests. It describes the integrated processes to be used for risk assessment
as well as the selection of risk management options. Some explanatory comments on
the scope of the IPPC with regard to environmental risks are given in Annex 1. Some
explanatory comments on the scope of the IPPC regarding PRA for LMOs are given
in Annex 2. Annex 3 deals with determining the potential for a living modified
organism to be a pest.
29.2.3.4 ISPM No. 21 Pest Risk Analysis for Regulated Non-quarantine

Pests (Adopted in 2004 and Published in 2019)
This standard provides guidelines for conducting pest risk analysis for regulated
non-quarantine pests. It describes the integrated processes to be used for risk
assessment and the selection of risk management options to achieve a pest tolerance
level.
Although ISPMs are internationally agreed to and adopted, they are meant to be
guidelines and their use is not mandatory within the framework of the IPPC. In
addition, their interpretation and application on a national level varies from country
to country. This variation is illustrated by the many different national systems and
procedures that exist for carrying out PRA. Countries often take slightly different
approaches to implement the standards while still staying true to their intent. A PRA
conducted according to international standards can provide the basis for our national
planners to allocate resources to problem areas posing the highest risk and thereby
enable them to better protect our agriculture and environment from the entry and
establishment of exotic pests.
29.2.3.5 Significance of PRA

Risk analysis may be defined as a science-based tool for decision-making; it is a
broad term, which encompasses activities including risk assessment, pathway anal-
ysis, risk management and communication, border controls and emergency pre-
paredness. International standards for these activities have been established and
may be applied for prevention, management of alien pests at the international,
regional or local levels.
International harmonization of phytosanitary measures can be successful only if
contracting parties implement standards. Control systems are needed to verify
whether this is done. If countries cannot resolve trade disputes bilaterally, a WTO
dispute procedure can be initiated. The PRA becomes important as compliance by
concerned countries with international standards is an important judgment criteria
for decisions by WTO dispute panels.
29.3 Role of Diagnostics in Quarantine Against Transboundary

Plant Pathogens
In the context of quality control, bulk samples need to be tested by drawing workable
samples as per the norms. The detection of pathogens is then carried out by the
approved or available techniques. Over the years a great variety of methods have
been developed that permit the detection and identification of pathogens. The
successful detection and control of pathogens in seed and other planting materials
depend upon the availability of rapid, reliable, robust, specific and sensitive methods
for detection and identification of pathogens.
Detection and diagnosis of pathogens are crucial for trade and for exchange of
germplasm. Early, sensitive, and accurate diagnosis is indispensable for certification
of seed and other planting materials under exchange. The selection of a diagnostic
Table 29.1 Summary of various techniques for detecting pathogens of quarantine significance
Techniques Fungi Bacteria Viruses Viroids Phytoplasma
Visual examination + + + + +
Seed washing test + +
Soaked seed test +
Whole embryo test +
Incubation tests + +
Phage sensitivity test +
Staining of inclusion bodies +
Electron microscopy + + +
Growing-on test + + + +
Infectivity test + + + + +
Enzyme-linked immunosorbent + + 2 2
assay (ELISA)
Dot-immunobinding assay (DIBA) + 2 2
Tissue blotting immunoassay + 2 2
Immunosorbent electron microscopy +
(ISEM)
Lateral flow strips + +
Polymerase chain reaction (PCR) + + + + +
Reverse transcription-PCR + 2 2
(RT-PCR)
Immunocapture-RT-PCR (IC-RT- + 2 2
PCR)
Real-time PCR + + + + +
Real-time RT-PCR + 2 2
Microarrays + + + + +
Loop-mediated isothermal + + + + +
amplification (LAMP)
Helicase-dependent amplification + + + + +
(HDA)
Next-generation sequencing (NGS) + + + + +
method for evaluating plant health depends on the host to be tested and the type of
pathogens that may be carried in the seed and other planting materials. The technique
should be reliable, reproducible within statistical limits, economical with regard to
time, labour and equipment and should be rapid for quarantine requirements.
The bulk samples of seed lots need to be tested by drawing workable samples as
per norms. The detection of pathogens is then carried out by the approved or
available techniques. Over the years a great variety of methods have been developed
that permit the detection and identification of pathogens and are summarized below
in Table 29.1.
29.4 Plant Quarantine Regulations
29.4.1 International Scenario
The recent trade-related developments in international activities and the thrust of the
WTO Agreements imply that countries need to update their quarantine or plant
health services to facilitate pest-free import/export.
The establishment of the WTO in 1995 has provided unlimited opportunities for
international trade of agricultural products. History has witnessed the devastating
effects resulting from diseases and insect pests introduced along with the interna-
tional movement of planting material, agricultural produce and products. It is only
recently, however, that legal standards have come up in the form of Sanitary and
Phytosanitary (SPS) measures for regulating the international trade. The WTO
Agreement on the Application of SPS measures concerns the application of food
safety and animal and plant health regulations. It recognizes government’s rights to
take SPS measures but stipulates that they must be based on science, should be
applied to the extent necessary to protect human, animal or plant life or health and
should not unjustifiably discriminate between members where identical or similar
conditions prevail (http: //www.wto.org).
The SPS Agreement aims to overcome health-related impediments of plants and
animals to market access by encouraging the “establishment, recognition and appli-
cation of common SPS measures by different members”. The primary incentive for
the use of common international norms is that these provide the necessary health
protection based on scientific evidence and improve trade flow at the same time.
SPS measures are defined as any measure applied within the territory of the
member state to protect animal or plant life or health from risks arising from the
entry, establishment or spread of pests, diseases, disease-carrying/causing
organisms; to protect human or animal life or health from risks arising from
additives, contaminants, toxins or disease-causing organisms in food, beverages or
foodstuffs; to protect human life or health from risks arising from diseases carried by
animals, plants or their products, or from the entry, establishment/spread of pests; or
to prevent or limit other damages from the entry, establishment or spread of pests.
The SPS Agreement explicitly refers to three standard-setting international
organizations commonly called as the “three sisters” whose activities are considered
to be particularly relevant to its objectives: International Plant Protection Convention
(IPPC) of Food and Agriculture Organization (FAO) of the United Nations, World
Organization for Animal Health (OIE) and Codex Alimentarius Commission of Joint
FAO/WHO. The IPPC develops the International Standards for Phytosanitary
Measures (ISPMs) which provide guidelines on pest prevention, detection and
eradication. To date, 43 ISPMs ((https://www.ippc.int/en/core-activities/standards-
setting/ispms/) have been developed and adopted (Annexure-I).
Prior to the establishment of WTO, governments on a voluntary basis could adopt
international standards, guidelines, recommendations and other advisory texts.
Although these norms shall remain voluntary, a new status has been conferred
upon them by the SPS Agreement. A WTO Member adopting such norms is
presumed to be in full compliance with the SPS Agreement.
29.4.2 National Scenario: Imports
Plant quarantine is defined as all activities designed to prevent the introduction

and/or spread of quarantine pests or to ensure their official control. Quarantine
pest is a pest of potential economic importance to the area endangered thereby and
not yet present there, or present but not widely distributed and being officially
controlled (FAO 2016).
As early as in 1914, the Government of India passed a comprehensive act, known
as Destructive Insects and Pests (DIP) Act, to regulate or prohibit the import of any
article into India likely to carry any pest that may be destructive to any crop, or from
one state to another. The DIP Act has since undergone several amendments. In
October 1988, New Policy on Seed Development was announced, liberalizing the
import of seeds and other planting materials. In view of this, Plants, Fruits and Seeds
(Regulation of import into India) Order (PFS Order) first promulgated in 1984 was
revised in 1989. The PFS Order was further revised in the light of World Trade
Organization (WTO) Agreements and the Plant Quarantine (Regulation of Import
into India) Order 2003 [hereafter referred to as PQ Order] came into force on 1st
January, 2004 to comply with the Sanitary and Phytosanitary Agreement (Khetarpal
et al. 2006). A number of amendments of the PQ Order were notified, revising
definitions, clarifying specific queries raised by quarantine authorities of various
countries, with revised lists of crops under the Schedules VI and VII and quarantine
weed species under Schedule VIII. The revised list under Schedules VI and VII now
includes 699 and 519 crops/commodities, respectively, and Schedule VIII now
includes 57 quarantine weed species. The Schedule IV includes 15 crops and
countries from where import is prohibited along with the name of pest(s). The PQ
Order ensures the incorporation of “Additional/Special Declarations” for import
commodities free from quarantine pests, on the basis of pest risk analysis (PRA)
following international norms, particularly for seed/planting material (http://www.
agricoop.nic.in/gazette.htm).
The Directorate of Plant Protection, Quarantine and Storage (DPPQS) under the
Ministry of Agriculture and Farmers Welfare is responsible for enforcing quarantine
regulations and for quarantine inspection and disinfestation of agricultural
commodities. The quarantine processing of bulk consignments of grain/pulses, etc.
for consumption and seed/planting material for sowing is undertaken by the 70 plant
quarantine stations located in different parts of the country and many pests were
intercepted in imported consignments (Sushil 2016; http://ppqs.gov.in/divisions/
plant-quarantine/strengthening-modernisation-plant-quarantine-facilities-india).
Import of bulk material for sowing/planting purposes is authorized only through six
regional plant quarantine stations viz., Amritsar, Chennai, Kolkata, Mumbai, New
Delhi and Bengaluru. There are 42 Inspection authorities who inspect the consign-
ment being grown in isolation in different parts of the country. Besides, DPPQS has
developed 22 standards on various phytosanitary issues such as on PRA, pest-free

areas for fruit flies and stone weevils, certification of facilities for treatment of wood-
packaging material, methyl bromide fumigation, etc. Also, two standard operating
procedures have been notified on export inspection and phytosanitary certification of
plants/plant products and other regulated articles and Post-entry quarantine inspec-
tion (www.plantquarantineindia.org/standards.htm).
The ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), the
nodal institution for exchange of plant genetic resources (PGR), has been
empowered under the PQ Order to handle quarantine processing of germplasm
including transgenic planting material imported for research purposes into the
country by both public and private sectors. ICAR-NBPGR has developed well-
equipped laboratories and post-entry quarantine green house complex. Keeping in
view the biosafety requirements, National Containment Facility of level-4 (CL-4)
has been established at ICAR-NBPGR to ensure that no viable biological material/
pollen/pathogen enters or leaves the facility during quarantine processing of
transgenics. Till date, >16,000 samples of transgenic crops comprising Arabidopsis
thaliana, Brassica spp., chickpea, corn, cotton, potato, rice, soybean, tobacco,
tomato and wheat with different traits imported into India for research purposes
were processed for quarantine clearance, wherein they are tested for associated
exotic pests, if any, and also for ensuring the absence of terminator gene technology
(embryogenesis deactivator gene) which are mandatory legislative requirements. At
ICAR-NBPGR, some of the important pathogens intercepted include fungi like
Fusarium nivale, Peronospora manshurica and Uromyces betae and bacterium
like Xanthomonas campestris pv. campestris (Bhalla et al. 2018a). In the last three
decades, by adopting a workable strategy such as PEQ growing in PEQ greenhouses/
containment facility and inspection, PEQ inspection at indenter’s site, electron
microscopy, enzyme-linked immunosorbent assay (ELISA) and reverse
transcription-polymerase chain reaction (RT-PCR), 45 viruses of great economic
and quarantine importance have been intercepted in exotic germplasm including
transgenics. The interceptions include 19 viruses not yet reported from India viz.,
Barley stripe mosaic virus (BSMV), Bean mild mosaic virus (BMMV), Bean pod
mottle virus (BPMV), Broad bean mottle virus (BBMV), Broad bean stain virus
(BBSV), Broad bean true mosaic virus (BBTMV), Cherry leaf roll virus (CLRV),
Cowpea mottle virus (CPMoV), Cowpea severe mosaic virus (CPSMV), Garlic
virus-C (GarV-C), Dioscorea latent virus (DLV), High plains virus (HPV), Maize
chlorotic mottle virus (MCMV), Pea enation mosaic virus (PEMV), Peanut stunt
virus (PSV), Pepino mosaic virus (PepMV), Raspberry ringspot virus (RpRSV),
Tomato ringspot virus (ToRSV) and Wheat streak mosaic virus (WSMV). Besides,
21 viruses not known to occur on particular host(s) in India have been intercepted
and these are also of quarantine significance for India. Twenty viruses have been
intercepted in germplasm imported from CGIAR centres (Chalam 2016, 2014;
Chalam et al. 2004, 2005a, 2007, 2008, 2009a, 2009b, 2012a, b, d, 2013b, c, d,
2014a, b, c, 2015a, b, 2016a, b, 2018a, b; Chalam and Khetarpal 2008; Chalam and
Maurya, 2018; Khetarpal et al. 1992, 1994, 2001; Kumar et al. 1991; Parakh et al.
1994, 2005, 2006, 2008; Prasada Rao et al. 1990, 2004, 2012; Singh et al. 2003;
Singh et al. 2015). Even though some of the intercepted viruses are not known to
occur in India, their potential vectors exist and so also the congenial conditions for
them to multiply, disseminate and spread the destructive exotic viruses/strains and
even native strains more efficiently. The risk of introduction of 45 viruses or their
strains in India was thus eliminated. All the plants infected by the viruses were
uprooted and incinerated.
The infected samples were salvaged by using suitable techniques (Singh and
Khetarpal 2005), and the disease-free germplasm was only used for further distribu-
tion and conservation. If not intercepted, some of the above quarantine pests could
have been introduced into our agricultural fields and caused havoc to our
productions. Thus, apart from eliminating the introduction of exotic pathogens
from our crop improvement programmes, the harvest obtained from disease-free
plants ensured conservation of pest-free exotic germplasm in the National
Genebank.
29.4.3 National Scenario: Exports
The Directorate of Plant Protection, Quarantine and Storage (DPPQS) under the
Ministry of Agriculture and Farmers Welfare, is responsible for enforcing quarantine
regulations and for quarantine inspection and disinfestation of agri-horticultural
commodities. All the materials meant for export should be accompanied by
Phytosanitary Certificate giving the details of the material and treatment in the
model certificate prescribed under the IPPC of FAO. The Ministry of Agriculture
and Farmers Welfare, Government of India, has notified 199 officers to grant
Phytosanitary Certificate for export of plants and plant material (http://
plantquarantineindia.nic.in/PQISPub/html/Export.htm).
The ICAR-NBPGR, the nodal institution for exchange of plant genetic resources
(PGR), is vested with the authority to issue Phytosanitary Certificate for seed
material and plant propagules of germplasm meant for export for research purposes
after getting approval from DARE. ICAR-NBPGR has developed well-equipped
laboratories and green house complex. ICAR-NBPGR undertakes detailed examina-
tion of germplasm meant for export for the presence of various pests using general
and pest-specific detection techniques and issues Phytosanitary Certificate giving the
details of the material and treatment in the model certificate prescribed under the
IPPC (Chalam and Mandal 2013; Jain and Chalam 2013).
29.4.4 National Domestic Quarantine
Domestic quarantine or internal quarantine is aimed to prevent the spread of

introduced exotic species or an indigenous key pest to clean (pest-free) areas within
the country and this has its provisions in the DIP Act, 1914, and is enforced by the
notification issued by the Central and State Governments. More than 30 pest species
seems to have been introduced in India while notifications have been issued against
the spread of nine introduced pests only namely fluted scale, San Jose scale, codling
moth, coffee berry borer, potato wart disease, potato cyst nematode, apple, BBTV
and banana mosaic virus (Khetarpal et al. 2006). According to notifications issued
under the DIP Act, an introduced pest, for example, BBTV, has been declared a pest
in states of Assam, Kerala, Orissa, Tamil Nadu (TN) and West Bengal (WB) and
banana plants, which come out of these states, have to be accompanied by a health
certificate from the state pathologist or other competent authorities that the plants are
free from it. However, due to the absence of domestic quarantine, BBTV has spread
to most banana growing areas in the country. The limitations and constraints of
domestic quarantine include lack of basic information on the occurrence and distri-
bution of major key pests in the country, in other words pest distribution maps are
lacking for most of the key pests; the absence of concerted action and enforcement of
internal quarantine regulations by the state governments; lack of interstate border
quarantine check-posts at rail and road lines greatly added to the free movement of
planting material across the states; lack of close cooperation and effective coordina-
tion between state governments and centre for timely notification of introduced
pests, organizing pest detection surveys for delineating the affected areas and
immediate launching of eradication campaigns in affected areas; lack of public
awareness; lack of rapid diagnostic tools/kits for quick detection/identification of
exotic pests at the field level; lack of rigorous seed/stock certification or nursery
inspection programmes to make available the pest-free seed/planting material for
farmers (Bhalla et al. 2014).
There is a need to review the status of existing domestic quarantine for establish-
ment of interstate quarantine check-posts for monitoring movement of viruses of
significance. Also, review and update the list of viruses to be regulated under
domestic quarantine. For example, BBTV and Banana mosaic virus (Cucumber
mosaic virus) need to be deleted as regulated pests under domestic quarantine as
they are widely spread in different parts of India.
There is a dire need to revisit the existing domestic quarantine scenario for
strengthening interstate quarantine check-posts and eventually for monitoring the
movement of viruses of significance. Also, review and update the list of viruses to be
regulated under domestic quarantine. For example, BBTV and Banana mosaic virus
(Cucumber mosaic virus) need to be removed as regulated pests under domestic
quarantine as they are widely spread across the country.
The following viruses are known to occur only in certain parts of the
country:
• Indian citrus ring spot virus: Known to occur in Haryana, Maharashtra (MH),
Punjab and Rajasthan
• Citrus mosaic virus: Known to occur in Andhra Pradesh (AP), Karnataka and
parts of TN
• Tomato spotted wilt virus: Reported from TN on Chrysanthemum
• Banana bract mosaic virus: Known to occur in AP, Karnataka, Kerala and TN
• Arabis mosaic virus: Known to occur in AP, Karnataka and parts of TN
• Red clover vein mosaic virus: Known to occur on rose in Palampur, Himachal
Pradesh (HP)
Thus, there is a need to consider the above viruses and others for inclusion as
regulated pests for domestic quarantine to prevent their spread to other parts of the
country, and there is also a need to effectively implement domestic quarantine. India
must develop organized services of plant quarantine at the state level parallel to
Australia and USA.
29.4.5 The Agricultural Biosecurity Bill, 2013
In order to meet the challenges of globalization and free trade, the Agricultural
Biosecurity Bill, 2013, was introduced in the Parliament of India on March 11, 2013.
The main provisions of the Bill is to set up an autonomous authority encompassing
the four sectors of agricultural biosecurity viz., plant health, animal health, living
aquatic resources (fisheries, etc.) and agriculturally important micro-organisms. It
provides for modernising the legal framework to regulate safe movement of plants
and animals within the country and in international trade, and harmonise the legal
requirements of the various sectors of agricultural biosecurity. The proposed legis-
lation is expected to ensure agricultural biosecurity of the country for common
benefit and for safeguarding the agricultural economy. The Bill repeals DIP Act,
1914, and the Livestock Importation Act, 1898, and will give direct powers to the
quarantine officers to deport or destroy or confiscate the consignment or lodge
complaints under the Indian Penal Code.
The Bill establishes the Agricultural Biosecurity Authority of India (Authority)
having functions such as (i) regulating the import and export of plants, animals and
related products; (ii) preventing the introduction of quarantine pests from outside
India; and (iii) implementing post-entry quarantine measures. The administrative
and technical control of existing Plant Quarantine Stations, Central Integrated Pest
Management Centres, and other laboratories under the DPPQS shall be transferred to
and vested in the Authority (http://www.indiaenvironmentportal.org.in/files/file/
Agricultural%20Biosecurity%20Bill.pdf).
29.5 Challenges in Diagnosis of Pathogens in Quarantine
The issues related to the quarantine methodology were analyzed/reviewed by

Khetarpal (2004) and Chalam and Khetarpal (2008). The challenge prior to import
is preparedness for pest risk analysis (PRA). PRA is now mandatory for import of
new commodities into India. The import permit will not be issued for the
commodities not covered under the Schedules V, VI and VII under the PQ Order.
Hence, for import of new commodities in bulk for sowing/planting, the importer
should apply to the Plant Protection Adviser to the Government of India for
conducting PRA. In case of germplasm, Import Permit shall be issued by the
Director, ICAR-NBPGR, after conducting PRA based on international standards
(http://agricoop.nic.in/Gazette/Psss2007.pdf).
The PRA process requires detailed information on pest scenario in both countries
importing and exporting the commodity. Database on all pests, including informa-
tion on host range, geographical distribution, strains, etc. should be made available
for its use as a ready reckoner by the scientists, extension workers and quarantine
personnel. ICAR-NBPGR has compiled pests of quarantine significance for cereals
(Dev et al. 2005; Chalam et al. 2005b), grain legumes (Chalam et al. 2012c), oilseeds
(Gupta et al. 2013; Chalam et al. 2013a) and tropical and sub-tropical fruit crops
(Bhalla et al. 2018b; Chalam et al. 2018b) for India. The Crop Protection Compen-
dium of CAB International, UK, is an useful asset to scan for global pest data (http://
www.cabi.org/cpc/).
As we face challenges to crops from intentional or unintentional introduction of
pests, speed and accuracy of detection become paramount. Intense efforts are
underway to improve detection techniques. The size of consignment received is
very critical in quarantine from the processing point of view. Bulk seed samples of
seed lots need to be tested by drawing workable samples as per norms. The
prescribed sampling procedures need to be followed strictly and there is a need to
develop/adapt protocols for batch testing, instead of individual seed analysis (Maury
et al. 1985). On the other hand, germplasm samples are usually received as a few
seeds/sample and thus it is often not possible to do sampling because of few seeds
and also because of the fact that a part of the seed is also to be kept as a voucher
sample in the National Genebank in India apart from the pest-free part that has to be
released. Hence, extreme precaution is needed to ensure that the result obtained in
the test did not denote a false positive or a false negative sample. Removal of exotic
viruses from the germplasm by growing in PEQ greenhouses inevitably causes a
delay in the release of seeds as it takes one crop season to release the harvest only
from the indexed virus-free plants. Samples received after the stipulated sowing time
would require the indenter to wait for another season. Non-destructive testing of the
seeds could shorten this time and therefore more attention needs to be given to
non-destructive techniques wherever possible (Khetarpal 2004; Chalam and
Khetarpal 2008).
29.6 Perspectives
The plant pathogens have great potential to spread locally and globally due to the
liberalized trade and exchange of research material and germplasm if stringent
quarantine measures are not followed as per SPS/WTO norms. There is also a
need to strengthen the domestic quarantine system to prevent the spread of viruses
with limited distribution within the country. The way forward to ensure biosecurity
is thus highlighted below:
• Strengthen Plant Quarantine Stations dealing with bulk samples in terms of

manpower, infrastructure (well-equipped laboratories, treatment facilities and
greenhouses) and expertise with special emphasis on advanced techniques for
detection of pathogens especially viruses/strains in bulk samples through regular

trainings.
• Initiate pre-import inspection and strengthen the post-entry quarantine (PEQ)
growing and inspection of the imported material. The inspection authorities
should be given adequate support in terms of manpower and funds for undertak-
ing the work. Also, impart training to Inspection Authorities on plant quarantine
issues with special emphasis on post-entry quarantine requirements and method-
ology as per the notified SOPs on the same.
• Regular survey and surveillance programme needs to be undertaken to get a
realistic picture of the status of pests in the country, and for authentic mapping of
endemic pests present in localized pockets and this in turn will help in identifica-
tion of pest-free areas and to include these pests under domestic quarantine. There
is a need for development of eradication strategies for recently introduced pests
and also for pests with limited distribution. This would give a boost to our exports
when the importing country is assured by a certification agency that the produce is
from pest-free areas.
• Detection and diagnosis of pests are crucial for application of mitigation
strategies, trade and for exchange of germplasm. There is a need to accredit
diagnostic laboratories at the central and state level for quick and accurate
identification of pests and also for National Certification Programme for Seed
Health in line with National Certification System for Tissue Culture-raised Plants,
Department of Biotechnology, Govt. of India and need to review seed certifica-
tion standards proposed in Seeds Act 1966. A National Repository of Diagnostics
for Diseases including antisera Bank, database of primers, seeds of indicator
hosts, reference collections, user-friendly diagnostics such as lateral flow strips/
dip sticks which can detect multiple viruses, multiplex RT-PCR protocols, LAMP
and HDA protocols for detection of viruses in the field and at ports of entry,
microarrays, DNA barcoding and ultimately, a cost-effective national biosecurity
chip for diagnosis of all current threats to crop plants would be the backbone for
strengthening the programme on biosecurity for pests. There is an urgent need to
develop a National Plant Pests Diagnostic and Certification Network linking
the research laboratories with seed/vegetative planting material testing
laboratories and quarantine stations, which would be the backbone for
strengthening the programme on biosecurity from plant pests including viruses
(Chalam et al. 2017).
• Review the national regulatory framework and develop a mechanism for distri-
bution or sale of pest-free seeds/ plants/ planting material within the country, be it
seed distribution for multi-location testing under All India Coordinated Research
Projects, inland supply of germplasm by ICAR-NBPGR or seed distribution by
the National/State Seed Corporations/private organizations. Also, need to
develop a national mechanism to monitor the movement of vegetatively
propagated material and tissue culture-raised plants across the states. Also,
strengthen the state certification mechanism to ensure the supply of pest-free
nursery material.
• Database on all pests, including information on host range, geographical distribu-

tion, strains, etc. should be made available for its use as a ready reckoner by the
scientists, extension workers and quarantine personnel.
• Establish proper authenticity for reports of new pests and deposition of reference
cultures in the national repositories may be made mandatory. The exports may
suffer due to wrong identification of pests and reporting the same as new reports.
• Use simulation models for developing an early warning system to predict
outbreaks of diseases and insect pests. Remote sensing may also be used for
the same.
Adopting reliable conventional, serological and molecular techniques with an

appropriate strategy for the detection of pathogens would go a long way in ensuring
the management through quarantine, pest-free trade and exchange of germplasm.
Besides preventing the introduction of exotic pathogens and insect pests, the role of
the diagnostics especially advanced biotechnological interventions in certification of
the planting material of the agri-horticultural crops against indigenous pests needs a
great impetus in boosting our production and trade.
References
Bhalla S, Chalam VC, Tyagi V, Lal A, Agarwal PC, Bisht IS (2014) Teaching manual on
germplasm exchange and plant quarantine. National Bureau of Plant Genetic Resources, New
Delhi. 340p+viii
Bhalla S, Chalam VC, Singh B, Gupta K, Dubey SC (2018a) Biosecuring plant genetic resources in
India: role of plant quarantine. ICAR-National Bureau of Plant Genetic Resources, New Delhi.
216 p +vi. ISBN 978-81-937111-1-8
Bhalla S, Chalam VC, Khan Z, Singh B, Dubey SC (2018b) Potential quarantine pests for India in
tropical and sub-tropical fruit crops. ICAR-National Bureau of Plant Genetic Resources, New
Delhi, p 510. +x ISBN 978-81-937111-0-1
Chalam VC (2014) Biotechnological interventions for biosecuring Indian agriculture from
transboundary plant viruses. In: Mishra SK (ed) Current trends and future challenges in
biotechnology and biomedicine for human welfare and sustainable development part
II. Department of Botany & biotechnology. Govt. New Science College, Rewa, Madhya
Pradesh, pp 16–24. 163p
Chalam VC (2016) Challenges for plant virus diagnosis in certification and transboundary move-
ment of planting material. In: Chalam VC, Akhtar J, Dubey SC (eds) Souvenir and abstracts of
zonal annual meeting of Delhi zone, Indian Phytopathological Society (IPS) & National
Symposium on “biosecurity in food value chain”, February 20, 2016, Delhi Zone, IPS and
ICAR-National Bureau of Plant Genetic Resources, New Delhi, pp 5–25. 101
Chalam VC, Khetarpal RK (2008) A critical appraisal of challenges in exclusion of plant viruses
during transboundary movement of seeds. Indian J Virol 19:139–149
Chalam VC, Mandal B (2013) Phytosanitary issues for export of ornamental plants. In:
Janakiram T, Prasad KV, Singh KP, Swaroop K, Raju DVS, Jain R, Export N (eds) Oriented
horticulture. Indian Agricultural Research Institute, New Delhi, pp 108–116. 140p
Chalam VC, Maurya AK (2018) Role of quarantine in ensuring biosecurity against transboundary
plant viruses. Agric Res J 55(4):612–626
Chalam VC, Pappu HR, Druffel KL, Khetarpal RK. (2004) Detection of potyviruses in legume
seeds by RT-PCR and real-time RT-PCR. In: Proceedings of National Symposium on Molecular
Diagnostics for the Management of Viral Diseases, October 14–16, 2004. Indian Agricultural
Research Institute and Indian Virological Society, New Delhi
Chalam VC, Khetarpal RK, Parakh DB, Maurya AK, Jain A, Singh S (2005a) Interception of seed-
transmitted viruses in French bean germplasm imported during 2002–03. Indian J Plant Prot
33:134–138
Chalam VC, Parakh DB, Singh S, Khetarpal RK (2005b) Viruses of quarantine significance in
cereals. In: Dev U, Khetarpal RK, Agarwal PC, Lal A, Kapur ML, Gupta K, Parakh DB (eds)
Potential quarantine pests for India: cereals. National Bureau of Plant Genetic Resources, New
Delhi, pp 82–92. 144p
Chalam VC, Khetarpal RK, Prasada Rao RDVJ, Parakh DB, Maurya AK (2007) Transboundary
movement of plant viruses: a case study of India. Indian J Virol 19(1):97–98
Chalam VC, Parakh DB, Khetarpal RK, Maurya AK, Jain A, Singh S (2008) Interception of seed-
transmitted viruses in cowpea and mungbean germplasm imported during 2003. Indian J Virol
19:12–16
Chalam VC, Parakh DB, Khetarpal RK, Maurya AK, Pal D (2009a) Interception of seed-
transmitted viruses in broad bean (Vicia faba L.) germplasm imported into India during
1996–2006. Indian J Virol 20:83–87
Chalam VC, Bhalla S, Gupta K, Singh B, Rajan GS, Chhabra R, Maurya AK, Randhawa GJ, Kapur
ML, Khetarpal RK (2009b) Methodology for processing transgenic germplasm in quarantine.
In: Tyagi RK, Mal B, Sharma SK, Khetarpal RK, Brahmi P, Singh N, Tyagi V, Archak S,
Gupta K, Agarwal A (eds) National Symposium on recent developments in the management of
plant genetic resources, December 17–18, 2009. Souvenir and Abstracts, Indian Society of Plant
Genetic Resources, New Delhi, pp 320–321. 370
Chalam VC, Arif M, Caasi DR, Fletcher J, Ochoa-Corona FM (2012a) Discrimination among
CLRV, GFLV and ToRSV using multiplex RT-PCR. Phytopathology 102(Supplement 4):21
Chalam VC, Arif M, Fletcher J, Ochoa-Corona FM (2012b) Detection of Bean pod mottle virus
using RT-PCR, RT-qPCR and isothermal amplification. Phytopathology 102(Suppl 4):21
Chalam VC, Bhalla S, Singh B, Rajan (eds) (2012c) Potential quarantine pests for India in grain
legumes. National Bureau of Plant Genetic Resources, New Delhi. 324 pp. + xii
Chalam VC, Parakh DB, Kaur H, Ajay K, Maurya AK, Khetarpal RK (2012d) Viruses of
quarantine significance in grain legumes. In: Chalam VC, Bhalla S, Singh B, Rajan (eds)
Potential quarantine pests for India in grain legumes. National Bureau of Plant Genetic
Resources, New Delhi, pp 174–211. 324p. + xii
Chalam VC, Parakh DB, Kumar A, Maurya AK (2013a) Viruses, viroids and phytoplasma of
quarantine significance in edible oilseeds. In: Gupta K, Singh B, Akhtar J, Singh MC (eds)
Potential quarantine pests for India in edible oilseeds. National Bureau of Plant Genetic
Resources, New Delhi, pp 175–212. 295p. + vii
Chalam VC, Parakh DB, Maurya AK, Khetarpal RK, Agarwal PC (2013b) Quarantine certification
of seed and other planting material for viruses. In: Proceedings of Asia Pacific Congress of
Virology, VIROCON 2013, December 17–20, 2013, Amity University, Noida. pp 67–69, 221p
Chalam VC, Parakh DB, Sharma R, Maurya AK (2013c) Maize chlorotic mottle virus: a quarantine
pest for India, Technical Bulletin. National Bureau of Plant Genetic Resources, New Delhi. 4p
Chalam VC, Parakh DB, Sharma R, Maurya AK (2013d) Wheat streak mosaic virus: a quarantine
pest for India, Technical bulletin. National Bureau of Plant Genetic Resources, New Delhi. 4p
Chalam VC, Parakh DB, Maurya AK (2014a) Biosecurity umbrella for Indian agriculture against
exotic plant viruses: A case study of quarantine of exotic germplasm. In: Proceedings of XXIII
National Conference (VIROCON 2014) on Recent trends in Virology Research in the Omics
Era, December 18–20 2014, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu
Chalam VC, Parakh DB, Maurya AK, Singh S, Khetarpal RK (2014b) Biosecuring India from seed-
transmitted viruses: the case of quarantine monitoring of legume germplasm imported during
2001-2010. Indian J Plant Prot. 42:270–279
Chalam VC, Parakh DB, Sharma R, Maurya AK (2014c) High plains viru: a quarantine pest for
India, Technical bulletin. National Bureau of Plant Genetic Resources, New Delhi. 4p
Chalam VC, Parakh DB, Maurya AK (2015a) Biosecuring Indian agriculture from transboundary
plant viruses: biotechnological interventions and phytosanitary issues. In: Proceedings of
National Symposium on Challenges and Management Approaches for Crop Diseases of
National Importance – Status and Prospects”, February 12–14, 2015, TNAU, Madurai
Chalam VC, Parakh DB, Sharma R, Maurya AK (2015b) Barley stripe mosaic virus: a quarantine
pest for India, Technical bulletin. National Bureau of Plant Genetic Resources, New Delhi. 4p
Chalam VC, Bhalla S, Gupta K, Singh B, Khan Z, Dubey SC (eds) (2016a) Generic Pest risk
analysis: import of transgenic soybean. ICAR-National Bureau of Plant Genetic Resources,
New Delhi, p 146. http://www.nbpgr.ernet.in/Downloadfile.aspx?EntryId¼7319
Chalam VC, Khetarpal RK, Agarwal PC (2016b) Addressing plant health management using
diagnostics and certification issues. In: Katti G, Kodaru A, Somasekhar N, Laha GS, Sarath
Babu B, Varaprasad KS (eds) Plant health Management for Food Security Issues and
Approaches. Astral International Pvt. Ltd., New Delhi, pp 39–59. 230 p
Chalam VC, Parakh DB, Maurya AK (2017) Role of viral diagnostics in quarantine for plant genetic
resources and preparedness. Indian J Plant Genet Resour 30:271–285
Chalam VC, Dubey SC, Murali Krishna C, Bhalla S, Singh K (eds) (2018a) Transboundary
movement of living modified organisms: strengthening capacities of enforcement agencies.
ICAR-National Bureau of Plant Genetic Resources and Ministry of Environment, Forest and
Climate Change, New Delhi. vi+159 p. ISBN 978-81-937111-2-5
Chalam VC, Parakh DB, Maurya AK, Priyadarshini C, Tripathi A, Sharma R (2018b) Viruses,
viroids, phytoplasma and spiroplasma of quarantine significance in tropical and sub-tropical
fruit crops. In: Bhalla S, Chalam VC, Khan Z, Kandan A, Dubey SC (eds) Potential quarantine
pests for India in tropical and sub-tropical fruit crops. ICAR-National Bureau of Plant Genetic
Resources, New Delhi, pp 277–373. 510 p
Dev U, Khetarpal RK, Agarwal PC, Lal A, Kapur ML, Gupta K, Parakh DB (eds) (2005) Pests of
quarantine significance in cereals. National Bureau of Plant Genetic Resources, New Delhi.
142p
FAO (2016) ISPM 5 Glossary of Phytosanitary Terms. https://www.ippc.int/static/media/files/
publication/en/2016/06/ISPM_05_2016_En_2016-06-03_c6w6Iq3.pdf
FAO (2019) Glossary of phytosanitary terms. International standard for phytosanitary measures
No. 5. Rome. Published by FAO on behalf of the Secretariat of the International Plant Protection
Convention (IPPC). 35 p. Licence: CC BY-NC-SA 3.0 IGO
Gupta K, Singh B, Akhtar J, Singh MC (eds) (2013) Potential quarantine pests for India in edible
oilseeds. National Bureau of Plant Genetic Resources, New Delhi. 295p. + vii http://www.cabi.
org/cpc/ Crop Protection Compendium, Wallingford, UK: CAB International. http://
plantquarantineindia.nic.in/PQISMain/Default.aspx Plant Quarantine Information System,
Directorate of Plant Protection, Quarantine and Storage, Ministry of Agriculture and Farmers
Welfare, Government of India. http://www.indiaenvironmentportal.org.in/files/file/Agricultural
%20Biosecurity%20Bill.pdf. Agricultural Biosecurity Bill 2013, Ministry of Agriculture and
Farmers Welfare, Government of India. https://www.ippc.int/en/core-activities/
standardssetting/ispms/ International Standards for Phytosanitary Measures. International
plant protection convention. Food and agriculture organization of the United Nations, Rome
Jain RK, Chalam VC (2013) Pests: Threat to seed exports. In: Souvenir of Indian seed congress,
February 8–9, 2013, 3–8. Gurgaon, India, National Seed Association of India, pp 3–8. 51 p
Khetarpal RK (2004) A critical appraisal of seed health certification and transboundary movement
of seeds under WTO regime. Indian Phytopathol 57:408–421
Khetarpal RK, Parakh DB, Singh S, Nath R (1992) ELISA detection of soybean mosaic virus in
testas, embryos and seedlings from mottled and unmottled seeds of imported soybean germ-
plasm. Indian J Virol 8:106–110
Khetarpal RK, Parakh DB, Singh S, Nath R, Jain RK, Varma A (1994) Bean common mosaic virus
detected by DAC-indirect ELISA in exotic Phaseolus vulgaris L. Indian J Virol 10:13–16
Khetarpal RK, Singh S, Parakh DB, Maurya AK, Chalam VC (2001) Viruses intercepted in exotic
germplasm during 1991-2000 in quarantine. Indian J Plant Genet Resour 14:127–129
Khetarpal RK, Lal A, Varaprasad KS, Agarwal PC, Bhalla S, Chalam VC, Gupta K (2006)
Quarantine for safe exchange of plant genetic resources. In: Singh AK, Srinivasan K,
Saxena S, Dhillon BS (eds) Hundred years of plant genetic resources Management in India.
National Bureau of Plant Genetic Resources, New Delhi, pp 83–108
Kumar CA, Singh S, Parakh DB (1991) Detection of pea seed borne mosaic virus (PSbMV) in pea
germplasm during quarantine processing. Indian Phytopathol 44:366–369
Maury Y, Duby C, Bossenec JM, Boudazin G (1985) Group analysis using ELISA: determination
of the level of transmission of soybean mosaic virus in soybean seed. Agronomie 5:405–415
Parakh DB, Khetarpal RK, Singh S, Ram N (1994) Post entry quarantine detection of soybean
mosaic virus by ELISA in soybean germplasm. Indian J Virol 10:17–21
Parakh DB, Chalam VC, Khetarpal RK, Maurya AK, Jain A, Singh S (2005) Interception of seed-
transmitted viruses in soybean germplasm imported during 2003 and 2004. Indian J Plant Prot
33:119–124
Parakh DB, Chalam VC, Maurya AK, Khetarpal RK, Singh S, Kaur A (2006) Interception of Pea
seed-borne mosaic virus in pea germplasm imported during 2003. Indian J Virol 17:35–38
Parakh DB, Khetarpal RK, Chalam VC (2008) Risk of seed-transmitted viruses associated with
exchange of soybean germplasm and the south Asian scenario. Indian J Virol 19:47–49
Plant quarantine (Regulation of Import into India) Order and amendments (2003) Department of
agriculture and cooperation, Ministry of Agriculture, India, http://www.agricoop.nic.in/gazette.
htm
Prasada Rao RDVJ, Reddy AS, Chakrabarty SK, Anitha K (2004) Interception of seed transmitted
viruses in peanut germplasm imported into India during 1986 to 2003. Indian J Plant Prot
32:102–104
Prasada Rao RDVJ, Anitha K, Chakrabarty SK, Girish AG, Sarath Babu B, Rameash K,
Abraham B, Varaprasad KS (2012) Quarantine pathogen interceptions on crop germplasm in
India during 1986-2010 and their possible economic impact. Indian J Agric Sci 82:436–441
Prasada RDVJ, Chakrabarty SK, Reddy AS (1990) Interception of Peanut stripe virus in imported
groundnut seed from Myanmar. FAO Plant Prot Bull 38:48–49
Singh B, Khetarpal RK (eds) (2005) Methods of salvaging germplasm infected with plant
pathogens. NBPGR, New Delhi. 94p
Singh B, Rajan BS, Chalam VC, Pandey BM, Singh SK, Kumar N, Khetarpal RK (2003)
Quarantine processing of imported transgenic planting material. Indian J Agric Sci 73:97–100
Singh B, Akhtar J, Dev U, Kandan A, Chand D, Kumar J, Agarwal PC (2015) Interception of
pathogens associated with imported plant genetic resources in India. Indian J Plant Prot.
43:68–74
Sushil SN (2016) Agricultural biosecurity system in India. In: Chalam VC, Akhtar J, Dubey SC
(eds) Souvenir and Abstracts of Zonal Annual Meeting of Delhi Zone, Indian Phytopathological
Society (IPS) & National Symposium on “Biosecurity in Food Value Chain”, February
20, 2016, Delhi Zone, IPS and ICAR-National Bureau of Plant Genetic Resources, New
Delhi, pp 26–35. 101 p
Remote Sensing Technology and Its
Applications in Plant Pathology 30
Ghada A. Khdery
Abstract
Early disease detection and plant health monitoring is a critical tool for reducing
the spread of diseases. Thanks to its great importance in detecting infection before
it occurs on plants, remote sensing is one of the modern science developments in
the monitoring of plant pathogens. Remote sensing techniques will be a very
useful tool for greatly tailoring the diagnostic results. Such innovative
technologies are unparalleled instruments for making agriculture healthier and
more sustainable and for reducing the unnecessary use of pesticides in crop
safety. This chapter discusses the importance of remote sensing in the control
of plant disease and its diagnostic methods, and some examples where remote
sensing was used to monitor plant diseases. Finally, this chapter discusses the
basic principles of hyper-spectrum measurements and the various types of
hyperspectral sensors for plant defense and plant disease detection in various
ranges.
Keywords
Plant pathology · Remote sensing · Hyperspectral technique · Spectral signature
30.1 Introduction
Innovative remote sensing technologies can supply new insights into host–pathogen
systems and have the chance to replace general destructive investigation methods
(Mutka and Bart 2014; Mahlein 2016). Among the various types of sensors, (chlo-
rophyll-fluorescence- thermography, RGB, hyperspectral and multispectral)
G. A. Khdery (*)
National Authority for Remote Sensing and Space Sciences (NARSS), Cairo, Egypt
e-mail: ghada.ali@narss.sci.eg

https://doi.org/10.1007/978-981-15-6275-4_30
684 G. A. Khdery
hyperspectral sensors have significant capabilities and various advantages for dis-
criminating plant diseases and host–pathogen interaction. Chlorophyll fluorescence
and thermography are able to reveal plant stress without determination of causes of
an agent. With hyperspectral technology, it is possible to identify the responsible
disease/pathogen (Bravo et al. 2004; Mahlein et al. 2010). Remote sensing can also
help in plant conservation from potential attacks of fungi, pests or bacteria. Remotely
sensed data and agricultural knowledge can give early warning and prevent disease
or a pest from affecting the crops, by taking suitable action at an early stage.
Detection of diseases at early stage is a lot easier less costly than currently used
impractical human scouting techniques.
Remote sensing is the most important tool for previous diagnosis of plant
diseases. It can provide a quick reaction compared to a manual scouting procedure
typically to determine the presence of the lesion (Moran et al. 1997). The theoretical
basis for remote sensing applications in the evaluation of crop diseases is that crop
diseases cause some physiological changes and severe damage to plant tissues. As a
result, infection from insect pests interferes with photosynthesis and plant structure
and thus affects the amount of light energy and modifies the reflection feature of the
plant (Hatfield and Pinter Jr. 1993). These changes are characterized as a significant
change in the plant spectral pattern. Therefore, as a first step, there is a great need to
determine the characteristics of spectral reflection in the case of infected and healthy
plants. Second, these characteristics will be used for pre-visual diagnosis of a
possible future infection. Many studies have been done about the ability of remote
sensing technology in diagnosis and detection of plant diseases such as (Mahlein
et al. 2010, 2012; Hillnhutter et al. 2011; Abdel et al. 2017). First of all we should
understand what is remote sensing? (Table 30.1)
30.2 Remote Sensing Definition
There are a number of definitions for remote sensing, including the following:
Remote Sensing (RS) is the science of identification, observation and measure-
ment of an object without direct contact with it. It is the science of deriving
information from spectral characteristics acquired at a distance about the earth and
water. Through aid of the eye, humans accomplish this mission. Car driving,
newspaper reading and watching in front of you are all remote sensing practices.
Many sensing devices record an object’s information by measuring electromagnetic
energy transmission from reflecting surfaces of an object, a camera is a remote
sensor because it measures the reflected light without touching the photographed
object. Besides, a camera is sensitive to certain light rays with its filters and
photographic emulsions and radar instruments (Aggarwal 2004).
30 Remote Sensing Technology and Its Applications in Plant Pathology 685
Table 30.1 Overview of plant pathosystems and plant diseases assessed by hyperspectral imaging
Early
Host–pathogen system Scale Detection detection Quantification References
Apple—Venturia inaequalis Leaf √ n.i. √ Delalieux
et al. (2009)
Barley—Blumeria graminis Tissue √ √ n.i. Kuska et al.
f.sp. hordei (2015)
Barley—Blumeria graminis Leaf √ √ √ Thomas
f.sp. hordei et al. (2017)
Barley—Blumeria graminis Leaf √ √ n.i. Wahabzada
f.sp. hordei, Puccinia et al. (2015)
hordei, Pyrenophora teres
Celery—Sclerotinia Canopy √ n.i. n.i. Huang and
sclerotiorum Apan (2006)
Cucumber—CMV, Leaf √ n.i. √ Berdugo
CGMMV, Sphaerotheca et al. (2014)
fuliginea
Sugar beet—Cercospora Leaf √ n.i. √ Bergstrasser
beticola et al. (2015)
Sugar beet—Cercospora Tissue √ n.i. √ Leucker
beticola et al. (2016)
Sugar beet Cercospora Leaf √ √ n.i. Rumpf et al.
beticola, Erysiphe betae, (2010)
Uromyces betae
Sugar beet—Cercospora Leaf √ √ √ Mahlein
beticola, Erysiphe betae, et al. (2010,
Uromyces betae 2012)
Sugar beet—Heterodera Canopy √ n.i. √ Hillnhutter
schachtiia, Rhizoctonia et al. (2011,
solani 2012)
Oilseed rape—Alternaria Leaf √ n.i. √ Baranowski
spp. et al. (2015)
Wheat—Blumeria graminis Canopy √ n.i. √ Cao et al.
f. sp. tritici (2013)
Wheat—Fusarium spp. Ear √ √ √ Bauriegel
et al. (2011)
Wheat—Puccinia Canopy √ n.i. √ Bravo et al.
striiformis (2003)
Wheat—Puccinia Canopy √ n.i. n.i. Bravo et al.
striiformis (2003)
Wheat—Puccinia Canopy √ n.i. √ Huang et al.
striiformis (2007)
Wheat—Puccinia Canopy √ √ n.i. Moshou
striiformis et al. (2005)
Wheat—Puccinia Leaf √ n.i. n.i. Devadas
striiformis, Puccinia et al. (2009)
graminis, Puccinia triticin
Source: Thomas et al. (2017)
n.i. indicates a non-investigated aspect
a
indicates nematodes
686 G. A. Khdery
Fig. 30.1 Steps of remote sensing process. (Source https://crisp.nus.edu.sg/~research/tutorial/

optical.htm)
30.2.1 Principles of Remote Sensing
Discrimination and identification of surface features or objects requires detection and

recording of radiant energy produced or reflected by materials or objects on the
surface (Fig. 30.1). Different objects depend on different amounts of energy in
different bands of the electromagnetic spectrum, incident to it. It depends on the
property of the substance (chemical, structural and physical), angle of incidence,
intensity, roughness of the surface and wavelength of radiant energy. The electro-
magnetic radiation spectrum ranges from small, high to long wavelengths of energy.
As an executor, the human eye only tests a fairly small segment of the spectrum from
0.4 to 0.7 nm in the visible field. The area between 0.4 and 5/Mm can be interpreted
as the wavelengths reflected. Reflection is that phenomenon in which an impinging
radiation beam of a specific wavelength is reflected back from the target without any
alteration. This can be contrasted with emittance, which is the emission of radiant
energy at a given wavelength due to an object’s temperature. Emittance provides
information which can be used for remote sensing applications to agricultural
problems (Aggarwal 2004).
30.2.2 Types of Remote Sensing (Fig. 30.2)
Active Remote Sensing: When remote sensing is performed with a man-made

source of radiation that is used to illuminate the body and reveal the shape of the
reflected signal, for example, radar and lidar remote sensing (Fig. 30.2).
Fig. 30.2 Types of remote sensing. (Source: https://grindgis.com/remote-sensing/active-and-pas

sive-remote-sensing)
Passive Remote Sensing: When remote sensing is performed with the help of
electromagnetic radiations (signals) reflected by a natural body (sun and earth). For
example, visible, NIR and microwave remote sensing (Fig. 30.2).
30.3 Spectral Signature
Spectral reflectance, as a function of wavelength, is the ratio of reflected energy to

incident energy. Each material on Earth’s surface has varying spectral properties.
Different surface materials exhibit different properties of spectral reflection. Spectral
reflection on a photograph of an object is responsible for the colour or tone. Trees
represent the length of the green waves, so trees appear green. The spectral reflec-
tance values of objects averaged over different, well-defined intervals of
wavelengths are the spectral signature of the objects or features they can be distin-
guished by. The spectral reflectance is wavelength dependent; it has different values
for a given terrain characteristic at different wavelengths, and its reflectance
properties are its content of moisture, organic matter content, texture, structure and
iron oxide. We can build a spectral signature for that object by measuring the energy
that is reflected by targets on the earth’s surface over a variety of different
wavelengths. And we may be able to distinguish between them by comparing the
response pattern of different features, which we may not be able to do if we only
compare them at a single wavelength. For instance, water and vegetation reflect in
688 G. A. Khdery
Generalised reflectance spectra of some earth surface materials

Visible NIR SWIR
50
45
40
Rellectance %
35 Soil Altered rocks

30 characteristic of a
25 mineralised zone
20
15
Clear water
10
Water with Healthy
5 vegetation
phytoplankton
0
0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
Wavelength in micrometeres
Clear water Water with phytoplankton Healthy vegetation

Soil Altered rocks characteristic of a mineralised zone
Fig. 30.3 Spectral signatures of water, vegetation and soil. (Source http://www.rsacl.co.uk/
images/base2.jpg)
the visible wavelength somewhat similarly but not in the infrared (Aggarwal 2004)
(Fig. 30.3).
30.3.1 Spectral Signature for Healthy and Infected Plant
Leaves have a low reflectance in the visible (0.4–0.7/m), a high reflectance in the
near-infrared (0.7–1.2 Mm) and a low reflectance in the middle and far infrared
(1.2 Mm) wavebands. This variability in the reflectance of the leaves has allowed the
separation of the leaves from the soil, which tends to show little difference in
reflectance across these wavelengths. As the leaf matures, reflectance tends to
increase in individual leaves; however, the changes depend on wavelength. These
variations derive from changes in the quality of intracellular water and chlorophyll.
Increased reflectance is also caused by lesions and decreased chlorophyll content
produced by a disease. Water stress increases the reflectance from an individual leaf
by reducing the inner water content. Information collected from individual leaves
provides a basic set of information about the process of changes occurring within a
plant; however, it must be generalized to a canopy or field level to be of practical
application. Leaves of green plants absorb most of the blue and red light to use in the
photosynthesis process, and most of the green light is reflected from the leaves of
plants. In Nir IR from 0.7 and 1.0 μm, there is also strong reflectance in the spongy
mesophyll cells, which are located in the internal part of a leaf in (SWIR) short wave
infrared. Water content is the major factor that affects spectral reflectance pattern.
The occurrence of higher water content in plant leaves leads to a decrease of
reflectance in short wave infrared zone (Gogoi et al. 2018).
Spectral characteristics of vegetation was discussed by (Sahoo et al. 2015) which

are determined by their biochemical and biophysical nature and attributes such as
senesced biomass, leaf area index, moisture content and pigment and structures. In
the visible region (VIS: 0.4–0.7 nm), pigments which absorb main light are chloro-
phyll a and b, xanthophylls, carotenoids and polyphenols. Chlorophyll a exhibits the
greatest absorption in the 0.41–0.43 and 0.60–0.69 nm regions, whereas Chlorophyll
b display maximum absorption in the 0.45–0.47 nm range. These strong absorption
bands induce a reflectance peak in the green zone at about 0.55 nm. In the near
infrared zone (NIR: 0.7–1.3 nm), reflectance and transmittance reach their greatest
values and absorption is very small. This is caused by interior diffusion at the cell–
air–water interfaces within the leaves. In shortwave-infrared (SWIR) (1.3–2.5 nm),
leaf properties are affected by other foliar constituents and water. The major bands of
absorption take place at 1.45, 1.94 and 2.7 nm and other processes at 0.96, 1.12,
1.54, 1.67 and 2.2 nm (Sahoo et al. 2015). Changes in reflectance arise from changes
in plant tissue’s biophysical and biochemical properties (Khdery et al. 2019; Gamal
et al. 2020a, b). When a plant is under stress, the chlorophyll production may
decrease resulting in less absorption of palisade cells in the blue and red bands. So
along with the green band, red and blue bands are also reflected. Therefore, in
stressed vegetation yellow or brown colour is developed. As a result, dark patches
are found in the image (Gogoi et al. 2018).
Basically, the visible region from 400 to 700 nm is related to the pigment
composition (Blackburn and Steele 1999; Gitelson et al. 2001) while (NIR region)
from 700 to 1100 nm is related to the water content, leaf traits and structure and
influence (Blackburn and Steele 1999; Gitelson et al. 2001).
30.4 Applications of Remote Sensing in Plant Diseases
Remote sensing can help in identifying, diagnosing and controlling plant diseases, as
well as the stress caused by a lack of water or nutrients. Remote sensing also helps
protect plants from any possible attack of bacteria, fungi or pests. Dimension
Remote sensing data can be combined with agricultural knowledge to provide
early warning to prevent plant from crop diseases, by taking appropriate action at
an early stage. The effects of such attack usually cause chlorophyll to break down,
and by remote sensing we can detect the reduced chlorophyll concentration in the
plants. In addition to chlorophyll loss, diseases and pests can cause destruction of
whole leaves. Besides the loss of chlorophyll, pests and diseases may cause the
destruction of entire leaves. This leads to a reduction in the total area of the leaf and,
ultimately, to a reduction in the photosynthesis ability of the plant. By defining the
leaf area index (LAI) for plant species, it is possible to identify an early pest attack
and educate farmers to take appropriate measures. The study by Apan et al. (2004)
demonstrated that Hyperion satellite hyperspectral imagery could be used to detect
orange rust (Puccinia kuehnii) disease in sugarcane.
Many researchers studying application of remote sensing technologies in detec-
tion of plant diseases (Abdel et al. 2017; He et al. 2019; Deleon et al. 2017; Piou and
Prévost 2013; Jiang et al. 2008; Hillnhutter et al. 2011; Mahlein et al. 2010, 2012).
690 G. A. Khdery
Remote Sensing Techniques Based on Different Sensors:

Based on sensors, the following two groups of remote sensing techniques are
used in monitoring plant diseases (Gogoi et al. 2018).
1. Imaging Approaches
(a) RGB Camera
(b) Multispectral imaging
(c) Hyperspectral imaging
(d) Thermal imaging
(e) Fluorescence imaging
Multiple studies using hyperspectral imaging stated that high spatial resolution is
crucial to avoid mixed spectral signals (Mahlein et al. 2012; Bravo et al. 2003; West
et al. 2010). These investigations focused on fungal-plant disease detection in the
field (Bravo et al. 2003; West et al. 2010) and in the laboratory (Mahlein et al. 2012).
In these studies, it was possible to detect and differentiate plant diseases, and in some
cases in early stages before they were visible to the human eye (Tables 30.2 and
30.3; (Rumpf et al. 2010)).
2. Non-Imaging Approaches
(a) VIS and IR spectroscopy
(b) Fluorescence spectroscopy (Table 30.4)
Table 30.2 Examples of studies on plant disease detection using imaging techniques
Optimum
Plant Disease Statistical methods spectral range References
Wheat Scab (Fusarium Step discrimination and 568,715 nm Delwiche and
head blight) discriminant analysis (550, 605, Kim (2000)
yellow rust, self-organizing 623, 660, and Moshou
nutrient map-neural network, 697 and et al. (2006)
deficiency quadratic discriminant 733 nm)
analysis, regression
analysis
Tomato Late blight Minimum noise fraction 700–750 nm, Huang et al.
disease transformation and 750–930 nm, (2007)
spectral angle mapping- 950–1030 nm
based classification and
1040–1130 nm
Grapefruit Citrus canker Principal component 553, 677, 718 Huang et al.
analysis and 858 nm (2007)
Sweet Blue mold, Difference in reflectance 540 and 680 nm Qin et al.
orange browning rot (2008)
Source: Sankaran et al. (2010)
Table 30.3 The specific wavelengths to identify the different infections

Samples Wavelengths (nm)
Healthy young leaves (548–557 nm) / (701–1387 nm)
Healthy old leaves (1574–1597 nm) / (1749–1775 nm)
Infected young leaves (cotton leaf warm) (542–559 nm) / (1580–1592 nm) / (1751–1763 nm)
Infected old leaves (cotton leaf warm) (350–698 nm) / (1944–2500 nm)
Infected young leaves (aphid) (1563–1567 nm) / (1785–1833 nm)
Infected old leaves (aphid) (1569–1580 nm) / (1764–1781 nm)
Infected young leaves (whiteflies) (1575–1579 nm) / (1764–1769 nm)
Infected old leaves (whiteflies) (1782–1814 nm)
Source: Yones et al. (2019b)
30.5 Case Studies on Plant Pathology from Egyptian

Environment
30.5.1 Case Study 1
Aboelghar and Abdel Wahab (2013) described a novel method for fungal character-
ization. The authors determined the spectral signatures of different B. cinerea
isolates as well as various fungal genera. A unique spectral pattern was investigated
at both the genus and isolate level. The short wave infrared II (2055–2315 nm)
provided the best discrimination between the fungal samples observed. Moreover,
the spectral analysis was performed on non-transformed data and investigated
significant differences among the fungal genera as well as B. cinerea isolates,
while the results investigated high similarity among replicates of the same isolate
of B. cinerea. The results of each spectral test were obtained reproducibly without an
expensive cost consumable during sample preparation and measurements. This
innovative approach would allow identifying, discriminating and classifying fungi
rapidly and inexpensively at the genus, species and isolating level (Figs. 30.4 and
30.5).
30.5.2 Case Study 2
Abd El Wahab et al. (2017) tested the capability of spectral measurement for
detecting the asymptomatic Botrytis infection. The two diagnostic applications,
spectroradiometer and qPCR, were evaluated to compare their reliability to distin-
guish Botrytis infected fruits from healthy ones. Both systems discriminated
between the healthy and infected strawberry fruits and demonstrated their accor-
dance in measurement results. Generally, the qPCR cycle and the spectral reflectance
values of healthy fruits were higher than those of infected ones along with the whole
sample collection. The different systems discriminated between healthy and infected
strawberry fruits and showed agreement in calculating results. The qPCR period and
692 G. A. Khdery
Table 30.4 Examples of studies on plant disease detection by different optical sensors. Source:
Mahlein (1)
Sensor Crop Disease/pathogen References
RGB Cotton Bacterial angular (Xanthomonas Camargo and Smith
campestris), Ascochyta blight (2009)
(Ascochyta gossypii)
Sugar Cercospora leaf spot (Cercospora Neumann et al.
beet beticola), sugarbeet rust (Uromyces (2014)
betae)
Grapefruit Citrus canker (X. axonopodis) Bock et al. (2008)
Tobacco Anthracnose (Colletotrichum Wijekoon et al.
destructivum) (2008)
Spectral Barley Net blotch (Pyrenophora teres), brown Kuska et al. (2015)
sensors rust (Puccinia hordei),
Wheat Head blight (Fusarium graminearum), Kuska et al. (2015)
yellow rust (Puccinia striiformis f. sp. and Moshou et al.
tritici) (2004)
Sugar Cercospora leaf spot (C. beticola), Mahlein et al. (2010
beet sugarbeet rust (U. betae) and Hillnhutter et al.
2012)
Tomato Late blight (Phytophthora infestans) Wang et al. (2008)
Tulip Tulip breaking virus (TBV) Polder et al. (2014)
Sugarcane Orange rust (Puccinia kuehnii) Apan et al. (2004)
Thermal Sugar Cercospora leaf spot (C. beticola) Chaerle et al. (2004)
beet
Cucumber Downy mildew (Pseudoperonospora Berdugo et al. (2014)
cubensis), powdery mildew and Oerke et al.
(Podosphaera xanthii) (2006)
Apple Apple scab (V. inequalis) Oerke et al. (2011)
Fluorescence Wheat Leaf rust (Puccinia triticina), powdery Burling et al. (2011)
imaging mildew (Blumeria graminis f.sp. tritici)
Sugar Cercospora leaf spot (C. beticola) Chaerle et al. (2007
beet and Konanz et al.
2014)
Bean Common bacterial blight (Xanthomonas Rousseau et al.
fuscans sub sp. fuscans) (2013)
stable fruit spectral reflectance values were generally higher than those of the
contaminated ones along with the entire collection of the samples. Spectral analysis
demonstrated a higher reflectance in healthy fruits than that of infected ones
throughout the visible near infrared (VNIR) spectral range, while the short wave
infrared (SWIR) spectral zone showed different degrees of gray mold infection.
Also, the results demonstrated that VNIR is the best spectral zone that would
discriminate between infected and healthy fruits, while SWIR-2 is the best spectral
zone to distinguish between population patterns of Botrytis within the infected fruits
(Fig. 30.6).
0.8
0.7
Reflectance (Unit)
Aspergillus sp.
0.6
Rhizoctonia sp.
0.5 Fusarium sp.
0.4 Penicillium digitatum
0.3 Penicillium italicum
0.2 Alternaria sp.
Botrytis cinerea
0.1
0
0 500 1000 1500 2000 2500
Wavelength (Nanometer (nm))
Fig. 30.4 Spectral reflectance pattern of different fungi using SWIR II at 2055–2315 nm. (Source:
Aboelghar and Abdel Wahab 2013)
Fig. 30.5 Spectral reflectance pattern of different isolates of B. cinerea. (Source: Aboelghar and
Abdel Wahab 2013)
694 G. A. Khdery
a 0.6 19 (Severe infection)

2 (Low infection)
0.5 A (Healthy)
0.4
Reflectance
0.3
0.2
0.1
0.0
0 500 1000 1500 2000 2500 3000
Wavelength (nm)
b 0.6
O (Severe infection)
0.5
D (Low infection)
0.4 A (Healthy)
Reflectance
0.3
0.2
0.1
0.0
0 500 1000 1500 2000 2500 3000
Wavelength (nm)
Fig. 30.6 Spectral reflectance pattern of healthy, low and severely infected strawberry fruits in two
varieties (a) Festival and (b) Sweet Charlie. (Source: Abd El Wahab et al 2017)
30.5.3 Case Study 3
Yones et al. (2019a) differentiated various infections (aphid, white fly, cotton leaf
worm, etc.) on leaves of sugar beet (old and young) using Field ASD
spectroradiometer in Egypt. For all young and old leaves the spectral outline of
reflection was identified. Results showed that the near infrared (NIR) and blue
regions were the best zones to identify the three infections spectrally on young
leaves. The results indicate that for the three infections on young leaves, near
infrared (NIR) and blue regions are the best areas for spectrum define. The results
suggest the possible use of remote sensing technologies for the identification of
pests, allowing for successive control and site-specific pest management sequencing.
Comparison of the reflectance of the plant infection shows the maximum reflectance
(1000 nm) in the infrared zone, comparatively low reflectance (1650 nm) and
minimum reflectance in the spectral zone (2200 nm). Discrimination results showed
the best wavelength to spectrally identify infected and healthy plant table results
(Figs. 30.7 and 30.8).
Fig. 30.7 The spectral reflectance pattern for young leaves of sugar beet plants with different kinds
of infestation (cotton leaf worm, aphid and whiteflies) with healthy plant. (Source: Yones et al.
2019b)
Fig. 30.8 The spectral reflectance pattern for old leaves of sugar beet plants with different kinds of
infestation (cotton leaf worm, aphid and whiteflies) with healthy plant. (Source: Yones et al. 2019b)
696 G. A. Khdery
Fig. 30.9 The spectral reflectance pattern for cotton bolls with different levels of infestation with
healthy bolls. (Source: Yones et al. 2019b)
30.5.4 Case Study 4
Yones et al. (2019b) developed a new approach to use the hyperspectral technique to
detect infested cotton plant with PBW with no losses to boll. A study was aimed to
identify the reflectance spectra of cotton plants with known PBW infestation and to
identify optical wavelengths that are sensitive to PBW damage. The spectral
measurements were performed using an ASD spectroradiometer in the spectral
range of 350–2500 nm. The study indicates that invasion of PBW can be detected
using hyperspectral data and its level identified, which could be used to monitor
trade and predictions. Comparison of the reflection of healthy and contaminated
cotton bollards reveals that the average spectral reflectance (1000 nm) in the infrared
spectral region was relatively low reflectance (1650 nm) and minimum spectral zone
reflectance (2200 nm) (Fig. 30.9).
30.5.5 Case Study 5
Yones et al. (2019c) used spectral data to differentiate between healthy- and pest-
infested three arid-land plants: citrus lemon trees, sweet almond and olives. The
reflection outline of the three healthy and infected plants was identified. The
optimum waveband and wavelength/s were determined to distinguish between
infected and healthy plants. The results showed that healthy plants gave higher
reflection values in the visible spectra compared to plants with olives; however,
0.8
0.7
0.6
Reflectance
0.5 Prunus dulcis

Prunus dulcis infected
0.4
Citrus limon
0.3 Citrus limon infected
0.2 Olea europaea

Infected Olea europaea
0.1
0
350
450
550
650
750
850
950
1050
1150
1250
1350
1450
1550
1650
1750
1850
1950
2050
2150
2250
2350
2450
Wavelength
Fig. 30.10 Spectral reflectance pattern for three cultivated plants (healthy and infected). (Source:
Yones et al. 2019c)
healthy plants showed a higher reflection than infected plants throughout the spec-
trum with other plants. Reflection measurements of the Prunus dulcis form showed a
decrease in chlorophyll at 550 nm) as mites attack almond trees, feed on leaves, and
remove chlorophyll. The spectral properties of C. lemon showed water stress, and
this figure is apparent at 950, 1150 and 1450 nm. Spectral scales for O. europaea, the
shape also indicates water stress, this is evident at 950, 1150 and 1450 nm
(Fig. 30.10 and Table 30.5).
30.6 Recommendations
The strong relationship between the results of the remote sensing analysis and the
pathology of the plant provides evidence of the value of hyperspectral reflectance
data for performing rapid evaluations of the plant health condition effectively and
without infection.
30.7 Conclusion
The chapter demonstrates the strong role remote sensing plays within the agricultural
sector. Remote sensing technology is a powerful tool used in monitoring plant
pathology. It can provide accurate and reliable information to guide decision-making
in crop protection and hence has great potential for use in control of plant diseases.
698 G. A. Khdery
Table 30.5 The optimal waveband to differentiate between healthy and infected plants
Species Optimal wavelength zones (nm)
Citrus limon 355-363-371-379-387-395-403-411-419-427-435-443-451-459-467-
475-483-491-499-507-515-523-531-539-547-555-563-571-579-587-
595-603-611-619-627-635-643-651-659-667-675-683-691-699-707-
715-723-731-739-747-755-763-771-779-787-795-803
Citrus limon 668-676-684-692-700-708-716-724-732-740-748-756-764-772-780-
infected 788-796-804
Infected Olea 350-358-366-390-398-422-430-438-462-470-478-494-518-526-574-
europaea 582-590-598-606-614-622-630-638-646-654-662-670-678-686-694-
702-710-718-726-734-742-750-758-766-774-782-790-798-806
Olea europaea 357-365-381-389-397-405-413-421-429-437-445-453
Prunusdulcis 529-537-545-553-561-569-577-585-593
Prunusdulcis 530-538-546-554-562-570-578-586-594-602
infected
Zizyphus vulgaris 351-359-367-375-383-391-399-407-415-423-431-439-447-455-463-
471-479-487-495-503-511-519-527-535-543-551-559-567-575-583-
591-599-607-615-623-631-639-647-655-663-671-679-687-695-703-
711-719-727-735-743-751-759-767-775-783-791-799
Zizyphus vulgaris 616-624-632-640-648-656-664-672-680-688-696-704-712-720-728-
(infected) 736-744-752-760-768-776-784-792-800-808
References
Abdel WH, Aboelghar M, Ali AM, Yones M (2017) Spectral and molecular studies on Gray Mold
in strawberry. Asian J Plant Pathol 11:167–173
Aboelghar M, Abdel Wahab H (2013) Spectral footprint of Botrytis cinerea, a novel way for fungal
characterization. Adv Biosci Biotechnol 4:374–382
Aggarwal S (2004) Principles of remote sensing. Satellite remote sensing and GIS applications in
agricultural meteorology, pp 23–38
Apan A, Held A, Phinn S, Markley J (2004) Detecting sugarcane ‘orange rust’ disease using EO-1
Hyperion hyperspectral imagery. Int J Remote Sens 25:489–498
Baranowski P, Jędryczka M, Mazurek W, Babula-Skowronska D, Siedliska A, Kaczmarek J (2015)
Hyperspectral and thermal imaging of oilseed rape (Brassica napus) response to fungal species
of the genus Alternaria. PLoS ONE 10:e0122913
Bauriegel E, Giebel A, Herppisch WB (2011) Hyperspectral and chlorophyll fluorescence imaging
to analyse the impact of Fusarium culmorum on the photosynthetic integrity of infected wheat
ears. Sensors 11:3765–3779
Berdugo CA, Zito R, Paulus S, Mahlein AK (2014) Fusion of sensor data for the detection and
differentiation of plant diseases in cucumber. Plant Pathol 63:1344–1356
Bergstrasser S, Fanourakis D, Schmittgen S, Cendrero-Mateo MP, Jansen M, Scharr H, Rascher U
(2015) Hyper ART: non-invasive quantification of leaf traits using hyperspectral absorption
reflectance- transmittance imaging. Plant Methods 11:1–17
Blackburn GA, Steele CM (1999) Towards the remote sensing of matorral vegetation physiology:
relationships between spectral reflectance, pigment and biophysical characteristics of semi-arid
bushland canopies. Remote Sens Environ 70:278–292
Bock CH, Parker PE, Cook AZ, Gottwald TR (2008) Visual rating and the use of image analysis for
assessing different symptoms of citrus canker on grapefruit leaves. Plant Dis 92:530–541
Bravo C, Moshou D, West J, McCartney A, Ramon H (2003) Early disease detection in wheat fields
using spectral reflectance. Biosyst Eng 84:137–145
Bravo C, Moshou D, Oberti R, West J, McCartney A, Bodria L, Ramon H (2004) Foliar disease
detection in the field using optical sensor fusion. International Commission of Agricultural
Engineering, Vol. VI Manuscript FP 04 008
Burling K, Hunsche M, Noga G (2011) Use of blue-green and chlorophyll fluorescence
measurements for differentiation between nitrogen deficiency and pathogen infection in
wheat. J Plant Physiol 168:1641–1648
Camargo A, Smith JS (2009) Image pattern classification for the identification of disease causing
agents in plants. Comput Electron Agric 66:121–125
Cao X, Luo Y, Zhou Y, Duan X, Cheng D (2013) Detection of powdery mildew in two winter
wheat cultivars using canopy hyperspectral reflectance. Crop Prot 45:124–131
Chaerle L, Lenk S, Hagenbeek D, Buschmann C, Straetena DVD (2004) Multicolor fluorescence
imaging for early detection of the hypersensitive reaction to tobacco mosaic virus. J Plant
Physiol 164:253–262
Chaerle L, Hagenbeek D, De Bruyne E, Van der Straeten D (2007) Chlorophyll fluorescence
imaging for disease-resistance screening of sugar beet. Plant Cell Tiss Org 91:97–106
Delalieux S, Somers B, Verstaeten WW, Vanaardt JAN, Keulemans W, Coppin P (2009)
Hyperspectral indices to diagnose leaf biotic stress on apple plants, considering leaf phenology.
Int J Remote Sens 30:1887–1912
Deleon L, Brewer MJ, Esquivel IL, Halcomb J (2017) Use of a geographic information system to
produce pest monitoring maps for south Texas cotton and sorghum land managers. Crop Prot
101:50–57
Delwiche SR, Kim MS (2000) Hyperspectral imaging for detection of scab in wheat. Biol Qual Prec
Agric II Proc SPIE 4203:13–20
Devadas R, Lamb DW, Simpfendorfer S, Backhouse D (2009) Evaluating ten spectral vegetation
indices for identifying rust infection in individual wheat leaves. Precis Agric 10:459–470
Gamal E, Khdery G, Morsy A, El-Sayed M, Hashim A, Saleh H (2020a) Hyperspectral indices for
discriminating plant diversity in Wadi AL-Afreet, Egypt. Plant Arch 20(suppl 2):3361–3371
Gamal E, Khdery G, Morsy A, El-Sayed M, Hashim A, Saleh H (2020b) Using GIS based
modelling to aid conservation of two endangered plant species (Ebenus Armitagei and Periploca
Angustifolia) at Wadi Al-Afreet, Egypt. Remote Sens Appl: Soc Environ 19:100336. https://doi.
org/10.1016/j.rsase.2020.100336
Gitelson AA, Merzlyak MN, Chivkunova OB (2001) Optical properties and nondestructive estima-
tion of anthocyanin content in plantm leaves. Photochem Photobiol 74:38–45
Gogoi NK, Deka B, Bora LC (2018) Remote sensing and its use in detection and monitoring plant
diseases: a review. Agric Res Commun Centre 39:307–313
Hatfield JL, Pinter PJ Jr (1993) Remote sensing for crop protection. Crop Prot 12:403–413
He Y, Kim SB, Balint-Kurti P (2019) A maize cytochrome b-c1 complex subunit protein
ZmQCR61 controls variation in the hypersensitive response. Planta 249:1477–1485
Hillnhutter C, Mahlein AK, Sikora RA, Oerke EC (2011) Remote sensing to detect plant stress
induced by Heterodera schachtii and Rhizoctonia solane in sugar beet fields. Field Crops Res
122:70–77
Hillnhutter C, Mahlein AK, Sikora RA, Oerke EC (2012) Use of imaging spectroscopy to
discriminate symptoms caused Heterodera schachtii and Rhizoctonia solane on sugar beet.
Precis Agric 13:17–32
Huang JF, Apan A (2006) Detection of Sclerotinia rot disease on celery using hyperspectral data
and partial least squares regression. J Spat Sci 51:129–142
Huang W, Lamb DW, Niu Z, Zhang Y, Liu L, Wang J (2007) Identification of yellow rust in wheat
using in-situ spectral reflectance measurements and airborne hyperspectral imaging. Precis
Agric 8:187–197
700 G. A. Khdery
Jiang JA, Tseng CL, Lu FM, Yang EC, Wu ZS, Chen CP et al (2008) A GSM-based remote wireless
automatic monitoring system for field information: a case study for ecological monitoring of
oriental fruit fly, Bactrocera dorsalis (Hendel). Comput Electron Agric 62:243–259
Khdery G, Frag E, Arafat S (2019) Natural vegetation cover discrimination using hyperspectral data
in Wadi Hagul, Egypt. Egypt J Remote Sens Space Sci 22:253–262
Konanz S, Kocsanyi L, Buschmann C (2014) Advanced multi-color fluorescence imaging system
for detection of biotic and abiotic stresses in leaves. Agriculture 4:79–95
Kuska M, Wahabzada M, Leucker M, Dehne HW, Kersting K, Oerke EC, Steiner U, Mahlein AK
(2015) Hyperspectral phenotyping on microscopic scale – towards automated characterization
of plant-pathogen interactions. Plant Methods 11:28
Leucker M, Mahlein AK, Steiner U, Oerke EC (2016) Improvement of lesion phenotyping in
Cercospora beticola-sugar beet interaction by hyperspectral imaging. Phytopathology
2:177–184
Mahlein AK (2016) Plant disease detection by imaging sensors-Parrels and specific demands for
precision agriculture and plant phenotyping. Plant Dis 100:241–251
Mahlein AK, Steiner U, Dehne HW, Oerke EC (2010) Spectral signatures of sugar beet leaves for
the detection and differentiation of diseases. Precis Agric 11:413–431
Mahlein AK, Steiner U, Hillnhütter C, Dehne HW, Oerke EC (2012) Hyperspectral imaging for
small-scale analysis of symptoms caused by different sugar beet disease. Plant Methods 8:3
Moran MS, Inoue Y, Barnes EM (1997) Opportunities and limitations for image-based remote
sensing in precision crop management. Remote Sens Environ 61:319–346
Moshou D, Bravo C, West J, Wahlen S, McCartney A, Ramon H (2004) Automatic detection of
‘yellow rust’ in wheat using reflectance measurements and neural networks. Comput Electron
Agric 44:173–188
Moshou D, Bravo C, Oberti R, West J, Bodria L, McCartney A, Ramon H (2005) Plant disease
detection based on data fusion of hyper-spectral and multi-spectral fluorescence imaging using
Kohonen maps. Real Time Imaging 11:75–83
Moshou D, Bravo C, Wahlen S, West J, McCartney A, De Baerdemaeker J, Ramon H (2006)
Simultaneous identification of plant stresses and diseases in arable crops using proximal optical
sensing and self-organising maps. Precis Agric 7:149–164
Mutka AM, Bart RS (2014) Image-based phenotyping of plant disease symptoms. Front Plant Sci
5:734
Neumann M, Hallau L, Klatt B, Kersting K, Bauckhage C (2014) Erosion band features for cell
phone image based plant disease classification. In: Proceeding of the 22nd International
Conference on Pattern Recognition (ICPR), Stockholm, Sweden, 24–28 August 2014, pp
3315–3320
Oerke EC, Steiner U, Dehne HW, Lindenthal M (2006) Thermal imaging of cucumber leaves
affected by downy mildew and environmental conditions. J Exp Bot 57:2121–2132
Oerke EC, Frohling P, Steiner U (2011) Thermographic assessment of scab disease on apple leaves.
Precis Agric 12:699–715
Piou C, Prévost E (2013) Contrasting effects of climate change in continental vs. oceanic
environments on population persistence and microevolution of Atlantic salmon. Glob Change
Biol Bioenergy 19:711–723
Polder G, van der Heijden GWAM, van Doorn J, Baltissen TAHMC (2014) Automatic detection of
tulip breaking virus (TBV) in tulip fields using machine vision. Biosyst Eng 117:35–42
Qin J, Burks TF, Kim MS, Chao K, Ritenour MA (2008) Citrus canker detection using
hyperspectral reflectance imaging and PCA-based image classification method. Sens &
Instrumen Food Qual 2:168–177
Rousseau C, Belin E, Bove E, Rousseau D, Fabre F, Berruyer R, Guillaumes J, Manceau C, Jaques
MA, Boureau T (2013) High throughput quantitative phenotyping of plant resistance using
chlorophyll fluorescence image analysis. Plant Methods 9:17
Rumpf T, Mahlein AK, Steiner U, Oerke EC, Dehne HW, Plümer L (2010) Early detection and
classification of plant diseases with support vector machines based on hyperspectral reflectance.
Sahoo RN, Ray SS, Manjunath KR (2015) Hyperspectral remote sensing of agriculture. Curr Sci
108:848–859
Thomas S, Wahabzada M, Kuska M, Rascher U, Mahlein AK (2017) Observation of plant–
pathogen interaction by simultaneous hyperspectral imaging reflection and transmission
measurements. Funct Plant Biol 44:23–34
Wahabzada M, Mahlein AK, Bauckhage C, Steiner U, Oerke EC, Kersting K (2015) Metro maps of
plant disease dynamics - automated mining of differences using hyperspectral images. PLoS
One. https://doi.org/10.1371/journal.pone.0116902
Wang X, Zhang M, Zhu J, Geng S (2008) Spectral prediction of Phytophthora infestans infection on
tomatoes using artificial neural network (ANN). Int J Remote Sens 29:1693–1706
West SJ, Bravo C, Oberti R, Moshou D, Ramon H, McCartney HA (2010) Detection of fungal
diseases optically and pathogen inoculum by air sampling. In: Oerke EC, Gerhards R, Menz G,
Sikora RA (eds) Precision crop protection—the challenge and use of heterogeneity. Springer,
Dordrecht, pp 135–150
WiJekoon CP, Goodwin PH, Hsiang T (2008) Quantifying fungal infection of plant leaves by
digital image analysis using scion image software. J Microbiol Method 74:94–101
Yones MS, Aboelghar M, Khdery GA, Dahi HF, Sowilem M (2019a) Spectral signature for
detecting pest infestation of some cultivated plants in the northern west coast of Egypt. Egypt
Acad J Biol Sci 12:73–38
Yones MS, Aboelghar M Khdery GA, Farag E, Ali AM, Salem NH, Ma’mon SAM (2019b)
Spectral measurements for monitoring of sugar beet infestation and its relation with production.
Asian J Agric Biol under press
Yones MS, Khdery GA, Dahi HF, Farg E, Arafat SM, Gamil WE (2019c) Early detection of pink
bollworm Pectinophora gossypiella (Saunders) using remote sensing technologies. Proc. SPIE
11149, Remote Sensing for Agriculture, Ecosystems, and Hydrology XXI, 111491C
(21 October)
Decision-Making Tools for Integrated
Disease Management 31
K. P. Singh, T. Aravind, Amit Kumar Srivastava, and C. S. Karibasappa
Abstract
The decision-making process is the core of any successful integrated disease

management programme. The complexity of decision-making process in IDM is
much higher as compared to conventional agriculture as it involves multiple
factors related to the host, pathogen and environment to be considered. Hence,
for taking the most efficient and economic decisions, a farmer or a scientist needs
the help of decision-making tools. This need has led to the development of four
such decision-making tools viz., warning services, expert systems, decision
support systems and onsite devices. They differ in their objective, scope, archi-
tecture and complexity of data that they can handle. But the prime objective of
these is to help the farming and the scientific community to take the best possible
decision regarding plant disease management. At present, their adoption is
limited and does not justify the cost and effort required for their development.
However, more efficient and user-friendly tools are being developed after
rectifying the drawbacks of the previous ones. Their efficient utilization will
help in successful plant disease management and lead to the concept of sustain-
able agriculture.
31.1 Introduction
To satisfy the ever-growing food demand, agricultural production must increase by

70% by 2050 globally. However, pests and crop diseases put global food supplies at
high risk (Carvajal-Yepes et al. 2019). Worldwide, yield losses caused by pests and
K. P. Singh (*) · T. Aravind · C. S. Karibasappa

Department of Plant Pathology, College of Agriculture, Pantnagar, Uttarakhand, India
A. K. Srivastava
Institute of Crop Science and Resource Conservation, Crop Science Group, Katzenburgweg 5,
University of Bonn, Bonn, Germany

https://doi.org/10.1007/978-981-15-6275-4_31
704 K. P. Singh et al.
diseases are estimated to average 21.5% in wheat, 30.0% in rice, 22.6% in maize,
17.2% in potato and 21.4% in soybean (Savary et al. 2019), which accounts for at
least half of the global human calorie intake (FAOSTAT 2018). Therefore,
quantifying the impacts of plant pests and associated diseases on crop production
and making timely interventions represents one of the most important research
questions for simulation models (Donatelli et al. 2017) for plant disease forecasting.
Plant disease forecasting is a management system used to predict the occurrence or
change in severity of plant diseases. At the field scale, these systems are used by
growers to make economic decisions about disease treatments for control. Often the
systems ask the grower a series of questions about the susceptibility of the host crop
and incorporate current and forecast weather conditions to make a recommendation.
Typically, a recommendation is made about whether disease treatment is necessary
or not. Forecasting systems are based on assumptions about the pathogen’s
interactions with the host and environment, the disease triangle (Fig. 31.1). The
objective is to accurately predict when the three factors – host, environment and
pathogen – all interact in such a fashion that disease can occur and cause economic
losses.
Moreover, a systems analysis approach is required to understand how pest and
disease problems arise and how they may be tackled because of the complex
relationships between crops and their pests and diseases and because of the dynamic
nature of pest populations. This has become more complex as climate change is
throwing new challenges due to change in temperatures and the rainfall amount and
pattern. The shift to a non-stationary climate indicates that current datasets are no
longer applicable to predict the behaviour of the production system. There is a
plethora of evidence now available that pathogens which for decades have had no
effect on crops are now becoming key determinants and affecting the crop yield
(Gramaje et al. 2016; Parker and Warmund 2011).
The FAO has defined sustainable agricultural development as “the management
and conservation of the natural resource base, and the orientation of technological
and institutional change in such a manner as to ensure the attainment and continued
Fig. 31.1 The plant disease

triangle represents the factors
necessary for disease to occur.
(Source: https://en.wikipedia.
org/wiki/Plant_disease_
forecasting)
31 Decision-Making Tools for Integrated Disease Management 705
satisfaction of human needs for present and future generations. Such development
conserves land, water, plant and animal genetic resources, is environmentally
non-degrading, technically appropriate, economically viable and socially accept-
able”. The concept of integrated pest management has emerged from this concept of
sustainability. It has shifted our focus from conventional chemical-based manage-
ment to an integrated approach for ensuring environmental safety and ecological
sustainability. Integrated disease management (IDM) can be viewed as harmonious
integration of different disease management strategies for the effective and econom-
ical management of plant diseases below the economic threshold level based on the
sound understanding of the whole crop ecosystem (Agrios 2005). It requires a
thorough understanding of the etiology and epidemiology of the disease, cost-
benefit analysis and deeper knowledge of the plant protection measures. IDM relies
upon the integration of different plant protection practices as compared to the sole
dependence on synthetic pesticides in conventional agriculture. The decision-
making process is the core of any IPM/IDM module (European commission
2009). Decisions are usually made based on a combination of empirical data,
analysis of the situation in hand and personal expertise on the subject. The complex-
ity in the decision-making process depends on the complexity of factors governing
the situation (McCown 2002). The simple task requires only a little knowledge and a
bit of experience in handling such a situation. However, more complex decisions
with greater consequences may require experience, judgment and quantitative anal-
ysis. In this regard, the decision making from an agricultural perspective is often
complicated as it involves complex interactions of many factors. It requires the
information and application knowledge from various interacting fields of science
starting from the knowledge on edaphic factors to the much complex economics and
marketing strategies (Reddy and Rao 1995). This type of complex decision-making
ability is usually lacking in the farming community, especially in those belonging to
the under-developed and developing countries.
The decisions taken in an agricultural farm can be broadly divided into three viz.,
strategic, tactical and operational (Rossi et al. 2012). Strategic decisions are taken
based on the consideration of the long-term plans (many years) of the whole farm. It
is taken by the owner/director of a farm and gives guidelines for the farm manage-
ment. The tactical decisions refer to the decisions having an impact for a few days to
weeks or a crop season and are taken by the manager in charge of the farm. The
operational decisions are taken by the employees and mainly deal with the imple-
mentation of the strategic or tactical decisions taken earlier (Rabbinge et al. 1993).
The chain of decision-making process starts with the identification and analysis of
the problem followed by the evaluation of possible solutions. The best possible
solution is selected and converted into action and the results are validated. It is a
continuous process and requires regular monitoring of onsite field problems and
analyzing the impact of the remedial measures adopted (March 1994).
Diverse control measures are currently available for the management of plant
diseases. These include various cultural, mechanical, physical, biological and chem-
ical methods (Odile et al. 2010). Of these, the use of chemical control is most popular
among the farming community. Since the advent of the concept of sustainable
agriculture, there is a common consensus among the scientific and farming

communities that the sole application of any of these management techniques will
be ineffective and unsustainable in the long run. Hence, integrated pest and disease
management is becoming more popular. Compared to disease management practices
in conventional agriculture, IDM involves more complex decision making (Rossi
et al. 2012).
For an individual farmer or a farm manager, it is difficult to make complex
decisions as it requires a thorough understanding of the multiple factors governing
the impact of decisions taken. Hence, they require tools that can help them in
selecting the best possible and effective decisions that can be adopted to yield
maximum economic benefits. Predictive systems are of much importance in this
regard for making decisions for integrated plant disease management. “Predictive
system” is used as a general term for formalized algorithms that assess disease risk
factors that inform the need for crop protection (Gent et al. 2013). The most
successful predictive systems take into account the whole of a crop ecosystem,
using simulation models, previously developed databases and decision-making
rules to analyze a situation and to provide the most appropriate action to be taken
(De Wolf and Isard 2007). The decisions are mostly taken based on the etiology and
epidemiology of the disease, prevailing weather parameters, crop growth stage,
previous history of the disease occurrence, etc. The different predictive systems
used in plant pathology include the warning services, expert systems, decision
support systems and on-site devices.
31.2 Warning Services
Warning services are based on general IPM/IDM guidelines or any of the disease-
forecasting models (Rossi et al. 2000). They do not take into consideration the
factors affecting the disease spread in an individual farm and are given on a regional
scale. They are usually carried out by the government agencies, NGOs or even the
private extension agents for free or on paid basis. The information is passed on to the
beneficiaries through mass media like radio, newspaper, television, etc. or personal
contacts through SMS/emails (Beta 2011).
31.3 Expert Systems
The complex agricultural decision requires the expertise and knowledge of all
aspects of agriculture. Such personal expertise is widely lacking in our farming
and scientific community. This necessitated the help of computer programmes that
are programmed to help the farmers in the quality decision regarding the diverse
agricultural processes, which lead to the evolution of the concept of Expert System
(ES). ES, sometimes called as Knowledge Base System, is defined as “a computer
programme designed to model the problem-solving ability of a human expert”
(Durkin 1994). It is intended to cater to the needs of making quality decisions as
well for technology dissemination. In agriculture, ES finds its applications in the

selection of crop varieties best suited for the locality, detection and diagnosis of pest
and diseases and their effective management, nutrient and water management, etc.
(Abu-Naser et al. 2008). It can be viewed as an excellent tool for relieving the
mounting pressure on limited expertise available in developing countries. It is
capable of gathering and analysing the vast data from many experts from multiple
fields of agriculture and allied fields (Chen et al. 2012). Moreover, it will help in
speeding up the process of pest identification and suggesting management strategies
for the same (Mahaman et al. 2003)
Shafinah et al. (2013) have described 13 diverse methodologies used to develop
ES. These include a rule-based system, knowledge-based system, neural network
system, fuzzy expert system, case-based reasoning, object-oriented methodology,
etc. In case of pest and disease detection and management, the rule-based ES is most
commonly employed. It employs IF (condition) and THEN (action) to solve the
problems. There are two types of rule-based ES viz., forward chaining (or data-
driven) and backward chaining (or goal-driven) expert systems. In the data-driven
ES, the process starts from the analysis of the initial facts and the conclusions are
drawn using the rules. In case of goal-driven ES, it begins with the highest level
rules. It searches and determines the rules that are allowed to be further sustained
(Shafinah et al. 2013).
The basic components of an ES include the knowledge base, inference protocol,
user interface and a knowledge acquisition mechanism (Feigenbaum 1992; Pham
and Pham 1988). The knowledge base encompasses the knowledge regarding the
problem like the facts, information and rules for judgment. The stores’ knowledge is
further analyzed and manipulated using the inference mechanism which is also
called as the control structure or reasoning mechanism. The user interface acts as a
communication bridge between the user and the ES. Knowledge acquisition domain
is used for further updating and upgrading the knowledge base and the ES. An
additional component, i.e. a memory component, used to store the temporary data
was suggested by Mahaman et al. (2003).
Some of the successful examples of ES in crop protection include:
(i) VEGES (Yialouris et al. 1997) – for the diagnosis of nutritional disorders, pests
and diseases of six greenhouse vegetables
(ii) AGRES (Ganesan 2006) – for the diagnosis of pest and diseases of major crops
in Kerala
(iii) RUBEXS -04 (Balasubramani and Lekshmi 2001) – for disease diagnosis in
rubber plants
31.4 Decision Support Systems
Decision support systems (DSS) are interactive computer-based systems that aid in
making quality decisions. Turban (1995) has defined DSS as “an interactive, flexible
and adaptable computer-based information system, specially developed for
supporting the solution of a non-structured management problem for improved

decision making. Apart from agriculture, DSS finds its application in a broad
range of areas like natural resource management, business management, health
sector and environmental studies (Eom and Kim 2006). In agriculture, DSS are
frequently developed and used in agronomy, soil science and crop protection studies
(Kumbhar and Singh 2013).
There are four important components of DSS viz., database management subsys-
tem, knowledge management subsystem, user interface and user (Mir and Quadri
2009). The first step in the creation of a DSS is the collection of the data on the
problem. For a DSS about plant disease, we need to collect data on the previous
occurrence of the disease, epidemiological factors governing the disease, survival
and spread of the pathogen, management measures adopted and the efficiency and
economy of the measures in managing the disease. The database creation is the most
critical step for any successful DSS. The database management subsystem manages
and provides access to the existing database through the Database Management
System (DBMS). DBMS creates and manages the database and regulates access to
the database. The knowledge-based management sub-system mostly consists of a
model or an inference protocol that solves the problem and provides a decision. The
accuracy of the decision making depends on the database and the efficiency of the
model used. Most DSS use “if-then” strategy to solve the problems. The user
interface is the part of the DSS that interacts with the end-user. It acts as a bridge
between the system and the end user. Mostly the user interface is a desktop or a
smartphone using which the end-user can feed the data and get desired results. The
user interface is also used for updating the database and the models already existing
with the system. The user can be a farmer, extension agent or a scientist who seeks
the right decision from the system (Fig. 31.2). According to Marakas (2003), the five
basic components of the DSS include data management system, model management
system, knowledge engine, user interface and the user. The model management
system performs the modelling and necessary data analysis, whereas the knowledge
engine is used for the problem recognition and finding out their solution.
In agriculture, DSS finds its application in entire aspects starting from the
selection of seeds to sale. It can be used as a decision making aid for plant protection,
integrated nutrient management agricultural extension, climate prediction, farm
mechanization, land use planning, etc. (Hansen 2002; Suarez de Cepadaet al.
2005; Jorgensen et al. 2006; Mosseddaq et al. 2005; Sodtke 2005). There is no
such thing called an ideal DSS. All the DSS developed has got its pros and cons. The
success of DSS is mainly assessed by the accuracy of the decision making and the
level of adoption by the end-users. Bhargava and Power (2001) have classified DSS
into five groups viz., model-driven, communication-driven, data-driven, document-
driven and the knowledge-driven DSS. The model-driven DSS relies on the access to
and manipulation of statistical, financial, optimization or simulation model. The
communication-driven DSS emphasizes on shared decision making based on com-
munication and collaboration. The knowledge-driven DSS, most commonly used in
plant disease diagnosis and management, is specialized for problem-solving based
Fig. 31.2 Components of a

decision support system User
User Interface
Knowledge Database
Base Management
Management Subsystem
Subsystem
on the stored database, facts and rules. The different types, categories and fields of
application of DSS in agriculture have been reviewed by Manos et al. (2004).
Many unique features of the plant protection measures make them an ideal area
for development and use of the DSS. They include a wide range of strategies
currently available for disease management, lack of needed expertise especially in
remote villages, complex interactions with the other crop production practices and
farming economics, etc. (Shtienberg 2013). Many a time, the scientific experts
become unavailable for solving the issues under real field conditions. Moreover,
the passage of human expertise from one expert to the successor is also fairly low. In
this aspect, DSS can play a vital role as the expertise of an individual stored as a
computer programme will be available at all times. It will help in bridging the gap
from lab to land and inconveniences caused due to the absence of the expert in
person. The apple scab predictive systems FAST and BLITECAST are the weather-
based disease prediction systems that are the predecessors of modern-day DSSs
(Jones et al. 1980; MacKenzie 1981; Madden et al. 1978). For successful adoption of
these systems, they have to be highly user-friendly, based on a scientific basis, farm
and user-specific and be able to provide simple, robust and most efficient solutions to
the problems (Cabrera 2012).
31.5 Onsite Devices
Onsite devices help in decision making on an individual farm. They are programmed
to collect data (e.g. weather data) from within the farm and give recommendations
regarding disease management practices based on the factors of the particular farm
where they are installed (Rossi et al. 2012). The best example of an onsite weather
data collection device is the μMETOS. The device can be operated via mobile phone,
personal computer or a notebook. The plugin devices used in precision agriculture
can also be used for collecting the onsite data for pest and disease management. At
present, attempts are being made to collect the weather data by linking with GIS and
GPS systems for accurate decision making. Such an onsite device has been devel-
oped for the accurate prediction of apple scab in the Garhwal Himalayas (Singh and
Kumar 2005).
31.6 Case Study 1: Late Blight of Potato
Late blight of is one of the most important diseases of potato causing widespread
crop loss worldwide. It is caused by the Oommycete pathogen Phytophthora
infestans (Mont.) de Bary (Kingdom Chromista; Phylum Oomycota, Class
Oomycetes; Order Pythiales; Family Pythiacea) (Singh 2009). The disease is preva-
lent in all the major potato growing regions of the world. The occurrence and
severity of the disease vary across years and from region to region depending
upon the climatic conditions. The pathogen survives in the soil as oospores and in
the infected tubers, which serve as the primary inoculum for the next season.
The disease is influenced by low temperature along with relative humidity, dew,
rainfall and wind velocity. Taking into consideration the economic importance of the
disease, the studies on the forecasting of the late blight epidemic had been
undertaken from 1926 by Van Everdingen (Dutch Rules). According to him, the
late blight can occur in severe form if the night temperature is below dew point for
atleast 4 hrs, minimum temperature of 10 C or slightly above, clouds on the next
day and rainfall during the next 24 hours of atleast 0.1 mm (Van Everdingen 1926).
Since then, a number of empirical and fundamental models have been developed to
forecast the epidemic (Table 31.1). The importance of weather parameters in the
development of late blight has been extensively reviewed well by Rotem et al.
(1971) and Harrison (1992).
Other than these basic models, certain decision support systems were also
developed for adopting the timely management strategies for the late blight of
potato. The first of these kind (BLITECAST) was developed by Krause and his
co-workers in 1975 at the Pennsylvania State University (Krause et al. 1975).
Basically it combined the concept of severity values (given by Wallin in 1962)
and blight favourable days (given by Hyre in 1947). The system gives the manage-
ment recommendations based on the weather parameters send by the growers for
their locality. This was further modified and incorporated into another programme
WISDOM (crop management programme) by Mac Hardy (1979). The other
Table 31.1 Various forecasting models developed for late blight of potato (Singh et al. 2012)
Sl. no. Fundamental models Sl. no. Emperical models
1. BLIGHTCAST (Krause et al. 1975) 1. Beaumont’s rules (Beaumont 1947)
2. Mac Hardy (1979) 2. Cook’s system (1949)
3. Fry et al. (1983) 3. Hyre’s system (1954)
4. Grunwald et al. (2000) 4. Smith’s system (1955)
5. Runno and Koppel (2002) 5. Wallin’s system (1962)
Table 31.2 Mills’ table and our university data to arrive at the incubation period based on
temperature and leaf wetness
Minimum wetting hours of
leaves for infection (approx.
hours)a
Average daily As per As per Days required (after infection for
temperature ( C) Mills’ table Univ. data symptom appearance)
25 11 9
16 and 24 9 6 9
15 10 8 12
14 10 8 13
13 11 9 13
12 11–15 9 14
11 12 10 15
10 14 13 16
9 15 13 17
a
The infection period is considered to start at the beginning of the rain
important decision support system for late blight of potato include PhytoPRE (Forrer
et al. 1993), SIMPHYT 1 and 2 (Gutsche 1993), NEGFRY (Hansen et al. 1995),
SIMBLIGHT (Benno kleinhenz et al. 2007), etc.
There are four major late blight forecasting models developed for the Indian
conditions:
1. Ten day moving graph – Chaudhary and Pal (1959)

2. Seven day moving precipitation model – Bhattacharyya et al. (1983)
3. JHULSACAST – Singh et al. (2000)
4. INDO-BLIGHTCAST – Singh et al. (2016a)
31.6.1 INDO-BLGHTCAST
It was developed by Singh and his colleagues at the Central Potato Research Institue,
Shimla, Himachal Pradesh, India. It was developed by using the late blight occur
dates and meteorological parameters at four different locations in the Indo gangetic
plains. It involves the calculation of night relative humidity (RH) for seven
consecuitive days and P-days (Physiological days – moving cumulative effective
temperature). Late blight is predicted to occur within 15 days if RH and P-days
exceed 525 and 52.5, respectively, for seven consecutive days.
31.7 Case Study 2: Apple Scab Prediction Model for Garhwal

Himalayas
Apple scab, caused by Venturia inequalis (Cke.) Aderh. is one of the most dreadful
diseases of crops leading to serious crop loss both under field conditions and post-
harvest stages. It was first time reported in India, in Kashmir, in 1935, followed by
Himachal Pradesh in 1977. Ever since its introduction through planting materials,
the disease has caused a series of the epidemic in the two states (Singh et al. 2015,
2016b). Over 60% of the area under sweet varieties of the total of 17,542 ha was
engulfed by apple scab during 2000 (Singh and Kumar 2008, 2009). The major
economic loss is caused in the form of scabbed apple fruits that are unfit for
marketing and consumption. Besides this, the disease also cause a decrease in
plant vigour, reduction in crop yield and lead to the gradual death of a tree.
According to Singh (2019), the average economic loss due to reduced marketability
because of scabbed fruit is 24%.
31.7.1 Symptomatology
Young plants are more susceptible to scab infections and the pathogen attack all the
above-ground plant parts. The symptoms of scab infection are first observed on the
under-surface of young leaves. Young lesions are olive green in colour which
gradually changes to velvety brown and later on to metallic black. Mycelium radiate
from the mature lesions. The leaf may develop a puckered appearance due to the
infection. On the fruits, many lesions coalesce to give a distorted appearance and
cracks may develop on such fruits (Fig. 31.3). Severe infection of the tree often leads
to heavy fruit drop. Primary infection lead to the development of isolated scattered
spots whereas the secondary infection cause severe leaf spotting and leaf distortion
(Singh et al. 2001).
31.7.2 Etiology of the Pathogen
The pathogen belongs to the kingdom Fungi, phylum Ascomycota, subphylum

Pezizomycotina, class Dothidiomycetes, order Pleosporales andfamily Venturiaceae
(Dube 2013). The conidial stage, Spiloceapomi, develops in the leaves and fruits on
the tree. In the host, the light-coloured mycelium remains sub-epidermal which later
on turns brownish. The reddish-brown conidia are mostly flame-shaped and one- or
Fig. 31.3 (a–d) Scab on leaves as velvety brown to olive spots; (e–g) Mousy black secondary scab
lesions and sign on the fruit surface of delicious apple
two-celled. The fungi produce pseudoothecia in the fallen leaves which produce
bicelledascospores that cause the primary infection (Singh 2009).
31.7.3 Survival and Spread
The pathogen survives in the dead and fallen infected leaves as saprophytes. In the
Garhwal Himalayas, the fungi start producing pseudothecia in November and mature
pseudothecia release infectious ascospores during May–June (Singh 2019). The
intermittent rains or heavy snowfall followed by dry climate are conducive for
pesudothecia development and ascospore release. Mature ascospores are discharged
into the air during periods of rain. This ascospore produces primary infections. The
conidial stage develops in the infected leaves and cause secondary infection in the
orchard. In apple scab development, the conidial stage has long been recognized as
an important phase, particularly for the rapid build up and spread of scab from tree to
tree within the orchard and from one orchard to another in the late spring and
summer. The optimum temperature and relative humidity regime for conidial pro-
duction and infection was 16–20 C and 90%, respectively (Singh et al. 2010).
31.7.4 Disease Cycle
The pathogen has two distinct phases viz., the saprophytic and the parasitic
(Fig. 31.4). The saprophytic stage (primary cycle) occurs in the fallen leaves on
the orchard floor whereas the parasitic phase (secondary cycle) is observed on the
leaves and fruits on the tree. The pathogen completes its sexual cycle during the
saprophytic stage whereas the parasitic phase is caused by the conidial stage of the
pathogen. The primary cycle starts production of pseudothecia in the overwintering
leaves in the late winter. Pseudothecia are spherical to sub-spherical and vary in size
from 90 to 160 μm with a prominent ostiole (Singh and Kumar 1999). It starts
producing the ascospores during the bud break stage of the tree which continues up
to the petal fall stage. The maximum ascospore release and primary infection occur
in May.
The secondary cycle begins with the induction of the asexual stage of the fungus
at the primary infection foci. The pathogen produces copious amounts of
conidiophores and conidia sub-epidermally. The conidiophores are around 90 μm
long and 5–6 μm thick with anolivaceous brown appearance. Conidia are produced
singly at the tip of conidiophores and then successively by proliferation through
scars of the fallen conidia that result in characteristic and distinct annellations on the
conidiophores. Conidia are 0–1 septate, and 12–30 6–10 μm, obpyriform to
Asci
AscosporesPrimary scab infection Germinated ascospore
Germinated conidia
Release of asci
and ascospore Conidium
from
pseudothecium
Scab symptom on leaves
Conidia on conidiophores and fruits
Dormancy
Mature pseudothecia
Pseudothecia on previous Scab on fallen leaves

Pseudothecia development
year fallen leaves
Fig. 31.4 Disease cycle of scab pathogen

obclavate and pale to mid-olivaceous brown. The discharge of conidia is mediated

by rains and the spread to adjacent trees/orchard is mediated by the winds.
31.7.5 Modification of Mills Rules for Garhwal Himalayas
In 1942, Mills proposed a rule for predicting the infection of V. inaequalis using the
leaf wetness period and temperature. According to him, if the product of leaf wetness
duration (in hours) and the minimum temperature exceeds 140, the infection is likely
to result. This does not hold good under all the apple-growing regions due to
variations in the other host, environment and pathogen-related factors. For example,
based on the published works of literature, MacHardy and Gadoury (1989) have
reported that the infection by the ascospores of the scab pathogen requires approxi-
mately three hours less than that was reported by Mills. Similarly, Sys and Soenen
(1970), Schwabe (1979), Olivier et al. (1983), Stensvand et al. (1997), Thakur and
Khosla (1999) and Belete and Boyraz (2017) have also revalidated the Mills table for
their localities. Using the apple scab predictor and μMETOS system, Singh (2005)
have developed an apple scab warning system for the efficient use of fungicides for
the management of the scab. μMETOS is an onsite weather data collecting system
and the apple scab predictor simulated the data on temperature, relative humidity,
rainfall and leaf wetness to a modified Mills table to give the intensity of infection
period and incubation period for the appearance of scab symptoms. Once the
infection is predicted and there is availability of mature ascospores, relevance of
these infection periods as predicted by the apple scab predictor and other weather
monitoring equipment can be seen under orchard conditions to judge whether this
Mills’ Infection Period Table is valid under Indian conditions or not (Singh 2019).
Series of trials were conducted in the Gangothri valley and the results revealed
that weather conditions were very favourable for the infection to occur with maxi-
mum infection period reported in May as the weather conditions are favourable and
the tree is at the most susceptible phonological stage, i.e. bloom to near petal fall
(Fig. 31.5). The Revised Mills’ Table revealed the minimum continued wetting
period (in hours) required for primary infection of apple leaves to occur. We defined
our minimum infection time as the minimum time required for successful infection
of any quality of tissue on trees. This observation indicated that at 10 C, according
to Mills’ table, the time needed for symptom appearance was 16 days. Our result
revealed that during April and May, 5–8 mild infection periods occurring could
initiate primary infection. Minimum 9–14 days were required for symptom expres-
sion under prevailing environmental conditions (Table 31.1 and Fig. 31.6).
31.7.6 Disease Prediction Model
An apple scab prediction model developed for the Uttarakhand Himalayas by Singh
and Kumar (2005).The model provides the same information on ascospore maturity
as a predictive degree-day model, but by tracking the future development of
Fig. 31.5 Occurrence of apple scab infection periods in the Gangotri valley of Uttarakhand hills
Fig. 31.6 μMETOS system
ascospores, it allows a grower to plan and schedule fungicide applications and to

integrate fungicide applications with insecticide and miticide applications with
greater efficiency. A degree day is calculated by subtracting the base temperature
from the average minimum and maximum temperature of each day. The total of each
day is added to the total of the previous day. The degree day and the ascospore
Fig. 31.7 Cumulative percentage of matured ascospore against degree days
discharge data of 15 years were subjected to regression analysis to obtain a mean-

ingful correlation to forecast the ascospore maturation based on accumulated degree
days (Singh 2019). The significant relationship was observed between the daily
temperature and ascospore maturity. Probit transformation was further carried out to
obtain the linear relationship between ascospore maturity and daily temperature.
Based on these data, two linear lines were developed, one for the use when the
cumulative degree days from 1 February to 15 May were <618 and another for use
when the cumulative degree days for those dates were > 618. Our data showed that
50% and 95% ascospore maturation occurs after 338 and 859 cumulative degree
days for the orchards situated at 1900–2200 m asl whereas it was >1167 for those
situated above 2200 m asl (Figs. 31.7 and 31.8). The need to quantify the ascosporic
inoculum has been met by a procedure that estimates potential ascospore dose
(PAD), i.e. the expected production of ascospores per m2 orchard floor.
PAD ¼ LD LLD PD AD n
Fig. 31.8 Linear relationship 160

between per cent ascospore y = 0.124x - 1.3944
Cumulative percent matured

140 R² = 0.9433
maturation and degree days in
different altitudes of 120
Uttaranchal Himalayas 1900-2200 m.
100
ascospores
asl.
80
60 >2200 m. asl.
40 y = 0.083x - 9.5571
20 R² = 0.9689
0
-20 0 500 1000 1500
Degree days (base 0 c)
Where,
PAD Potential ascospore dose

LD Lesion density, number of lesions on leaves per square meter of orchard
floor at leaf fall
LLD Leaf litter density, the proportion of the orchard floor covered by leaf litter at
bud break
PD Pseudothecial density, number of mature pseudothecia/visible lesion
multiplied by a lesion fertility factor
AD Ascus density, number of asci/pseudothecium
n Number of ascospores/ascus
31.7.7 Scab Prediction System
Based on the findings of the study, an onsite prediction system, which provides
infection alert and disease forecast, was developed for the major apple-growing
region in Garhwal Himalayas by Singh and Kumar (1999, 2005, 2008, 2009). The
onsite weather data is automatically recorded by the μMETOS system installed at
different locations in the region of the Garhwal Himalayas. Each of these systems
also contains an inbuilt model that simulates the inoculum production and the
infection process. Based on the onsite weather parameters, cultivar susceptibility,
host phenological stages and the pathogen parameters specific to the orchard, the
disease forecast will be generated (Singh and Kumar 2009). The forecast thus
developed was passed on to the growers using All India Radio, local newspapers,
SMS alerts, etc. which helped the orchardists to accordingly plan the plant protection
measures against the apple scab.
31.7.7.1 Upscaling Disease Forecasting System at the Regional–

National Scale: a Systems Analysis
A comprehensive and detailed analysis of the production systems at the national
level, which is currently resource and data limited, is the need of time. However,
emerging issues like impacts of climate change on crop diseases and their eventual
effect on crop production could be addressed via model-based, spatially explicit
scenario analysis of the national production system. One major output would be the
identification of potential hotspots of vulnerability to climate change. Due to the fact
that a national assessment of the food production systems may require a high level of
generalization, which would not be able to account for the specificity of local or
regional production systems, a two-phase approach is required. The approach is
based on a finer resolution analysis of local/regional production systems using
cropping system-integrated disease models combined with farm-scale models to
represent locally relevant bio-physical and bio-economic drivers. Then the results
of the first-phase analysis can be integrated into national food system models taking
into account economic and physical equilibrium of demand and supply at the
national scale. A possible pathway for such a two-phase analysis is presented in
Fig. 31.9, showing possible feedback to the supply and demand module of the
nation-wide model integration chain. In a biophysical analysis of disease occurrence
at the level of landscape units, generalization from the production systems as
analyzed above, and integration of the information about the percentage of areas
used by a crop, soil typologies topography, climate and cropping intensity (popula-
tion density could also be used as a proxy for cropping intensity but only after
validation), would produce multiple sub-grid-scale simulation results per unit area.
At the regional scale, this would not be useful only to the bio-economic analysis to
explore different production systems, but would also create a key input to the
development of a modelling layer to build a link between simulation and statistics.
The production constraints due to prices can be successfully accounted for via the
linking of bio-physical disease models and economic models at the national level.
Structural constraints instead are context-specific, and must be considered in the
regional analysis outlined above.
The whole concept of crop yield forecasting and envisaged modelling capacity
would in turn combat detrimental effects of climate change as well and would be
helpful in:
• Informing the farmers and consultants with real-time information about their
crops, giving risk-assessment information and monitoring decision support rele-
vant to farm management.
• Strengthening resilience and adaptive capacity to climate-related hazards on
crops.
• Improving institutional capacity on climate change mitigation, adaptation, impact
reduction and early warning.
• Integrating climate change measures into national policies, strategies and
planning.
720
Fig. 31.9 Schematic presentation of a two-way approach of production systems analysis

K. P. Singh et al.
Addressing such issues would, therefore, ensure sustainable food production

systems and implement resilient agricultural practices that increase productivity
and production, which strengthen capacity for adaptation to climate change.
31.8 Conclusion
Thus, from the available literature sources cited above, it can be concluded that the
decision-making tools offer a viable option for taking appropriate disease manage-
ment decision. Though the present adoption rate of these technologies is limited
among both the scientific and farming community, they have got great potential to
cater to the needs of both in the near future. For this, however, the flaws in these
systems need to be rectified to increase their trust and acceptance among the
end-users. The apple scab warning service based on the onsite devices in the
Garhwal Himalayas offers an exciting example for the successful implementation
of such technologies for the benefit of the farmers. The adoption of these tools will
help to improve the decision making in IDM programmes, which will lead to better
management of the plant diseases and take us one step closer to achieving the goal of
sustainable agriculture.
References
Abu-Naser SS, Kashkash KA, Fayyad M (2008) Developing an expert system for plant disease
diagnosis. J Artif Intell 1:78–85
Agrios GN (2005) Plant pathology, 5th edn. Elsevier Academic Press, London, p 4
Balasubramani N, Lekshmi PSS (2001) Perceptions of rubber growers on information technology
enabledrubber expert system. Indian Res J Ext Edu 8:82–88
Beaumont A (1947) The dependence on the weather of the dates of outbreak of potato blight
epidemics. Trans Br Mycol Soc 31:45–53
Belete T, Boyraz N (2017) Critical review on apple scab (Venturiainaequalis) biology, epidemiol-
ogy, economic importance, management and defense mechanisms to the causal agent. J Plant
Physiol Pathol 5:2–11
Beta (2011) Progettocercospora: un concretoaiuto per avviaretrattamenti al momento giusto. Beta
News 5:2
Bhargava H, Power DJ (2001) Decision support systems and web technologies: a status report. In:
America’s Conference on Information Systems, 3–5 August 2001, Boston
Bhattacharyya SK, Phadatare SG, Khanna RN, Srivastava DS, Prasad B (1983) Efficacy of some
fungicides in controlling late blight of potato in India. Indian J Agric Sci 53:153–157
Cabrera VE (2012) DairyMGT: a suite of decision support systems in dairy farm management
Carvajal-Yepes M, Cardwell K, Nelson A, Garrett KA, Giovani B, Saunders DGO, Kamoun S,
Legg JP, Verdier V, Lessel J, Neher RA, Day R, Pardey P, Gullino ML, Records AR, Bextine B,
Leach JE, Staiger S, Tohme J (2019) A global surveillance system for crop diseases. Science
364:1237–1239
Chaudhary SD, Pal SC (1959) Forecasting late blight of potatoes in the hills of West Bengal. Am
Potato J 36:284–287
Chen Y, Hsu CY, Liu L, Yang S (2012) Constructing a nutrition diagnosis expert system. Expert
Syst Appl 39:2132–2156
De Wolf ED, Isard SA (2007) Disease cycle approach to plant disease prediction. Annu Rev
Donatelli M, Magarey RD, Bregaglio S, Willocquet L, Whish JPM, Savary S (2017) Modelling the
impacts of pests and diseases on agricultural systems. Agric Syst 155:213–224
Dube HC (2013) An introduction to fungi. Scientific Publishers, New Delhi
Durkin J (1994) Expert systems: design and development, 1st edn. Prentice Hall, Englewood cliffs.
ISBN. 0-02-330970-9
Eom S, Kim E (2006) A survey of decision support system applications (1995-2001). J Oper Res
Soc 57:1264–1278
European Commission (2009) Draft guidance document for establishing IPM principles. Supple-
ment to the Final Report 07.0307/2008/504015/ETU/B3, 49 pp.
FAOSTAT (2018) Food and Agriculture Organization of the United Nations, FAOSTAT Statistics
Database: www.fao.org/faostat/en/
Feigenbaum EA (1992) Personal view of expert systems: looking back and looking ahead,
knowledge systems laboratory, Department of Computer Science, Stanford University, Report
No. KSL 92-41, 1–20
Forrer HR, Gujer HO, Fried PM (1993) PhytoPRE - a comprehensive information and decision
support system for late blight in potatoes. In: Workshop on computer based decision support
system (DSS) in crop protection. Italy
Fry WE, Apple AE, Bruhn JA (1983) Evaluation of potato late blight forecast modified to
incorporatehost resistance and fungicide weathering. Phytopathology 73:1054–1059
Ganesan V (2006) Decision support system “Crop-9-DSS” for identified crops. In: Proceedings of
world academy of science, engineering and technology vol. 12 ISSN 1307- 6884 PWASET
Volume
Gent DH, Mahaffee WF, McRoberts N, Pfender WF (2013) The use and role of predictive systems
in disease management. Annu Rev Phytopathol 51:267–289
Gramaje D, Baumgartner K, Halleen F, Mostert I, Sosnowski MR, Urbez-Torres JR, Armengol J
(2016) Fungal trunk diseases: a problem beyond grapevines. Plant Pathol 65:355–356
Grunwald NJ, Rubio-Covarrubias OA, Fry WE (2000) Potato late blight management in Toluca
valley: forecasts and resistant cultivars. Plant Dis 84:410–416
Gutsche V (1993) PROGEB- a model aided forecasting service for pest management in cereals and
potatoes. EPPO Bull 23:577–581
Hansen JE, Andersson B, Hermansen A (1995) NEGFRY-A system for scheduling chemical
control of late blight in potatoes. In: Dowley LJ et al (eds) Phytophthora infestans 150. Boole
Press Ltd., Dublin, pp 201–208
Hansen JW (2002) Applying seasonal climate prediction to agricultural production. Agric Syst
74:305–307
Harrison JG (1992) Effects of the aerial environment on late blight of potato foliage – a review.
Jones AL, Lillevik SL, Fisher PD, Stebbins TC (1980) A microcomputer-based instrument to
predict primary apple scab infection periods. Plant Dis 64:69–72
Jorgensen LN, Noe E, Langrad AM, Jensen JE, Orum JE, Rydahl P (2006) Decision support
systems: barriers and farmer’s need for support. Paper presented at the EPPO conference on
‘computer aids for plant protection’ in Wageningen, 17–19 October, 2006
Kleinhenz B, Kristina F, Joachim K, Dietma R (2007) SIMBLIGHT1 – a new model to predict first
occurrence of potato late blight. EPPO Bull 37:339–343
Krause RA, Massie LB, Hyre RA (1975) BLITECAST: a computerized forecast of potato late
blight. Plant Dis Rep 59:95–98
Kumbhar V, Singh PT (2013) A comprehensive study of application of decision support system in
agriculture in Indian context. Int J Comp Appl 63:6–11
Mac Hardy WE (1979) A simplified, non computerized program forecasting potato late blight
(Phytophthora infestans). Plant Dis Rep 63:21–25
MacHardy WE, Gadoury DM (1989) A revision of mills criteria for predicting apple scab infection
periods. Phytopathology 79:304–310
MacKenzie DR (1981) Scheduling fungicide application for potato late blight with BLITECAST.
Plant Dis 65:394–399
Madden L, Pennypacker SP, MacNab AA (1978) FAST, a forecast system for Alternariasolanion
tomato. Phytopathology 68:1354–1358
Mahaman BD, Passam HC, Sideridis ABYialouris CP. (2003) DIARES-IPM: a diagnostic advisory
rule-based expert system for integrated pest management in Solanaceous crop systems. Agric
Syst 76:1119–1135
Manos B, Ciani A, Bournaris T, Vassiliadou I, Papathanasiou J (2004) A taxonomic survey of
decision support systems in agriculture. Agric Econ Rev 5:80–94
Marakas GM (2003) Decision support systems in the 21st century. Prentice Hall, Upper Saddle
River. ISBN: 9780130922069
March JG (1994) Primer on decision making: how decisions happen, vol 289. The Free Press,
New York
McCown RL (2002) Changing systems for supporting farmers’ decisions: problems, paradigms,
and prospects. Agric Syst 74:179–220
Mir SA, Quadri SMK (2009) Climate change, intercropping, Pest control and beneficial
microorganisms. Climate change, intercropping, Pest control and beneficial microorganisms
(January 1970). https://doi.org/10.1007/978-90-481-2716-0
Mosseddaq F, Dnidane S, Lahlou M (2005) Integrated wheat N nutrition management in morocco: a
decision support model paper presented at EFITA/WCCA, 2005, 25–28 July 2005, Vila Real,
Portugal
Odile C, David-Mathieu T, Tristan J, Anne Sophie W (2010) Disease decision support systems:
their impact on disease management and durability of fungicide effectiveness. Fungicides, (May
2014). https://doi.org/10.5772/13335
Olivier JM, Lambert C, Lefeuvre M (1983) Application du thermohumectographe KIT- INRA
etude des risqué de tavelure du pommier a l’echelle du Maine-et-Loire (France). Bull OEPP
13:47–56
Parker M, Warmund M (2011) Effect of temperature on apple trees – eXtension. Extension. http://
articles.extension.org/pages/60619/effect-of-temperature-on-apple-trees
Pham DT, Pham PTN (1988) Expert systems in mechanical and manufacturing engineering. Int J
Adv Manuf Tech 3:3–21
Rabbinge R, Rossing WAH, Van Der Werf W (1993) Systems approaches in epidemiology and
plant disease management. Neth J Plant Pathol 99:161–171
Reddy MN, Rao NH (1995) GIS Based decision support systems in agriculture. National Academy
of Agricultural Research Management, Rajendranagar, pp 1–11
Rossi V, Ponti I, Cravedi P (2000) The status of warning services for plant pests in Italy. EPPO Bull
30:19–29
Rossi V, Caffi T, Salinari F (2012) Helping farmers face the increasing complexity of decision-
making for crop protection. Phytopathol Mediterr 51:457–479
Rotem J, Cohen Y, Putler J (1971) Relativity of limiting and optimum inoculum loads, wetting
duration and temperature for infection by Phytophthora infestans. Phytopathology 61:275–278
Runno E, Koppel M (2002) Validation of potato late blight control system NEGFRY in estonian
conditions. Latvia University of Agriculture, Latvia, pp 61–64
Savary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A (2019) The global
burden of pathogens and pests on major food crops. Nat Ecol Evol 3:430–439
Schwabe WFS (1979) Changes in scab susceptibility of apple leaves as influenced by age.
Phytophylactica 11:53–56
Shafinah K, Sahari N, Sulaiman R, Yusoff MM, Ikram MM (2013) A framework of an expert
system for crop pest and disease management. J Theor Appl Inf Technol 58:182–190
Shtienberg D (2013) Will decision-support systems be widely used for the management of plant
diseases. Annu Rev Phytopathol 51:1–16
Singh KP, Kumar J (2005) IPM of apple diseases. Techanical Bulletin, GBPUAT, CFHA 6:1–48
Singh RS (2009) Plant disease, 9th edn. Oxford and IBH Publishing Co. Pvt. Ltd, New Delhi, pp
271–286
Singh KP, Kumar J (1999) Studies on ascospore maturity of venturia inaequalis, the apple scab
pathogen, in central himalayas of India. J Mycol Plant Pathol 29:408–415
Singh KP, Kumar J (2005) IPM of apple diseases. Tech Bull GBPUAT CFHA 6:1–48
Singh KP, Kumar J (2008) Disease warning system for scab of apple: a field study. GBPUAT
CFHA 22:1–18
Singh KP, Kumar J (2009) Potential ascospore dose of apple scab fungus, Venturiainaequalis, from
Indian Himalayas. Indian J Agric Sci 79:184–189
Singh BP, Islam A, Sharma VC, Shekhawat GS (2000) JHULSACAST: a computerized forecast of
potato late blight in Western Uttar Pradesh. J Indian Potato Assoc 27:25–34
Singh KP, Kumar J, Singh HB (2001) Curative and protective action of ergosterol-biosynthesis
inhibiting fungicides in relation to infection periods against apple scab in Uttaranchal
Himalayas. Indian J Plant Path 19:34–38
Singh KP, Kumar J, Kumar B (2010) GBPUAT and apple disease research in the Gangotri valley
region of India. In: Singh KP, Shahi DK (eds) Microbial diversity and plant disease manage-
ment. VDM Verlag, Saarbrücken, pp 276–301
Singh VK, Shailbala, Pundhir VS (2012) Forecasting models: an effective toos for potato late blight
management. In: Singh VK, Singh Y, Singh A (eds) Eco-friendly Innovative Approaches in
Plant Disease Management. International Book Distributors and Publisher, New Delhi, pp
102–112
Singh KP, Singh A, Singh UP (2015) Phenolic acid content of some apple cultivars with varying
degrees of resistance to apple scab. Int J Fruit Sci 15:267–280
Singh BP, Govindakrishnan PM, Islam A, Rawat S, Sharma S, Sreekumar J (2016a) INDO-
BLIGHTCAST – a model for forecasting late blight across agroecologies. Int J Pest Manag.
https://doi.org/10.1080/09670874.2016.1210839
Singh KP, Kumar J, Singh A, Prasad RK, Singh RP, Prasad D (2016b) Maturation, ascospores
discharge pattern and relevance of mills criteria for predicting apple scab infection period in
India. Plant Path J 15:108–123
Singh KP (2019) Aerobiology, epidemiology and management strategies in apple scab: science and
its applications. Indian Phytopathol 72:381–408
Sodtke RM (2005) A Multi-objective DSS for cover crop management processing fuzzy expert
knowledge. Paper presented at EFITA/WCCA, 2005, 25–28 July 2005, Vila Real, Portugal
Stensvand A, Gadoury DM, Amundsen T, Semb L, Seem RC (1997) Ascospore release and
infection of apple leaves by conidia and ascospores of Venturiaina equalis at low temperature.
Sys S, Soenen A (1970) Investigations on the infection criteria of scab on apple with respect to the
table of Mills and La Plante. Agriculture, Heverlee, Belgium 18:3–8
Thakur VS, Khosla K (1999) Relevance of Mills infection periods to apple scab (Venturiaina
equalis) prediction and rescheduling fungicide applications in Himanchal Pradesh. Indian J
Agric Sci 69:152–156
Turban E (1995) Decision support and expert systems: management support systems. In: Prentice
Hall. NJ, Englewood cliffs
Van Everdingen E (1926) Het.Verband tusschen de weergesteldhied en de aarolppelziekte,
Phytopthorainfestans (the relation between weather conditions and potato blight, Phytophthora
infestans) Tijdschr. Plan Theory 32:129–140
Wallin JR (1962) Summary of recent progress in predicting late blight epidemics in United States
and Canada. Am Potato J. 39:306–312
Yialouris C, Passam HC, Sideridis A, Metin C (1997) VEGES—A multilingual expert system for
the diagnosis of pests, diseases and nutritional disorders of six greenhouse vegetables. Comput
Electron Agric 19:55–67
Bioinformatics in Plant Pathology
32
Aamir Khan, Sakshi Singh, and Vinay Kumar Singh
Abstract
Unprecedented success and availability of enormous next-generation sequencing

data of host-pathogen in the public domain give us opportunities to understand
the disease system biologically. The availability of genome data of host-pathogen
in popular depository systems provides strong and proper help to retrieve,
annotate, analyze and identify the functional elements for characterization at
gene and genome levels for application development. The primary goal of
bioinformatics is to enhance the understanding of biological processes using
sequence pattern recognition, biological data mining, machine learning
algorithms for biological datasets and visualization of biological data and
molecules. Significant research efforts in the field include databases, software
and tools development, genome analysis, anthropology, forensic genetics,
sequence alignment, gene finding, genome assembly, drug design, drug discov-
ery, protein structure alignment, protein structure prediction, gene expression
analysis, microarray data analysis, protein–protein interactions and genome-
wide association studies. Scientists, Paulien Hogeweg and Ben Hesper coined
the term in 1970 to refer to the study of biological information processes in biotic
systems. Margaret Oakley Dayhoff, the mother and father of bioinformatics
compiled one of the first protein sequence databases. Elvin A. Kabat, the scientist
A. Khan
Division of Crop Improvement and Biotechnology, Indian Institute of Vegetable Research,
S. Singh
Department of Molecular and Human Genetics, Institute of Science, Banaras Hindu University,
V. K. Singh (*)
Centre for Bioinformatics, School of Biotechnology, Institute of Science, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
e-mail: vinaysingh@bhu.ac.in

https://doi.org/10.1007/978-981-15-6275-4_32
726 A. Khan et al.
who pioneered biological sequence analysis, developed the approach in 1970.

Bioinformatics tools, techniques and databases can be used to identify potential
genes, and target protein for host–pathogen interaction, drug designing and
discovery and harvesting biological information from the plant genomes and
their genes. Bioinformatics applications can be very beneficial in the improve-
ment of crops and helpful for the development of designer crops.
Keywords
Bioinformatics · Plant pathology · Designer crops · Plant genomics · Next-
generation sequencing
32.1 Introduction
Quality and quantity based designer crops and disease-free crops are in demand today.
For that, crop improvement and protection is the first priority, in which computational
biology approach for sequenced plant genomes plays a very important role and helps
in crop improvement by maximizing the yield, quality-based fruits and grains produc-
tion and disease resistant crops varieties (Chen and Chen 2008; King 2004; Mochida
and Shinozaki 2010; Batley and Edwards 2016; Moody 2004). Development of
sequence markers based on single nucleotide polymorphism and simple sequence
repeat identification has now become feasible method for crop improvement. Lots of
techniques, databases, tools and software have been developed to understand and
analyze the biological system fully. Here standard bioinformatics techniques with
specific tools and software are described.
32.2 Bioinformatics Techniques
32.2.1 Comparative Analysis
A comparative analysis is a field of biological sequence analysis in which the

genomic sequence features of different organisms are compared. The genomic
features may include the DNA sequence, regulatory region sequence genes and
gene order. The major principle of comparative analysis is that to identify the
common features between homologous sequences, it will often be encoded within
the DNA that is evolutionarily conserved between them or differ region which are
involved in diversity (Hardison 2003; Ong et al. 2016; Gebhardt et al. 2005; Sayers
et al. 2019) (Fig. 32.1).
32.2.2 Sequence Analysis
Sequence analysis is the process of subjecting a DNA, RNA or protein homologous

gene (orthologous and paralogous genes) sequence to understand its evolution,
32 Bioinformatics in Plant Pathology 727
Fig. 32.1 Genome availability details in the NCBI database for retrieval and comparison of
sequences
function, structure or features based on sequence alignment and searches against

biological sequence databases like reference genes, proteins, UniProtKB/swiss-prot,
protein data bank, etc. Sequence analysis includes the comparison of common region
homologous sequences in order to find similarity and dissimilarity; identification of
intrinsic features of the sequence such as active sites, post-translational modification
sites, gene-structures, reading frames and distributions of introns and exons and
regulatory elements; identification of sequence differences and variations such as
point mutations, single nucleotide variants (SNV) and single nucleotide
polymorphisms (SNPs) in order to get the genetic marker, revealing the evolution
and genetic diversity of sequences and organisms and identification of molecular
structure from sequence alone. A basic local alignment tool is the best tool for
revealing the evolutionary and genetic diversity of sequences and organisms and
identification of molecular structure from sequence (Aljanabi 2001; Bolger et al.
2018; Martinez 2013; Demuth and Hahn 2009; Lyons and Freeling 2008; Altschul
et al. 1990; McClure et al. 1994; Pirovano and Heringa 2008; Bawono et al. 2017)
(Fig. 32.2).
32.2.3 Gene Identification
Gene hunting, gene finding or gene prediction refers to the process of identifying the
regions of genomic DNA that encode genes. Gene identification is one of the first
and most important steps in understanding the gene and genome of organisms once
they are sequenced and available to the public domain. Gene finding is one of the key
steps in genome annotation, following genome sequence assembly and the filtering
of non-coding (intronic) regions and coding (exonic) regions (Alioto 2012; Wang
et al. 2004; Mochida and Shinozaki 2010) (Fig. 32.3).
728 A. Khan et al.
Fig. 32.2 Basic local alignment search tool web page for sequence similarity analysis
Fig. 32.3 Softberry server is a collection of software tools for genomic research focused on
computational methods for high throughput biomedical data analysis
32.2.4 Phylogenetic Analysis
Phylogenetic analysis is the study of the evolutionary relationships among groups of

homologous genes from organisms (e.g. species or populations). These phylogenetic
relationships are discovered based on phylogenetic inference methods (distance-
matrix methods: Neighbor-Joining (NJ), UPGMA (Unweighted Pair Group Method
with Arithmetic mean) and WPGMA (Weighted Pair Group Method with Arithmetic
mean), Fitch–Margoliash method, using outgroups, etc.; Maximum parsimony:
Branch and bound, Sankoff-Morel-Cedergren algorithm, MALIGN and POY; Max-
imum likelihood; Bayesian inference) using sequence or morphological data. A
phylogenetic tree is a branching tree diagram that represents the evolutionary
relationships among selected biological organisms or species. The phylogeny

inferences based on similarities and differences in their genetic or physical
characteristics. Phylogenetic analyses have become central to understanding
genomes, diversity, evolution and ecology (Thompson et al. 1994, 2002).
32.2.5 Protein–Protein Interaction
Protein–protein interactions (PPIs) are the physical contacts between two or more
protein molecules with high specificity based on biochemical events directed by
hydrophobic effect and electrostatic forces. In STRING database known interactions
based on curated databases or experimentally determined, predicted interactions
based on gene neighbourhood or gene fusions or gene co-occurrence and other
interactions based on textmining or co-expression or protein homology (De Las
Rivas and Fontanillo 2010; Kozakov et al. 2017; Szklarczyk et al. 2019) (Figs. 32.4
and 32.5).
32.2.6 Microarray Data Analysis
NCBI developed the Gene Expression Omnibus (GEO) database in 2000 for high-
throughput gene expression data. Microarray data analysis is used to infer informa-
tion from the data generated from DNA, RNA and protein microarray experiments;
these information allows researchers to investigate the expression level of a huge
number of genes of the entire organism genome in a single experiment. Gene
Expression Omnibus (GEO) is a public database using MIAME (Minimum Infor-
mation About a Microarray Experiment) compliant data submissions. Sequence and
Fig. 32.4 STRING is a database for functional protein association networks

730 A. Khan et al.
Fig. 32.5 ClusPro server is a web-based server for the direct docking of two interacting proteins
Fig. 32.6 Gene Expression Omnibus (GEO) is a database repository of high throughput gene
expression data and microarrays
array-based data are accepted by the repository. Techniques and tools are available
to help researchers query and download experimental datasets and gene expression
profiles. GEO has collected repository and it consists freely available microarray
data, next-generation sequencing data, and other high-throughput functional geno-
mics data submitted by the scientific community (Clough and Barrett 2016). Due to
the complexity of data which are generated by experiments are analyzed by
bioinformaticians and bio scientists with specialized softwares. GEO has developed
many tools for data query, analysis and visualization that can be analyzed directly on
the GEO server (Fig. 32.6).
32.2.7 Structure Prediction and Refinement
Protein structure prediction is the construction of the three-dimensional

(3D) structure of a protein from its amino acid sequence. In three-dimensional
structure, the 3D prediction contains folds and secondary and tertiary structures
from its primary sequence. It is highly important in drug designing and in the
designing of 3D novel enzymes (Krieger et al. 2003; Xiang 2006; França 2015;
Cavasotto and Phatak 2009; Xu et al. 2000).
32.2.8 Molecular Docking Calculation
Molecular docking is the interaction of two or more molecules to provide a stable

complex structure. Based on the binding properties of the ligand and target, it
generates a three-dimensional structure complex. Molecular docking is an approach
to predict the orientation of one molecule to second molecule in the bound structure,
which forms a stable complex. Knowledge of the active site orientation in turn may
be useful in predicting the binding strength or binding affinity between receptor-
ligand molecules using scoring functions. Molecular docking is a prominent method
for structure-based drug design, due to the prediction of the binding-conformation of
molecular ligands to the target receptor binding site. Characterization of the active
binding behaviour plays an important role in rational design of novel pesticides,
herbicides, insecticides and fungicides (Ferreira et al. 2015; Guedes et al. 2014;
Morris and Lim-Wilby 2008; Meng et al. 2011; de Ruyck et al. 2016; Pagadala et al.
2017; Zhao and Caflisch 2015; Kroemer 2007; Sousa et al. 2006; Jones and Willett
1995; Lybrand 1995; Goodsell et al. 1996; Gschwend et al. 1996; Trosset and Cavé
2019).
32.3 Bioinformatics Databases
Biological Data Model

Biological data model is a library of biological life sciences information and
biological databases; it has a collection of computational analysis tools, literature
and high-throughput experimental data. Biological database contains information
from research areas including genomics, phylogenetics, proteomics, metabolomics
microarray gene expression and phenomics. Information contained in biological
databases includes gene structure and function, macromolecular structure, cellular
and chromosomal localization and SNP and mutations in sequences and structures
(Wheeler et al. 2005; Galperin and Fernández-Suárez 2012). NCBI is a data model
that contains popular search engine Entrez. Entrez is NCBI’s retrieval system and pri-
mary text search that integrates the PubMed and PMC database of biomedical
literature with so many molecular databases including genome, gene, DNA, genetic
variation, gene expression, protein sequence and structure.
732 A. Khan et al.
32.3.1 NCBI
NCBI stands for the National Center for Biotechnology Information and is strongly
associated with the National Library of Medicine (NLM) and National Institutes of
Health (NIH), Bethesda, Maryland. The NCBI was founded in 1988 by Senator
Claude Pepper. NCBI resources contain chemicals and bioassays data, data and
software, DNA and RNA sequence data, domains and structures, genes and expres-
sion data, genetics and medicine, genomes and maps, homology data, literature,
protein sequence and structure, sequence analysis, taxonomy, training and tutorials
data and variation data (NCBI Resource Coordinators 2016; Wheeler et al. 2005)
(Figs. 32.7, 32.8, and 32.9).
Fig. 32.7 National Center for Biotechnology Information web page
Fig. 32.8 NCBI genome details page1 (The genome information can search by different
kingdoms, groups, subgroups, organism name present in the NCBI database)
Fig. 32.9 NCBI genome details page2 (The genome information of eukaryota kingdom, plants
group with their subgroups)
32.3.2 DDBJ
DDBJ (DNA Data Bank of Japan), founded in 1986, is a biological databank that
mainly contains DNA sequence information. DDBJ is located at National Institute of
Genetics (NIG), Shizuoka prefecture, Japan. It is also a member of INSDC (Interna-
tional Nucleotide Sequence Database Collaboration). The INSDC consists of a joint
effort to collect and share DNA and RNA sequence data with GenBank (USA) and
the European Nucleotide Archive (UK). DDBJ Sequence Read Archive (DRA),
NCBI Sequence Read Archive (SRA) and EBI Sequence Read Archive (ERA) share
new data and updated data on nucleotide sequences, and each of the three databases
(DDBJ, NCBI and EMBL) are synchronized on a daily basis through continuous
interaction between the staff at each of the collaborating organizations (Kodama
et al. 2012) (Fig. 32.10).
32.3.3 EMBL
European Molecular Biology Laboratory (EMBL) is a research institution supported

by 25 member states. EMBL was founded in 1974 and is a molecular biology
research organization funded by public money from its member states conducted
by approximately 85 independent groups. The web-based submission systems
include WebIn at EMBL-EBI, Sakura (“cherry blossoms”) at DDBJand BankIt at
the NCBI (Madeira et al. 2019) (Fig. 32.11).
734 A. Khan et al.
Fig. 32.10 DNA Data Bank of Japan web homepage
Fig. 32.11 European Molecular Biology Laboratory web page
32.3.4 Ensembl Plants
Ensembl Plants is an integrative database containing genome-scale information of

plants. Ensembl Plants database includes genome sequence, gene models, polymor-
phic loci and functional annotation and various tools for analysis of sequence data. It
contains various additional information, such as variation data, individual genotype
data, linkage, population structure and phenotype data (Bolser et al. 2016, 2017)
(Fig. 32.12).
Fig. 32.12 Ensembl Plants front page for genome-scale information of plant species
Fig. 32.13 PlantGDB database for the comparative plant genomics information
32.3.5 PlantGDB
PlantGDB is a resource for comparative genomics and a database of molecular

sequence data for plant genomes. PlantGDB contains assembled unique transcripts
(PUT), genome survey sequence assemblies (GSS), genome browsers and workflow
Management (Dong et al. 2004; Duvick et al. 2008) (Fig. 32.13).
736 A. Khan et al.
32.3.6 Phytozome
Phytozome is a comparative hub for plant genomes and gene family’s data and
analysis. Phytozome provides a view of genome organization, gene family, gene
structure and the evolutionary history of gene at the level of sequence. It also
provides access to the sequences and functional annotations of plant genomes and
genes (Goodstein et al. 2012) (Fig. 32.14).
32.3.7 UNIPROT
UniProt database is a freely accessible database for protein sequence and functional
annotation information, many entries being derived from different genome sequenc-
ing projects. UniProt contains a large amount of biological function of protein
information derived from the literature mining. The main aim of UniProt is to
provide a freely accessible resource, comprehensive and high-quality information
of protein sequence and functional annotation information to scientific community
(UniProt Consortium 2018) (Fig. 32.15).
32.3.8 PDB
PDB (Protein Data Bank) is a databank for the three-dimensional (3D) structural data
of a large number of biological molecules, such as nucleic acids and proteins. The
structural data is typically obtained by X-ray crystallography, NMR spectroscopy
and cryo-electron microscopy. They are submitted by structural biologists from all
around the world and are freely accessible on the net via website URLs. PDBmain
Fig. 32.14 Homepage of Phytozome database

Fig. 32.15 UniProt database
Fig. 32.16 Protein Data Bank homepage
member organizations are PDBe, PDBj, RCSB and BMRB. The PDB is overseen by
an international organization called the Worldwide Protein Data Bank, wwPDB
(Berman et al. 2000; Berman 2008; Laskowski et al. 1997) (Fig. 32.16).
32.3.9 MMDB
The Molecular Modeling Database (MMDB) is a three-dimensional biomolecular

structure database of experimentally determined macromolecules and hosted by the
National Center for Biotechnology Information (Chen et al. 2003) (Fig. 32.17).
738 A. Khan et al.
Fig. 32.17 Molecular modeling database of NCBI
Fig. 32.18 Gene Expression Omnibus database of deposited high-throughput gene expression
profiling data
32.3.10 GEO
GEO (Gene Expression Omnibus) is a gene expression database that archives and
freely distributes microarray datasets, next-generation sequencing analysis details
and other high-throughput functional genomics datasets deposited by the research
community. The main goals of GEO are to provide versatile and robust database in
which researchers can efficiently store high-throughput functional genomic data,
offer simple submission procedures and formats to the research community that
supports complete and well-annotated data deposits and provide user-friendly
mechanisms to researchers that allow users to review, query, locate and download
studies and gene expression profiles of interest for query and analysis (Clough and
Barrett 2016) (Fig. 32.18).
32.4 Bioinformatics Tools and Software
32.4.1 BiGGEsTS
BiclusterinG Gene Expression Time Series (BiGGEsTS) is a free tool and graphical
application based on bi-clustering algorithms mainly developed for analysis of gene
expression time series data (Gonçalves et al. 2009) (Fig. 32.19).
32.4.2 HCE
HCE (Hierarchical Clustering Explorer) consists of hierarchical clustering algorithm

to enable researchers to determine the grouping of data with informative dendrogram
and colour mosaic visual feedback and dynamic query controls (Seo et al. 2006)
(Fig. 32.20).
32.4.3 ClustVis
ClustVis is a web tool which allows researchers to upload their data and create Heat
maps and PCA (Principal Component Analysis) plots. Data can be uploaded as a file
or by pasting data to the text box (Metsalu and Vilo 2015) (Fig. 32.21).
Fig. 32.19 BiclusterinG Gene Expression Time Series

740 A. Khan et al.
Fig. 32.20 Hierarchical Clustering Explorer
Fig. 32.21 ClustVis web tool
32.4.4 BLAST
BLAST (Basic Local Alignment Search Tool) finds regions of similarity and
dissimilarity between sequences. The BLAST programme compares nucleotide or
protein sequences to sequence databases and calculates identity with statistical
significance (Altschul et al. 1990; Mount 2007) (Fig. 32.22).
Fig. 32.22 Basic Local Alignment Search Tool
Fig. 32.23 Clustal series homepage
32.4.5 Clustal
Clustal omega, Clustalw and Clustalx (Clustal series) are widely used programmes
for multiple sequence alignment (Higgins et al. 1996; Chenna et al. 2003; Sievers
and Higgins 2014) (Fig. 32.23).
32.4.6 Bioedit
BioEdit is a free sequence alignment editor for editing and manipulation of sequence
alignment data (Tippmann 2004) (Fig. 32.24).
742 A. Khan et al.
Fig. 32.24 BioEdit is a biological sequence alignment editor tool
Fig. 32.25 Molecular evolutionary genetic analysis
32.4.7 MEGA
MEGA is a tool for manual and automatic sequence alignment, phylogenetic tree
preparation, estimating rates of molecular evolution, web-based database mining and
testing evolutionary hypotheses (Kumar et al. 2018) (Fig. 32.25).
32.4.8 Figtree
Figtree is a graphical viewer of phylogenetic tree visualization and for producing

publication-ready figures of phylogenetic trees (Rambaut 2012) (Fig. 32.26).
32.4.9 Circos
Circos server is basically for identification and analysis of similarities and dissimi-
larity/differences generated from gene and genome comparisons (Krzywinski et al.
2009) (Fig. 32.27).
32.4.10 Prosite
PROSITE server is protein database that consists of protein families, functional

domains and functional signature sites and amino acid profiles and patterns in
sequence (Sigrist et al. 2002) (Fig. 32.28).
32.4.11 CDD
Conserved Domain Database (CDD) is a protein database that consists of well-

annotated multiple sequence alignments as position-specific score matrices (PSSMs)
for identification of conserved domains via RPS-BLAST. CDD includes NCBI-
curated functional domains based on 3D-structure information to define domain
boundaries and provide functional insights into sequence/structure/function
Fig. 32.26 FigTree server

744 A. Khan et al.
Fig. 32.27 Circos server
Fig. 32.28 PROSITE server
relationships, using Pfam, SMART, COG, PRK and TIGRFAMs databases

(Marchler-Bauer et al. 2017) (Fig. 32.29).
32.4.12 Interproscan
InterProScan is a server to annotate protein families and domains automatically.

InterPro provides functional signature analysis of proteins by classifying them into
families, domains and important sites (Mitchell et al. 2019) (Fig. 32.30).
Fig. 32.29 Conserved Domain Database
Fig. 32.30 InterProScan server
32.4.13 EasyModeller
EasyModeller is a graphical user interface programme used for homology modeling

for predicting models of protein tertiary structures (Kuntal et al. 2010) (Fig. 32.31).
32.4.14 RAMPAGE/PROCHECK
PROCHECK server checks the stereochemical quality of a protein structure model;

it produces Ramachandran plot to analyze the overall and residue-by-residue geom-
etry (Laskowski et al. 2017; Lovell et al. 2003) (Figs. 32.32 and 32.33).
746 A. Khan et al.
Fig. 32.31 EasyModeller
Fig. 32.32 RAMPAGE server
32.4.15 VERIFY3D
VERIFY3D server is used for determination of an atomic model (3D) with its amino
acid sequence, by assigning a structural class based on alpha, beta, loop, polar,
non-polar, etc. location and comparing the results to template structures (Eisenberg
et al. 1997) (Fig. 32.34).
32.4.16 YASARA
YASARA (Yet Another Scientific Artificial Reality Application) is a computer

programme for molecular vizualization, modeling and docking (Krieger and Vriend
2014) (Fig. 32.35).
Fig. 32.33 PDBSum
Fig. 32.34 SAVES server
32.4.17 BIOVIA Discovery Studio 2019
BIOVIA Discovery Studio contains BIOVIA Pipeline Pilot used for simulations,
macromolecule design and analysis, antibody modeling, structure-based design,
pharmacophore and ligand-based design, QSAR, ADMET and predictive toxicol-
ogy, X-ray and visualization (Fig. 32.36).
748 A. Khan et al.
Fig. 32.35 Yet Another Scientific Artificial Reality Application
Fig. 32.36 BIOVIA Discovery Studio
32.4.18 Patchdock
The PatchDock server performs protein–protein docking and generates protein-small

molecule complexes (Schneidman-Duhovny et al. 2005) (Fig. 32.37).
Fig. 32.37 PatchDock server
Fig. 32.38 Hex server
32.4.19 Hex
Hex tool/server is a graphics programme for docking calculation and visualizing

docking modes of pairs of protein and DNA molecules. Hex is also useful for
calculation of protein-ligand docking; it can superpose molecules (Macindoe et al.
2010) (Fig. 32.38).
750 A. Khan et al.
32.5 Plant and Pathogen Genomics
Five main types of pathogenic organisms that cause plant diseases are viruses,
bacteria, fungi, protozoa and worms/nematodes, which can lead from damage to
death. The genome availability of plants and pathogens gives us opportunities to
understand the bio systems and disease mechanisms (Tables 32.1, 32.2, and
32.3).
Table 32.1 List of important plant diseases with their causing organism, in which most of
pathogen genomes are available in the NCBI database
Disease Causing organism (pathogen)
Bacterial leaf Pseudomonas syringae subsp. syringae
blight
Aster yellows Phytoplasma
Bacterial wilt Erwinia tracheiphila
Bacterial blight Xanthomonas campestris, Xanthomonas axonopodis, Pseudomonas syringae
Crown gall Agrobacterium tumefaciens
Bacterial soft rot Erwinia, Pectobacterium and Pseudomonas
Scab Venturia inaequalis, Streptomyces scabies
Anthracnose Colletotrichum
Black knot Dibotryon morbosum or Apiosporina morbosa
Blight Cryphonectria parasitica, Cochliobolus heterostrophus, Colletotrichum
capsici
Chestnut blight Cryphonectria parasitica
Late blight Phytophthora infestans
Canker Sirococcusclavigignenti-juglandacearum, Seiridiumcardinale,
Gibberellabaccata, Diplodiaquercina, Leptosphaeria coniothyrium,
Cryptosporella umbrina, Colletotrichum coccodes
Clubroot Plasmodiophora brassicae
Damping-off Pythium
Dutch elm Claviceps purpurea
disease
Fusarium wilt Fusarium oxysporum
Panama disease
Leaf blister Taphrina caerulescens
Downy mildew Pseudoperonospora cubensis
Powdery mildew Podosphaera xanthii, Erysiphe cichoracearum
Oak wilt Ceratocystis fagacearum
Rot Oomycota
Basal rot Botrytis, Fusarium, and Penicillium
Graymold rot Botrytis cinerea
(continued)

Disease Causing organism (pathogen)
Rust Phragmidium spp.
Blister rust Cronartium ribicola
Cedar-apple rust Gymnosporangium juniperi-virginianae
Coffee rust Hemileia vastatrix
Scab Venturia inaequalis
Smut Sporisorium scitamineum
Bunt Tilletia tritici
Corn smut Ustilago maydis
Sooty mold Cladosporium and Alternaria
Verticillium wilt Verticillium
Curly top (Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus,
Grablovirus, Mastrevirus, Topocuvirus, Turncurtovirus)
Mosaic Tobacco mosaic virus
Psorosis Citrus psorosisophiovirus
Spotted wilt Tomato spotted wilt virus
Root-knot Meloidogyne
nematodes
Witchweed Pratylenchus
752 A. Khan et al.
Table 32.2 List of important plant pathogen genome details

Organism name Organism groups Size(Mb) Assemblies
Abutilon Brazil virus Viruses; Geminiviridae 0.005271 1
Abutilon golden mosaic virus Viruses; Geminiviridae 0.002629 1
Abutilon mosaic Bolivia virus Viruses; Geminiviridae 0.005399 1
Abutilon mosaic Brazil virus Viruses; Geminiviridae 0.005282 1
Abutilon mosaic virus Viruses; Geminiviridae 0.005217 1
African cassava mosaic Burkina Faso Viruses; Geminiviridae 0.00277 1
virus
African cassava mosaic virus Viruses; Geminiviridae 0.005503 1
Ageratum enation virus Viruses; Geminiviridae 0.00276 3
Ageratum leaf curl Cameroon virus Viruses; Geminiviridae 0.002792 1
Ageratum leaf curl virus – [G52] Viruses; Geminiviridae 0.002735 1
Ageratum yellow vein China virus – OX1 Viruses; Geminiviridae 0.002739 1
Ageratum yellow vein Hualian virus Viruses; Geminiviridae 0.002756 2
Ageratum yellow vein Sri Lanka virus Viruses; Geminiviridae 0.002748 1
Ageratum yellow vein virus Viruses; Geminiviridae 0.002768 12
Agrobacterium tumefaciens Bacteria; 7.2733 69
Proteobacteria;
Alphaproteobacteria
Alfalfa leaf curl virus Viruses; Geminiviridae 0.002745 1
Allamanda leaf curl virus Viruses; Geminiviridae 0.002755 1
Allamanda leaf mottle distortion virus Viruses; Geminiviridae 0.005462 1
Alternanthera yellow vein virus Viruses; Geminiviridae 0.002745 3
Alternaria alternata Eukaryota; Fungi; 32.9908 6
Ascomycetes
Alternaria arborescens Eukaryota; Fungi; 33.9434 5
Ascomycetes
Alternaria atra Eukaryota; Fungi; 35.1121 1
Ascomycetes
Alternaria brassicae Eukaryota; Fungi; 34.1411 1
Ascomycetes
Alternaria brassicicola Eukaryota; Fungi; 31.0365 2
Ascomycetes
Alternaria consortialis Eukaryota; Fungi; 34.2409 1
Ascomycetes
Alternaria gaisen Eukaryota; Fungi; 34.3469 1
Ascomycetes
Alternaria gansuensis Eukaryota; Fungi; 75.0519 1
Ascomycetes
Alternaria solani Eukaryota; Fungi; 32.7791 2
Ascomycetes
Alternaria sp. MG1 Eukaryota; Fungi; 34.6956 1
Ascomycetes
Alternaria tenuissima Eukaryota; Fungi; 35.7042 7
Ascomycetes
(continued)

Andrographis yellow vein leaf curl virus Viruses; Geminiviridae 0.002754 1
Aster yellow witches’-broom Bacteria; 0.72397 1
phytoplasma Terrabacteriagroup;
Tenericutes
Asystasia mosaic Madagascar virus Viruses; Geminiviridae 0.005404 1
Axonopus compressus streak virus Viruses; Geminiviridae 0.002858 1
Bean calico mosaic virus Viruses; Geminiviridae 0.005175 1
Bean chlorosis virus Viruses; Geminiviridae 0.005279 1
Bean dwarf mosaic virus Viruses; Geminiviridae 0.005191 1
Bean golden mosaic virus Viruses; Geminiviridae 0.005197 2
Bean golden yellow mosaic virus Viruses; Geminiviridae 0.005255 2
Bean leaf crumple virus Viruses; Geminiviridae 0.002598 1
Bean leaf curl Madagascar virus Viruses; Geminiviridae 0.002754 1
Bean white chlorosis mosaic virus Viruses; Geminiviridae 0.005163 1
Bean yellow dwarf virus Viruses; Geminiviridae 0.002561 1
Bean yellow dwarf virus Viruses; Geminiviridae 0.002561 1
Bean yellow mosaic Mexico virus Viruses; Geminiviridae 0.002641 1
Beet curly top Iran virus Viruses; Geminiviridae 0.002859 4
Beet curly top virus Viruses; Geminiviridae 0.002994 8
Bhendi yellow vein Bhubhaneswar virus Viruses; Geminiviridae 0.002757 1
Bhendi yellow vein Delhi virus [2004: Viruses; Geminiviridae 0.002751 1
New Delhi]
Bhendi yellow vein Haryana virus Viruses; Geminiviridae 0.00274 1
Bhendi yellow vein India virus [India: Viruses; Geminiviridae 0.002739 1
Dharwad OYDWR2:2006]
Bhendi yellow vein mosaic virus Viruses; Geminiviridae 0.002747 6
Bipolaris maydis Eukaryota; Fungi; 32.9292 2
Ascomycetes
Bitter gourd yellow vein virus Viruses; Geminiviridae 0.005453 1
Blainvillea yellow spot virus Viruses; Geminiviridae 0.0053 1
Blechum interveinal chlorosis virus Viruses; Geminiviridae 0.005285 1
Boerhavia yellow spot virus Viruses; Geminiviridae 0.002621 1
Botrytis cinerea Eukaryota; Fungi; 42.6301 4
Ascomycetes
‘Brassica napus’ phytoplasma Bacteria; Terrabacteria 0.743598 1
group; Tenericutes
Bretziellafagacearum Eukaryota; Fungi; 26.782 1
Ascomycetes
Bromuscatharticus striate mosaic virus Viruses; Geminiviridae 0.002797 1
Cabbage leaf curl Jamaica virus Viruses; Geminiviridae 0.005069 1
Cabbage leaf curl virus Viruses; Geminiviridae 0.005096 1
Candidatus Phytoplasma Bacteria; Terrabacteria 0.687137 1
group; Tenericutes
(continued)
754 A. Khan et al.

Candidatus Phytoplasma aurantifolia Bacteria; Terrabacteria 0.474669 1
group; Tenericutes
Candidatus Phytoplasma australiense Bacteria; Terrabacteria 0.959779 2
group; Tenericutes
Candidatus Phytoplasma mali Bacteria; Terrabacteria 0.601943 1
group; Tenericutes
Candidatus Phytoplasma oryzae Bacteria; Terrabacteria 0.533195 2
group; Tenericutes
Candidatus Phytoplasma phoenicium Bacteria; Terrabacteria 0.541091 2
group; Tenericutes
Candidatus Phytoplasma pini Bacteria; Terrabacteria 0.474136 1
group; Tenericutes
Candidatus Phytoplasma pruni Bacteria; Terrabacteria 0.598511 1
group; Tenericutes
Candidatus Phytoplasma solani Bacteria; Terrabacteria 0.821322 3
group; Tenericutes
Candidatus Phytoplasma ziziphi Bacteria; Terrabacteria 0.750803 1
group; Tenericutes
Capraria yellow spot Yucatan virus Viruses; Geminiviridae 0.005208 1
Cassava mosaic Madagascar virus Viruses; Geminiviridae 0.00551 1
‘Catharanthus roseus’ aster yellows Bacteria; Terrabacteria 0.603949 1
phytoplasma group; Tenericutes
Catharanthus yellow mosaic virus Viruses; Geminiviridae 0.002752 1
Centrosema yellow spot virus Viruses; Geminiviridae 0.002675 1
Chayote yellow mosaic virus Viruses; Geminiviridae 0.002787 1
Chenopodium leaf curl virus Viruses; Geminiviridae 0.002626 1
Chickpea chlorosis Australia virus Viruses; Geminiviridae 0.002572 1
Chickpea chlorosis virus Viruses; Geminiviridae 0.002603 3
Chickpea chlorosis virus-A Viruses; Geminiviridae 0.002582 1
Chickpea chlorotic dwarf virus Viruses; Geminiviridae 0.002587 5
Chickpea redleaf virus Viruses; Geminiviridae 0.002605 1
Chickpea yellow dwarf virus Viruses; Geminiviridae 0.002547 1
Chickpea yellows virus Viruses; Geminiviridae 0.002557 1
Chilli leaf curl Ahmedabad virus-India Viruses; Geminiviridae 0.002744 1
[India/Ahmedabad/2014]
Chilli leaf curl India virus Viruses; Geminiviridae 0.002755 1
Chilli leaf curl Kanpur virus Viruses; Geminiviridae 0.002754 1
Chilli leaf curl Vellanad virus Viruses; Geminiviridae 0.002788 1
Chilli leaf curl virus Viruses; Geminiviridae 0.002858 7
Chino del tomate Amazonas virus Viruses; Geminiviridae 0.002615 1
Chino del tomate virus Viruses; Geminiviridae 0.005213 3
Chloris striate mosaic virus Viruses; Geminiviridae 0.00275 1
‘Chrysanthemum coronarium’ Bacteria; Terrabacteria 0.739592 1
(continued)

Chrysanthemum yellows phytoplasma Bacteria; Terrabacteria 0.659699 1
group; Tenericutes
Citrus psorosis virus Viruses; Aspiviridae 0.011278 1
Cladosporium cladosporioides Eukaryota; Fungi; 33.2257 1
Ascomycetes
Cladosporium phlei Eukaryota; Fungi; 32.816 1
Ascomycetes
Cladosporium sp. SL-16 Eukaryota; Fungi; 35.8569 1
Ascomycetes
Cladosporium sphaerospermum Eukaryota; Fungi; 26.8942 1
Ascomycetes
Claviceps purpurea Eukaryota; Fungi; 32.0914 3
Ascomycetes
Cleome golden mosaic virus Viruses; Geminiviridae 0.002566 1
Cleome leaf crumple virus Viruses; Geminiviridae 0.005386 1
Clerodendron golden mosaic virus Viruses; Geminiviridae 0.005524 1
Clerodendron yellow mosaic virus Viruses; Geminiviridae 0.00276 1
Clerodendrum golden mosaic China Viruses; Geminiviridae 0.005515 2
virus
Clerodendrum golden mosaic Jiangsu Viruses; Geminiviridae 0.002753 1
virus
Cnidoscolus mosaic leaf deformation Viruses; Geminiviridae 0.005254 1
virus
Coccinia mosaic Tamil Nadu virus Viruses; Geminiviridae 0.00544 1
Colletotrichum acutatum Eukaryota; Fungi; 52.1291 2
Ascomycetes
Colletotrichum chlorophyti Eukaryota; Fungi; 52.387 1
Ascomycetes
Colletotrichum coccodes Eukaryota; Fungi; 50.122 2
Ascomycetes
Colletotrichum falcatum Eukaryota; Fungi; 48.1864 1
Ascomycetes
Colletotrichum fioriniae Eukaryota; Fungi; 50.1509 3
Ascomycetes
Colletotrichum fructicola Eukaryota; Fungi; 55.9157 3
Ascomycetes
Colletotrichum gloeosporioides Eukaryota; Fungi; 61.9165 6
Ascomycetes
Colletotrichum godetiae Eukaryota; Fungi; 35.0343 1
Ascomycetes
Colletotrichum graminicola Eukaryota; Fungi; 51.6443 2
Ascomycetes
Colletotrichum higginsianum Eukaryota; Fungi; 50.7161 3
Ascomycetes
Colletotrichum incanum Eukaryota; Fungi; 53.2546 2
Ascomycetes
(continued)
756 A. Khan et al.

Colletotrichum lentis Eukaryota; Fungi; 56.1001 1
Ascomycetes
Colletotrichum lindemuthianum Eukaryota; Fungi; 99.1667 2
Ascomycetes
Colletotrichum musae Eukaryota; Fungi; 49.1188 1
Ascomycetes
Colletotrichum nymphaeae Eukaryota; Fungi; 49.9563 1
Ascomycetes
Colletotrichum orbiculare Eukaryota; Fungi; 89.7483 1
Ascomycetes
Colletotrichum orchidophilum Eukaryota; Fungi; 48.5565 1
Ascomycetes
Colletotrichum salicis Eukaryota; Fungi; 48.3734 1
Ascomycetes
Colletotrichum sansevieriae Eukaryota; Fungi; 51.2013 1
Ascomycetes
Colletotrichum shisoi Eukaryota; Fungi; 69.6677 1
Ascomycetes
Colletotrichum siamense Eukaryota; Fungi; 55.9616 1
Ascomycetes
Colletotrichum sidae Eukaryota; Fungi; 86.8278 1
Ascomycetes
Colletotrichum simmondsii Eukaryota; Fungi; 50.4742 1
Ascomycetes
Colletotrichum sp. JS-367 Eukaryota; Fungi; 87.1965 1
Ascomycetes
Colletotrichum spinosum Eukaryota; Fungi; 82.7349 1
Ascomycetes
Colletotrichum sublineola Eukaryota; Fungi; 64.8486 2
Ascomycetes
Colletotrichum tanaceti Eukaryota; Fungi; 57.9125 1
Ascomycetes
Colletotrichum tofieldiae Eukaryota; Fungi; 52.7196 5
Ascomycetes
Colletotrichum trifolii Eukaryota; Fungi; 109.66 1
Ascomycetes
Colletotrichum truncatum Eukaryota; Fungi; 57.9128 2
Ascomycetes
Common bean mottle virus Viruses; Geminiviridae 0.005235 1
Common bean severe mosaic virus Viruses; Geminiviridae 0.00519 2
Corchorus golden mosaic virus Viruses; Geminiviridae 0.005352 2
Corchorus yellow spot virus Viruses; Geminiviridae 0.005195 1
Corchorus yellow vein mosaic virus Viruses; Geminiviridae 0.002743 1
Corchorus yellow vein virus – Viruses; Geminiviridae 0.005415 1
[HoaBinh]
Cotton chlorotic spot virus Viruses; Geminiviridae 0.00532 1
(continued)

Cotton leaf crumple virus Viruses; Geminiviridae 0.00518 2
Cotton leaf curl Alabad virus Viruses; Geminiviridae 0.002744 4
Cotton leaf curl Allahabad virus [India: Viruses; Geminiviridae 0.002744 1
Karnal:OY77:2005]
Cotton leaf curl Bangalore virus Viruses; Geminiviridae 0.002751 1
Cotton leaf curl Gezira virus Viruses; Geminiviridae 0.00278 9
Cotton leaf curl Kokhran virus Viruses; Geminiviridae 0.002759 3
Cotton leaf curl Multan virus Viruses; Geminiviridae 0.002754 5
Cotton leaf curl Shahdadpur virus Viruses; Geminiviridae 0.002748 1
Cotton leaf curl virus Viruses; Geminiviridae 0.002753 1
Cotton yellow mosaic virus Viruses; Geminiviridae 0.005482 2
Cowpea golden mosaic virus Viruses; Geminiviridae 0.002728 1
Crassocephalum yellow vein virus – Viruses; Geminiviridae 0.002745 1
Jinghong
Cronartiumribicola Eukaryota; Fungi; 94.3329 1
Basidiomycetes
Croton yellow vein mosaic virus Viruses; Geminiviridae 0.002757 2
Croton yellow vein virus Viruses; Geminiviridae 0.002744 1
Cucurbit leaf crumple virus Viruses; Geminiviridae 0.005232 1
‘Cynodondactylon’ phytoplasma Bacteria; Terrabacteria 0.483935 1
group; Tenericutes
Dalechampia chlorotic mosaic virus Viruses; Geminiviridae 0.005214 1
Datura leaf curl virus Viruses; Geminiviridae 0.002782 1
Datura leaf distortion virus Viruses; Geminiviridae 0.005163 1
Deinbollia mosaic virus Viruses; Geminiviridae 0.005461 1
Desmodium leaf distortion virus Viruses; Geminiviridae 0.005083 1
Desmodium mottle virus Viruses; Geminiviridae 0.00548 1
Dicliptera yellow mottle virus Viruses; Geminiviridae 0.005204 2
Digitariaciliaris striate mosaic virus Viruses; Geminiviridae 0.002816 2
Digitariadidactyla striate mosaic virus Viruses; Geminiviridae 0.002762 1
Digitaria streak virus Viruses; Geminiviridae 0.002701 1
Digitaria streak virus Viruses; Geminiviridae 0.002701 1
Dolichos yellow mosaic virus Viruses; Geminiviridae 0.005494 2
Dragonfly-associated mastrevirus Viruses; Geminiviridae 0.00265 2
Duranta leaf curl virus Viruses; Geminiviridae 0.002759 1
East African cassava mosaic Cameroon Viruses; Geminiviridae 0.005543 1
virus
East African cassava mosaic Kenya Viruses; Geminiviridae 0.005573 1
virus
East African cassava mosaic Malawi Viruses; Geminiviridae 0.005558 2
virus
East African cassava mosaic virus Viruses; Geminiviridae 0.005576 4
East African cassava mosaic Zanzibar Viruses; Geminiviridae 0.005548 1
virus
(continued)
758 A. Khan et al.

‘Echinacea purpurea’ witches’-broom Bacteria; Terrabacteria 0.545427 1
Eclipta yellow vein virus Viruses; Geminiviridae 0.002748 2
Emilia sonchifolia yellow vein Thailand Viruses; Geminiviridae 0.002746 1
virus
Emilia yellow vein virus-[Fz1] Viruses; Geminiviridae 0.002725 1
Eragrostiscurvula streak virus Viruses; Geminiviridae 0.002754 2
Eragrostis minor streak virus Viruses; Geminiviridae 0.002689 1
Eragrostis streak virus Viruses; Geminiviridae 0.002746 1
Erectites yellow mosaic virus Viruses; Geminiviridae 0.002751 1
Erwinia tracheiphila Bacteria; 4.71727 3
Proteobacteria;
Gammaproteobacteria
Eupatorium yellow vein mosaic virus Viruses; Geminiviridae 0.002778 1
Eupatorium yellow vein virus Viruses; Geminiviridae 0.002767 5
Euphorbia caput-medusae latent virus Viruses; Geminiviridae 0.002683 2
Euphorbia leaf curl Guangxi virus Viruses; Geminiviridae 0.002747 1
Euphorbia leaf curl virus Viruses; Geminiviridae 0.002746 1
Euphorbia mosaic Peru virus Viruses; Geminiviridae 0.0026 1
Euphorbia mosaic virus Viruses; Geminiviridae 0.005215 1
Euphorbia yellow leaf curl virus Viruses; Geminiviridae 0.002731 1
Euphorbia yellow mosaic virus Viruses; Geminiviridae 0.005187 2
Exomismicrophylla associated virus Viruses; Geminiviridae 0.002974 1
French bean leaf curl virus Viruses; Geminiviridae 0.002741 1
French bean severe leaf curl virus Viruses; Geminiviridae 0.002771 1
Fusarium oxysporum Eukaryota; Fungi; 61.3869 129
Ascomycetes
Golovinomyces cichoracearum Eukaryota; Fungi; 65.8869 3
Ascomycetes
Gossypium darwinii symptomless virus Viruses; Geminiviridae 0.00274 1
Gossypium punctatum mild leaf curl Viruses; Geminiviridae 0.005462 1
virus
Grapevine red blotch virus Viruses; Geminiviridae 0.003206 2
Hedyotisuncinella yellow mosaic virus Viruses; Geminiviridae 0.002749 1
Hemidesmus yellow mosaic virus Viruses; Geminiviridae 0.002825 1
Hemileiavastatrix Eukaryota; Fungi; 543.605 2
Basidiomycetes
Hollyhock leaf crumple virus Viruses; Geminiviridae 0.002755 1
Hollyhock leaf curl virus Viruses; Geminiviridae 0.002748 1
Hollyhock yellow vein mosaic Islamabad Viruses; Geminiviridae 0.002741 2
virus
Hollyhock yellow vein mosaic virus Viruses; Geminiviridae 0.00275 2
Honeysuckle yellow vein Kagoshima Viruses; Geminiviridae 0.002762 1
virus
(continued)

Honeysuckle yellow vein mosaic virus Viruses; Geminiviridae 0.002759 2
Honeysuckle yellow vein virus Viruses; Geminiviridae 0.002784 15
Horsegram yellow mosaic virus Viruses; Geminiviridae 0.005405 1
Horseradish curly top virus Viruses; Geminiviridae 0.00308 1
Indian cassava mosaic virus Viruses; Geminiviridae 0.00546 4
Ipomoea yellow vein virus Viruses; Geminiviridae 0.002791 1
Italian clover phyllody phytoplasma Bacteria; Terrabacteria 0.597245 1
group; Tenericutes
Jacquemontia mosaic Yucatan virus Viruses; Geminiviridae 0.005193 1
Jacquemontia yellow mosaic virus Viruses; Geminiviridae 0.005189 1
Jacquemontia yellow vein virus Viruses; Geminiviridae 0.002585 1
Jatropha leaf crumple virus Viruses; Geminiviridae 0.002735 1
Jatropha leaf curl Gujarat virus Viruses; Geminiviridae 0.002758 1
Jatropha leaf curl virus Viruses; Geminiviridae 0.002844 2
Jatropha leaf yellow mosaic Viruses; Geminiviridae 0.002744 1
Katarniaghat virus
Jatropha mosaic India virus Viruses; Geminiviridae 0.00274 1
Jatropha mosaic Nigeria virus Viruses; Geminiviridae 0.002781 1
Jatropha mosaic virus Viruses; Geminiviridae 0.005198 1
Jatropha yellow mosaic virus Viruses; Geminiviridae 0.002757 1
Kenaf leaf curl virus-[India: Viruses; Geminiviridae 0.00274 1
Bahraich:2007]
Kudzu mosaic virus Viruses; Geminiviridae 0.005403 1
Leonurus mosaic virus Viruses; Geminiviridae 0.002652 1
Linderniaanagallis yellow vein virus Viruses; Geminiviridae 0.00274 1
Lisianthus enation leaf curl virus Viruses; Geminiviridae 0.002759 1
Ludwigia yellow vein Vietnam virus Viruses; Geminiviridae 0.002751 1
Ludwigia yellow vein virus Viruses; Geminiviridae 0.002758 1
Luffa yellow mosaic virus Viruses; Geminiviridae 0.005455 1
Lycianthes yellow mosaic virus Viruses; Geminiviridae 0.005456 1
Macroptilium bright mosaic virus Viruses; Geminiviridae 0.002636 1
Macroptilium common mosaic virus Viruses; Geminiviridae 0.00523 1
Macroptilium golden mosaic virus Viruses; Geminiviridae 0.005158 1
Macroptilium golden yellow mosaic Viruses; Geminiviridae 0.005224 1
virus
Macroptilium mosaic Puerto Rico virus Viruses; Geminiviridae 0.005186 1
Macroptilium yellow mosaic Florida Viruses; Geminiviridae 0.005247 1
virus
Macroptilium yellow mosaic virus Viruses; Geminiviridae 0.005223 2
Macroptilium yellow net virus Viruses; Geminiviridae 0.005197 1
Macroptilium yellow spot virus Viruses; Geminiviridae 0.00266 1
Macroptilium yellow vein virus Viruses; Geminiviridae 0.002656 1
Maize bushy stunt phytoplasma Bacteria; Terrabacteria 0.576118 1
group; Tenericutes
(continued)
760 A. Khan et al.

Maize streak Reunion virus Viruses; Geminiviridae 0.002882 1
Maize streak Reunion virus Viruses; Geminiviridae 0.002882 1
Maize streak virus Viruses; Geminiviridae 0.002701 12
Maize striate mosaic virus Viruses; Geminiviridae 0.002746 2
Malachra yellow mosaic virus Viruses; Geminiviridae 0.002739 1
Malvastrum bright yellow mosaic virus Viruses; Geminiviridae 0.005213 1
Malvastrum leaf curl Guangdong virus Viruses; Geminiviridae 0.002767 1
Malvastrum leaf curl Philippines virus Viruses; Geminiviridae 0.002742 1
Malvastrum leaf curl virus Viruses; Geminiviridae 0.002745 1
Malvastrum yellow mosaic Helshire Viruses; Geminiviridae 0.002609 1
virus
Malvastrum yellow mosaic Jamaica Viruses; Geminiviridae 0.005192 1
virus
Malvastrum yellow mosaic virus Viruses; Geminiviridae 0.002728 1
Malvastrum yellow vein Baoshan virus Viruses; Geminiviridae 0.002745 2
Malvastrum yellow vein Cambodia virus Viruses; Geminiviridae 0.002737 1
Malvastrum yellow vein Changa Manga Viruses; Geminiviridae 0.002754 1
virus
Malvastrum yellow vein Honghe virus Viruses; Geminiviridae 0.00274 1
Malvastrum yellow vein virus Viruses; Geminiviridae 0.002731 1
Malvastrum yellow vein Yunnan virus Viruses; Geminiviridae 0.002747 1
Melochia mosaic virus Viruses; Geminiviridae 0.005213 1
Melochia yellow mosaic virus Viruses; Geminiviridae 0.005288 1
Meloidogyne arenaria Eukaryota; Animals; 284.032 3
Roundworms
Meloidogyne enterolobii Eukaryota; Animals; 162.967 1
Roundworms
Meloidogyne floridensis Eukaryota; Animals; 74.846 2
Roundworms
Meloidogyne graminicola Eukaryota; Animals; 38.185 1
Roundworms
Meloidogyne hapla Eukaryota; Animals; 53.013 1
Roundworms
Meloidogyne incognita Eukaryota; Animals; 183.532 3
Roundworms
Meloidogyne javanica Eukaryota; Animals; 150.345 2
Roundworms
Melon chlorotic leaf curl virus Viruses; Geminiviridae 0.005325 3
Merremia mosaic Puerto Rico virus Viruses; Geminiviridae 0.005225 1
Merremia mosaic virus Viruses; Geminiviridae 0.005085 2
Mesta yellow vein mosaic Bahraich virus Viruses; Geminiviridae 0.002737 1
Mesta yellow vein mosaic virus Viruses; Geminiviridae 0.002752 2
Milkweed yellows phytoplasma Bacteria; Terrabacteria 0.583806 1
group; Tenericutes
(continued)

Mimosa yellow leaf curl virus Viruses; Geminiviridae 0.002757 1
Mirabilis leaf curl virus Viruses; Geminiviridae 0.002778 1
Miscanthus streak virus Viruses; Geminiviridae 0.002672 1
Miscanthus streak virus Viruses; Geminiviridae 0.002672 1
Mungbean yellow mosaic India virus Viruses; Geminiviridae 0.005361 1
Mungbean yellow mosaic virus Viruses; Geminiviridae 0.005398 1
New Jersey aster yellows phytoplasma Bacteria; Terrabacteria 0.652092 1
group; Tenericutes
Oat dwarf virus Viruses; Geminiviridae 0.00274 1
Oat dwarf virus Viruses; Geminiviridae 0.00274 1
Okra enation leaf curl virus Viruses; Geminiviridae 0.002738 1
Okra enation leaf curl virus [India: Viruses; Geminiviridae 0.002724 1
Munthal EL37:2006]
Okra leaf curl Cameroon virus Viruses; Geminiviridae 0.002764 1
Okra leaf curl India virus [India:Sonipat Viruses; Geminiviridae 0.002723 1
EL14A:2006]
Okra leaf curl Oman virus Viruses; Geminiviridae 0.002788 1
Okra leaf curl virus Viruses; Geminiviridae 0.002386 1
Okra mottle virus Viruses; Geminiviridae 0.005313 1
Okra yellow crinkle virus Viruses; Geminiviridae 0.002795 3
Okra yellow mosaic Mexico virus Viruses; Geminiviridae 0.005194 1
Onion yellows phytoplasma Bacteria; Terrabacteria 0.853092 1
group; Tenericutes
Ophiognomoniaclavigignenti- Eukaryota; Fungi; 52.5149 3
juglandacearum Ascomycetes
Oxalis yellow vein virus Viruses; Geminiviridae 0.002661 1
Panicum streak virus Viruses; Geminiviridae 0.002736 9
Papaya leaf crumple virus-Panipat Viruses; Geminiviridae 0.002736 1
8 [India:Panipat:Papaya:2008]
Papaya leaf curl China virus Viruses; Geminiviridae 0.002751 5
Papaya leaf curl Guandong virus Viruses; Geminiviridae 0.002764 2
Papaya leaf curl virus Viruses; Geminiviridae 0.002769 12
Paspalum dilatatum striate mosaic virus Viruses; Geminiviridae 0.002806 1
Paspalum striate mosaic virus Viruses; Geminiviridae 0.002816 2
Passionfruit leaf distortion virus Viruses; Geminiviridae 0.005172 1
Passionfruit severe leaf distortion virus Viruses; Geminiviridae 0.005316 1
Pavonia mosaic virus Viruses; Geminiviridae 0.005367 1
Pavonia yellow mosaic virus Viruses; Geminiviridae 0.005378 1
Pea leaf distortion virus Viruses; Geminiviridae 0.002738 1
Peanut witches’-broom phytoplasma Bacteria; Terrabacteria 0.566694 1
group; Tenericutes
Pectobacterium actinidiae Bacteria; 4.92217 3
Proteobacteria;
Gammaproteobacteria
(continued)
762 A. Khan et al.

Pectobacterium aquaticum Bacteria; 4.46724 6
Proteobacteria;
Gammaproteobacteria
Pectobacterium atrosepticum Bacteria; 5.10459 11
Proteobacteria;
Gammaproteobacteria
Pectobacterium betavasculorum Bacteria; 4.68521 2
Proteobacteria;
Gammaproteobacteria
Pectobacterium brasiliense Bacteria; 5.02631 28
Proteobacteria;
Gammaproteobacteria
Pectobacterium carotovorum Bacteria; 4.86291 33
Proteobacteria;
Gammaproteobacteria
Pectobacterium fontis Bacteria; 4.15156 1
Proteobacteria;
Gammaproteobacteria
Pectobacterium odoriferum Bacteria; 5.4726 15
Proteobacteria;
Gammaproteobacteria
Pectobacterium parmentieri Bacteria; 5.2273 19
Proteobacteria;
Gammaproteobacteria
Pectobacterium peruviense Bacteria; 4.87102 5
Proteobacteria;
Gammaproteobacteria
Pectobacterium polaris Bacteria; 5.00842 7
Proteobacteria;
Gammaproteobacteria
Pectobacterium polonicum Bacteria; 4.83613 1
Proteobacteria;
Gammaproteobacteria
Pectobacterium punjabense Bacteria; 4.73253 1
Proteobacteria;
Gammaproteobacteria
Pectobacterium versatile Bacteria; 4.94937 1
Proteobacteria;
Gammaproteobacteria
Pectobacterium wasabiae Bacteria; 5.1493 4
Proteobacteria;
Gammaproteobacteria
Pectobacterium zantedeschiae Bacteria; 5.094 3
Proteobacteria;
Gammaproteobacteria
Pedilanthus leaf curl virus Viruses; Geminiviridae 0.002764 3
Pepper golden mosaic virus Viruses; Geminiviridae 0.005208 3
(continued)

Pepper huasteco yellow vein virus Viruses; Geminiviridae 0.00522 1
Pepper leaf curl Bangladesh virus Viruses; Geminiviridae 0.002754 3
Pepper leaf curl Lahore virus Viruses; Geminiviridae 0.00274 1
Pepper leaf curl Lahore Virus- Viruses; Geminiviridae 0.002747 1
[Pakistan:Lahore1:2004]
Pepper leaf curl virus Viruses; Geminiviridae 0.00276 3
Pepper leaf curl Yunnan virus-[YN323] Viruses; Geminiviridae 0.002747 1
Pepper leafroll virus Viruses; Geminiviridae 0.002568 1
Pepper yellow dwarf virus – Mexico Viruses; Geminiviridae 0.002971 1
Pepper yellow dwarf virus – New Mexico Viruses; Geminiviridae 0.002959 1
Pepper yellow leaf curl Indonesia virus Viruses; Geminiviridae 0.005476 1
Pepper yellow leaf curl Thailand virus Viruses; Geminiviridae 0.005474 2
Pepper yellow leaf curl virus Viruses; Geminiviridae 0.006028 2
Pepper yellow leaf curl virus PSSWS-14 Viruses; Geminiviridae 0.002748 1
Pepper yellow vein Mali virus Viruses; Geminiviridae 0.002786 1
Periwinkle leaf yellowing phytoplasma Bacteria; Terrabacteria 0.824596 1
group; Tenericutes
Phytophthora infestans Eukaryota; Protists; 228.544 2
Other Protists
Plantago lanceolata latent virus Viruses; Geminiviridae 0.002832 1
Plasmodiophora brassicae Eukaryota; Protists; 24.5596 7
Other Protists
Poinsettia branch-inducing phytoplasma Bacteria; Terrabacteria 0.63144 1
group; Tenericutes
Potato yellow mosaic Panama virus Viruses; Geminiviridae 0.005126 1
Potato yellow mosaic virus Viruses; Geminiviridae 0.00514 4
Pouzolzia golden mosaic virus Viruses; Geminiviridae 0.002725 2
Pouzolzia mosaic Guangdong virus Viruses; Geminiviridae 0.002739 1
Premna leaf curl virus Viruses; Geminiviridae 0.002753 1
Prunus latent virus Viruses; Geminiviridae 0.003174 1
Pseudomonas syringae Bacteria; 6.0937 376
Proteobacteria;
Gammaproteobacteria
Pseudoperonospora cubensis Eukaryota; Protists; 64.3328 1
Other Protists
Pumpkin yellow mosaic Malaysia virus Viruses; Geminiviridae 0.002724 1
Pythium aphanidermatum Eukaryota; Protists; 35.8768 1
Other Protists
Pythium arrhenomanes Eukaryota; Protists; 44.6726 1
Other Protists
Pythium brassicum Eukaryota; Protists; 50.0694 1
Other Protists
Pythium guiyangense Eukaryota; Protists; 110.178 1
Other Protists
(continued)
764 A. Khan et al.

Pythium insidiosum Eukaryota; Protists; 53.239 12
Other Protists
Pythium irregulare Eukaryota; Protists; 42.9681 2
Other Protists
Pythium iwayamai Eukaryota; Protists; 43.1992 1
Other Protists
Pythium oligandrum Eukaryota; Protists; 41.9689 3
Other Protists
Pythium periplocum Eukaryota; Protists; 35.8865 1
Other Protists
Pythium splendens Eukaryota; Protists; 53.361 1
Other Protists
Radish leaf curl virus Viruses; Geminiviridae 0.002759 2
Ramie mosaic virus Viruses; Geminiviridae 0.005446 1
Ramie mosaic Yunnan virus Viruses; Geminiviridae 0.002759 1
Rhynchosia golden mosaic Havana Viruses; Geminiviridae 0.005151 1
virus-[Cuba:Havana:28:2007]
Rhynchosia golden mosaic Sinaloa virus Viruses; Geminiviridae 0.005103 1
Rhynchosia golden mosaic virus Viruses; Geminiviridae 0.005174 3
Rhynchosia golden mosaic Yucatan Viruses; Geminiviridae 0.005139 1
virus
Rhynchosia mild mosaic virus Viruses; Geminiviridae 0.005162 1
Rhynchosia rugose golden mosaic virus Viruses; Geminiviridae 0.005186 1
Rhynchosia yellow mosaic India virus Viruses; Geminiviridae 0.005406 1
Rhynchosia yellow mosaic virus Viruses; Geminiviridae 0.005379 1
Rice latent virus 1 Viruses; Geminiviridae 0.002757 2
Rice latent virus 2 Viruses; Geminiviridae 0.002843 1
Rice orange leaf phytoplasma Bacteria; 0.599264 1
Terrabacteriagroup;
Tenericutes
Rose leaf curl virus Viruses; Geminiviridae 0.002741 1
Saccharum streak virus Viruses; Geminiviridae 0.002744 1
Sauropus leaf curl virus Viruses; Geminiviridae 0.002762 1
Senecio yellow mosaic virus Viruses; Geminiviridae 0.002746 1
Senna leaf curl virus Viruses; Geminiviridae 0.002742 1
Sida angular mosaic virus Viruses; Geminiviridae 0.005349 1
Sida bright yellow mosaic virus Viruses; Geminiviridae 0.005348 1
Sida chlorotic mottle virus Viruses; Geminiviridae 0.002601 1
Sida chlorotic vein virus Viruses; Geminiviridae 0.005145 1
Sidaciliaris golden mosaic virus Viruses; Geminiviridae 0.002638 1
Sida common mosaic virus Viruses; Geminiviridae 0.002687 1
Sida golden mosaic Braco virus Viruses; Geminiviridae 0.0026 1
Sida golden mosaic Brazil virus Viruses; Geminiviridae 0.002659 1
(continued)

Sida golden mosaic Buckup virus- Viruses; Geminiviridae 0.005199 1
[Jamaica:St. Elizabeth:2004]
Sida golden mosaic Costa Rica virus Viruses; Geminiviridae 0.005192 1
Sida golden mosaic Florida virus Viruses; Geminiviridae 0.005186 2
Sida golden mosaic Honduras virus Viruses; Geminiviridae 0.005192 1
Sida golden mosaic Lara virus Viruses; Geminiviridae 0.002633 1
Sida golden mosaic virus Viruses; Geminiviridae 0.005227 1
Sida golden mottle virus Viruses; Geminiviridae 0.005184 1
Sida golden yellow spot virus Viruses; Geminiviridae 0.002813 1
Sida golden yellow vein virus Viruses; Geminiviridae 0.002603 1
Sida golden yellow vein virus-[Jamaica: Viruses; Geminiviridae 0.00515 1
Liguanea2:2008]
Sida leaf curl virus Viruses; Geminiviridae 0.002757 1
Sida micrantha mosaic virus Viruses; Geminiviridae 0.005331 4
Sida mosaic Alagoas virus Viruses; Geminiviridae 0.005292 1
Sida mosaic Bolivia virus 1 Viruses; Geminiviridae 0.005348 1
Sida mosaic Bolivia virus 2 Viruses; Geminiviridae 0.005316 1
Sida mosaic Sinaloa virus Viruses; Geminiviridae 0.005182 1
Sida mottle Alagoas virus Viruses; Geminiviridae 0.002649 1
Sida mottle virus Viruses; Geminiviridae 0.002668 1
Sida yellow blotch virus Viruses; Geminiviridae 0.002664 1
Sida yellow leaf curl virus Viruses; Geminiviridae 0.002664 1
Sida yellow mosaic Alagoas virus Viruses; Geminiviridae 0.00269 1
Sida yellow mosaic China virus Viruses; Geminiviridae 0.002751 1
Sida yellow mosaic virus Viruses; Geminiviridae 0.002661 1
Sida yellow mosaic Yucatan virus Viruses; Geminiviridae 0.005197 1
Sida yellow mottle virus Viruses; Geminiviridae 0.005222 1
Sida yellow net virus Viruses; Geminiviridae 0.002676 1
Sida yellow vein Madurai virus Viruses; Geminiviridae 0.002753 1
Sida yellow vein virus Viruses; Geminiviridae 0.005205 1
Sida strum golden leaf spot virus Viruses; Geminiviridae 0.002666 1
Siegesbeckia yellow vein Guangxi virus Viruses; Geminiviridae 0.002784 1
Siegesbeckia yellow vein virus Viruses; Geminiviridae 0.002768 1
Solanum mosaic Bolivia virus Viruses; Geminiviridae 0.005196 1
South African cassava mosaic virus Viruses; Geminiviridae 0.00556 1
Soybean blistering mosaic virus Viruses; Geminiviridae 0.002605 1
Soybean chlorotic blotch virus Viruses; Geminiviridae 0.005355 1
Soybean chlorotic spot virus Viruses; Geminiviridae 0.005208 1
Soybean mild mottle virus Viruses; Geminiviridae 0.002768 1
Spilanthes yellow vein virus Viruses; Geminiviridae 0.002761 1
Spinach curly top Arizona virus Viruses; Geminiviridae 0.00286 1
Spinach severe curly top virus Viruses; Geminiviridae 0.003065 1
Spinach yellow vein Sikar virus Viruses; Geminiviridae 0.002753 1
(continued)
766 A. Khan et al.

Sporisorium scitamineum Eukaryota; Fungi; 20.0676 4
Basidiomycetes
Sporobolus striate mosaic virus 1 Viruses; Geminiviridae 0.002789 1
Squash leaf curl China virus Viruses; Geminiviridae 0.002756 3
Squash leaf curl China virus – [B] Viruses; Geminiviridae 0.005455 1
Squash leaf curl Philippines virus Viruses; Geminiviridae 0.005444 1
Squash leaf curl virus Viruses; Geminiviridae 0.005241 1
Squash leaf curl Yunnan virus Viruses; Geminiviridae 0.002714 1
Squash mild leaf curl virus Viruses; Geminiviridae 0.00519 1
Sri Lankan cassava mosaic virus Viruses; Geminiviridae 0.005466 2
Stachytarpheta leaf curl virus Viruses; Geminiviridae 0.002749 1
Streptomyces scabiei Bacteria; Terrabacteria 10.1487 17
group; Actinobacteria
Sugarcane chlorotic streak virus Viruses; Geminiviridae 0.002757 1
Sugarcane streak Egypt virus Viruses; Geminiviridae 0.002706 1
Sugarcane streak Reunion virus Viruses; Geminiviridae 0.00274 2
Sugarcane streak virus Viruses; Geminiviridae 0.002758 2
Sugarcane striate virus Viruses; Geminiviridae 0.002749 2
Sugarcane white streak virus Viruses; Geminiviridae 0.00283 1
Sunn hemp leaf distortion virus Viruses; Geminiviridae 0.002774 1
Sweet potato golden vein associated Viruses; Geminiviridae 0.002824 1
virus
Sweet potato golden vein Korea virus Viruses; Geminiviridae 0.002807 1
Sweet potato leaf curl Bengal virus Viruses; Geminiviridae 0.002823 1
Sweet potato leaf curl Canary virus Viruses; Geminiviridae 0.002837 2
Sweet potato leaf curl China virus Viruses; Geminiviridae 0.002771 1
Sweet potato leaf curl Georgia virus Viruses; Geminiviridae 0.002773 1
Sweet potato leaf curl Guangxi virus Viruses; Geminiviridae 0.002831 1
Sweet potato leaf curl Henan virus Viruses; Geminiviridae 0.002785 2
Sweet potato leaf curl Lanzarote virus Viruses; Geminiviridae 0.002814 1
Sweet potato leaf curl Sao Paulo virus Viruses; Geminiviridae 0.002782 1
Sweet potato leaf curl Shanghai virus Viruses; Geminiviridae 0.002834 1
Sweet potato leaf curl Sichuan virus 1 Viruses; Geminiviridae 0.002764 1
Sweet potato leaf curl Sichuan virus 2 Viruses; Geminiviridae 0.002786 1
Sweet potato leaf curl South Carolina Viruses; Geminiviridae 0.002782 1
virus
Sweet potato leaf curl Spain virus Viruses; Geminiviridae 0.00278 1
Sweet potato leaf curl Uganda virus- Viruses; Geminiviridae 0.002799 1
[Uganda:Kampala:2008]
Sweet potato leaf curl virus Viruses; Geminiviridae 0.002844 15
Sweet potato mosaic virus Viruses; Geminiviridae 0.002803 2
Sweet potato symptomless virus 1 Viruses; Geminiviridae 0.002886 2
(continued)

Sweet potato symptomless virus 1 Viruses; Geminiviridae 0.002886 2
Switchgrass mosaic-associated virus 1 Viruses; Geminiviridae 0.002739 1
Synedrella leaf curl virus Viruses; Geminiviridae 0.002749 1
Synedrella yellow vein clearing virus Viruses; Geminiviridae 0.002751 1
Telfairia golden mosaic virus Viruses; Geminiviridae 0.002742 1
Tilletia caries Eukaryota; Fungi; 29.5409 2
Basidiomycetes
Tobacco curly shoot virus Viruses; Geminiviridae 0.002743 1
Tobacco leaf curl Comoros virus Viruses; Geminiviridae 0.002755 1
Tobacco leaf curl Cuba virus Viruses; Geminiviridae 0.005176 2
Tobacco leaf curl Japan virus Viruses; Geminiviridae 0.002761 1
Tobacco leaf curl Pusa virus Viruses; Geminiviridae 0.002707 1
Tobacco leaf curl Thailand virus Viruses; Geminiviridae 0.002752 1
Tobacco leaf curl virus Viruses; Geminiviridae 0.002762 1
Tobacco leaf curl Yunnan virus Viruses; Geminiviridae 0.00275 1
Tobacco leaf curl Zimbabwe virus Viruses; Geminiviridae 0.002767 1
Tobacco leaf rugose virus Viruses; Geminiviridae 0.002622 1
Tobacco mosaic virus Viruses; Virgaviridae 0.006395 1
Tobacco mottle leaf curl virus Viruses; Geminiviridae 0.002634 1
Tobacco yellow crinkle virus Viruses; Geminiviridae 0.005154 1
Tobacco yellow dwarf virus Viruses; Geminiviridae 0.00258 1
Tobacco yellow dwarf virus Viruses; Geminiviridae 0.00258 1
Tomato bright yellow mosaic virus Viruses; Geminiviridae 0.002619 1
Tomato bright yellow mottle virus Viruses; Geminiviridae 0.002639 1
Tomato chino La Paz virus Viruses; Geminiviridae 0.002632 3
Tomato chlorotic leaf distortion virus- Viruses; Geminiviridae 0.00523 1
[Venezuela:Zulia:2004]
Tomato chlorotic mottle Guyane virus Viruses; Geminiviridae 0.005234 1
Tomato chlorotic mottle virus Viruses; Geminiviridae 0.005195 3
Tomato common mosaic virus Viruses; Geminiviridae 0.005058 1
Tomato curly stunt virus Viruses; Geminiviridae 0.002766 1
Tomato dwarf leaf virus Viruses; Geminiviridae 0.005034 1
Tomato enation leaf curl virus Viruses; Geminiviridae 0.002756 1
Tomato golden leaf distortion virus Viruses; Geminiviridae 0.00263 1
Tomato golden leaf spot virus Viruses; Geminiviridae 0.002669 1
Tomato golden mosaic virus Viruses; Geminiviridae 0.005096 1
Tomato golden mottle virus Viruses; Geminiviridae 0.005172 1
Tomato golden vein virus Viruses; Geminiviridae 0.005095 1
Tomato interveinal chlorosis virus Viruses; Geminiviridae 0.002617 1
Tomato latent virus Viruses; Geminiviridae 0.002746 1
Tomato leaf curl Anjouan virus Viruses; Geminiviridae 0.002781 1
Tomato leaf curl Arusha virus Viruses; Geminiviridae 0.002766 2
Tomato leaf curl Bangalore virus Viruses; Geminiviridae 0.002759 5
(continued)
768 A. Khan et al.

Tomato leaf curl Bangladesh virus Viruses; Geminiviridae 0.002761 1
Tomato leaf curl Barka virus Viruses; Geminiviridae 0.002753 1
Tomato leaf curl Burkina Faso virus Viruses; Geminiviridae 0.002784 1
Tomato leaf curl Cameroon virus Viruses; Geminiviridae 0.002808 1
Tomato leaf curl Cebu virus Viruses; Geminiviridae 0.002723 1
Tomato leaf curl China virus Viruses; Geminiviridae 0.002738 4
Tomato leaf curl China virus – OX2 Viruses; Geminiviridae 0.002744 1
Tomato leaf curl Comoros virus Viruses; Geminiviridae 0.002765 1
Tomato leaf curl Cotabato virus Viruses; Geminiviridae 0.00275 1
Tomato leaf curl Diana virus Viruses; Geminiviridae 0.002745 1
Tomato leaf curl Gandhinagar virus Viruses; Geminiviridae 0.00276 1
Tomato leaf curl Ghana virus Viruses; Geminiviridae 0.002803 2
Tomato leaf curl Guangdong virus Viruses; Geminiviridae 0.002744 1
Tomato leaf curl Guangxi virus Viruses; Geminiviridae 0.002752 1
Tomato leaf curl Gujarat virus Viruses; Geminiviridae 0.005445 1
Tomato leaf curl Hainan virus Viruses; Geminiviridae 0.002756 3
Tomato leaf curl Hanoi virus Viruses; Geminiviridae 0.00274 1
Tomato leaf curl Iran virus Viruses; Geminiviridae 0.002763 1
Tomato leaf curl Java virus Viruses; Geminiviridae 0.002752 2
Tomato leaf curl Joydebpur virus Viruses; Geminiviridae 0.002798 2
Tomato leaf curl Karnataka virus Viruses; Geminiviridae 0.002772 7
Tomato leaf curl Kerala virus Viruses; Geminiviridae 0.002767 2
Tomato leaf curl Kumasi virus Viruses; Geminiviridae 0.002794 1
Tomato leaf curl Laos virus Viruses; Geminiviridae 0.002748 1
Tomato leaf curl Liwa virus Viruses; Geminiviridae 0.002761 1
Tomato leaf curl Madagascar virus Viruses; Geminiviridae 0.002775 1
Tomato leaf curl Madagascar virus- Viruses; Geminiviridae 0.002777 1
Menabe [Madagascar:
Morondova:2001]
Tomato leaf curl Malaysia virus Viruses; Geminiviridae 0.002754 1
Tomato leaf curl Mali virus Viruses; Geminiviridae 0.002773 1
Tomato leaf curl Mayotte virus Viruses; Geminiviridae 0.002768 1
Tomato leaf curl Mindanao virus Viruses; Geminiviridae 0.002761 1
Tomato leaf curl Moheli virus Viruses; Geminiviridae 0.002756 1
Tomato leaf curl Namakely virus Viruses; Geminiviridae 0.002772 2
Tomato leaf curl New Delhi virus Viruses; Geminiviridae 0.005435 8
Tomato leaf curl New Delhi virus 2 Viruses; Geminiviridae 0.002735 1
Tomato leaf curl New Delhi virus 4 Viruses; Geminiviridae 0.002739 1
Tomato leaf curl Nigeria virus- Viruses; Geminiviridae 0.002784 1
[Nigeria:2006]
Tomato leaf curl Oman virus Viruses; Geminiviridae 0.002763 1
Tomato leaf curl Palampur virus Viruses; Geminiviridae 0.005481 4
(continued)

Tomato leaf curl Patna virus Viruses; Geminiviridae 0.002752 1
Tomato leaf curl Philippines virus Viruses; Geminiviridae 0.002755 3
Tomato leaf curl Pune virus Viruses; Geminiviridae 0.002756 1
Tomato leaf curl purple vein virus Viruses; Geminiviridae 0.002629 1
Tomato leaf curl Rajasthan virus Viruses; Geminiviridae 0.002758 1
Tomato leaf curl Ranchi virus Viruses; Geminiviridae 0.002762 1
Tomato leaf curl Seychelles virus Viruses; Geminiviridae 0.002742 1
Tomato leaf curl Sinaloa virus Viruses; Geminiviridae 0.005173 1
Tomato leaf curl Sri Lanka virus Viruses; Geminiviridae 0.002756 1
Tomato leaf curl Sudan virus Viruses; Geminiviridae 0.002782 4
Tomato leaf curl Sulawesi virus Viruses; Geminiviridae 0.002751 1
Tomato leaf curl Taiwan virus Viruses; Geminiviridae 0.002743 4
Tomato leaf curl Toliara virus Viruses; Geminiviridae 0.002764 1
Tomato leaf curl Uganda virus Viruses; Geminiviridae 0.002747 1
Tomato leaf curl Vietnam virus Viruses; Geminiviridae 0.002745 1
Tomato leaf curl virus Viruses; Geminiviridae 0.002766 5
Tomato leaf deformation virus Viruses; Geminiviridae 0.002591 1
Tomato leaf distortion virus Viruses; Geminiviridae 0.002645 1
Tomato mild mosaic virus Viruses; Geminiviridae 0.005371 1
Tomato mild yellow leaf curl Aragua Viruses; Geminiviridae 0.005168 1
virus
Tomato mosaic Havana virus Viruses; Geminiviridae 0.005206 1
Tomato mosaic Trujillo virus Viruses; Geminiviridae 0.002637 1
Tomato mottle leaf curl virus Viruses; Geminiviridae 0.005229 2
Tomato mottle Taino virus Viruses; Geminiviridae 0.005159 1
Tomato mottle virus Viruses; Geminiviridae 0.005145 1
Tomato mottle wrinkle virus Viruses; Geminiviridae 0.005124 1
Tomato pseudo-curly top virus Viruses; Geminiviridae 0.002861 1
Tomato rugose mosaic virus Viruses; Geminiviridae 0.005194 1
Tomato rugose yellow leaf curl virus Viruses; Geminiviridae 0.005305 1
Tomato severe leaf curl virus Viruses; Geminiviridae 0.002755 4
Tomato severe rugose virus Viruses; Geminiviridae 0.005164 1
Tomato spotted wilt tospovirus Viruses; Tospoviridae 0.016634 1
Tomato yellow leaf curl Axarquia virus Viruses; Geminiviridae 0.002763 1
Tomato yellow leaf curl China virus Viruses; Geminiviridae 0.002741 7
Tomato yellow leaf curl Guangdong Viruses; Geminiviridae 0.002744 1
virus
Tomato yellow leaf curl Indonesia virus- Viruses; Geminiviridae 0.002762 1
[Lembang]
Tomato yellow leaf curl Kanchanaburi Viruses; Geminiviridae 0.005504 1
virus
Tomato yellow leaf curl Malaga virus Viruses; Geminiviridae 0.002782 1
(continued)
770 A. Khan et al.

Tomato yellow leaf curl Mali virus Viruses; Geminiviridae 0.002796 3
Tomato yellow leaf curl Sardinia virus Viruses; Geminiviridae 0.002773 1
Tomato yellow leaf curl Saudi virus Viruses; Geminiviridae 0.002775 1
Tomato yellow leaf curl Shuangbai Viruses; Geminiviridae 0.002748 1
virus – [Y4536]
Tomato yellow leaf curl Thailand virus Viruses; Geminiviridae 0.005488 5
Tomato yellow leaf curl Vietnam virus Viruses; Geminiviridae 0.002745 1
Tomato yellow leaf curl virus Viruses; Geminiviridae 0.00279 6
Tomato yellow leaf curl Yunnan virus Viruses; Geminiviridae 0.002754 1
Tomato yellow leaf distortion virus Viruses; Geminiviridae 0.005219 1
Tomato yellow margin leaf curl virus Viruses; Geminiviridae 0.005118 1
Tomato yellow mottle virus Viruses; Geminiviridae 0.005121 1
Tomato yellow spot virus Viruses; Geminiviridae 0.0053 1
Tomato yellow vein streak virus Viruses; Geminiviridae 0.00513 1
Triumfetta yellow mosaic virus Viruses; Geminiviridae 0.005277 1
Turnip curly top virus Viruses; Geminiviridae 0.002981 4
Turnip curly top virus Viruses; Geminiviridae 0.002981 4
Turnip leaf roll virus Viruses; Geminiviridae 0.002965 1
Turnip leaf roll virus Viruses; Geminiviridae 0.002965 1
TYLCAxV-Sic1-[IT:Sic2/2:04] Viruses; Geminiviridae 0.002771 1
Urochloa streak virus Viruses; Geminiviridae 0.002736 1
Urochloa streak virus Viruses; Geminiviridae 0.002736 1
Ustilago maydis Eukaryota; Fungi; 19.6644 7
Basidiomycetes
Vaccinium witches’-broom phytoplasma Bacteria; 0.647754 1
Terrabacteriagroup;
Tenericutes
Velvet bean golden mosaic virus Viruses; Geminiviridae 0.002767 1
Velvet bean severe mosaic virus Viruses; Geminiviridae 0.00539 1
Venturia inaequalis Eukaryota; Fungi; 72.7916 85
Ascomycetes
Vernonia crinkle virus Viruses; Geminiviridae 0.002791 1
Vernonia yellow vein Fujian virus Viruses; Geminiviridae 0.002739 1
Vernonia yellow vein virus Viruses; Geminiviridae 0.002745 1
Verticillium albo-atrum Eukaryota; Fungi; 36.4685 1
Ascomycetes
Verticillium alfalfae Eukaryota; Fungi; 32.863 2
Ascomycetes
Verticillium dahliae Eukaryota; Fungi; 33.9003 13
Ascomycetes
Verticillium isaacii Eukaryota; Fungi; 35.6909 1
Ascomycetes
Verticillium klebahnii Eukaryota; Fungi; 36.0824 1
Ascomycetes
(continued)

Verticillium longisporum Eukaryota; Fungi; 99.1892 2
Ascomycetes
Verticillium nonalfalfae Eukaryota; Fungi; 31.7515 3
Ascomycetes
Verticillium nubilum Eukaryota; Fungi; 37.9116 1
Ascomycetes
Verticillium tricorpus Eukaryota; Fungi; 36.0604 2
Ascomycetes
Verticillium zaregamsianum Eukaryota; Fungi; 37.1319 1
Ascomycetes
Vigna yellow mosaic virus Viruses; Geminiviridae 0.002602 1
Vinca leaf curl virus Viruses; Geminiviridae 0.002776 1
Watermelon chlorotic stunt virus Viruses; Geminiviridae 0.005498 1
West African Asystasia virus 1 Viruses; Geminiviridae 0.005388 2
West African Asystasia virus 2 Viruses; Geminiviridae 0.002744 1
Wheat blue dwarf phytoplasma Bacteria; 0.611462 1
Terrabacteriagroup;
Tenericutes
Wheat dwarf India virus Viruses; Geminiviridae 0.002783 1
Wheat dwarf virus Viruses; Geminiviridae 0.00275 6
Wheat dwarf virus Viruses; Geminiviridae 0.00275 6
Whitefly-associated begomovirus 1 Viruses; Geminiviridae 0.002609 1
Wissadula golden mosaic virus Viruses; Geminiviridae 0.0052 1
Wissadula yellow mosaic virus Viruses; Geminiviridae 0.002621 1
Xanthomonas axonopodis Bacteria; 5.41058 14
Proteobacteria;
Gammaproteobacteria
Xanthomonas campestris Bacteria; 5.07619 71
Proteobacteria;
Gammaproteobacteria
Table 32.3 Plant genome sequence details
772
Organism name Organism groups Assembly Level Size (Mb) WGS Scaffolds CDS
Abrus precatorius Eukaryota; Plants; GCA_003935025.1 Scaffold 347.23 QYUI01 160 40048
Land Plants
Acer yangbiense Eukaryota; Plants; GCA_008009225.1 Chromosome 665.888 VAHF01 280 28320
Land Plants
Actinidia chinensis Eukaryota; Plants; GCA_000467755.1 Contig 604.217 AONS01 26721 0
Land Plants
Actinidia chinensis var. Eukaryota; Plants; GCA_003024255.1 Chromosome 553.842 NKQK01 1234 33115
chinensis Land Plants
Actinidia eriantha Eukaryota; Plants; GCA_004150315.1 Chromosome 690.611 QOVS01 1735 0
Land Plants
Aegilops tauschii Eukaryota; Plants; GCA_000347335.2 Chromosome 4310.35 AOCO02 112210 0
Land Plants
Aegilops tauschii Eukaryota; Plants; GCA_002105435.1 Chromosome 247.197 LYXL01 1 0
Land Plants
Aegilops tauschii subsp. Eukaryota; Plants; GCA_002575655.1 Chromosome 4224.92 NWVB01 109583 0
strangulata Land Plants
Aegilops tauschii subsp. Eukaryota; Plants; GCA_001957025.1 Contig 4327.32 MCGU01 68538 55713
tauschii Land Plants
Aethionema arabicum Eukaryota; Plants; GCA_000411095.1 Scaffold 192.488 ASZG01 18312 0
Land Plants
Alloteropsis semialata Eukaryota; Plants; GCA_004135705.1 Chromosome 747.772 QPGU01 688 0
Land Plants
Alnus glutinosa Eukaryota; Plants; GCA_003254965.1 Scaffold 611.874 QAOD01 167345 0
Land Plants
Amaranthus hypochondriacus Eukaryota; Plants; GCA_000753965.1 Scaffold 502.148 JPXE01 117340 0
Land Plants
Amaranthus tuberculatus Eukaryota; Plants; GCA_000180655.1 Contig 4.34798 ACQK01 15440 0
Land Plants
A. Khan et al.
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Amborella trichopoda Eukaryota; Plants; GCA_000471905.1 Scaffold 706.495 AWHE01 5746 31494
Land Plants
Ananas comosus Eukaryota; Plants; GCA_902162155.1 Scaffold 315.839 CABGUK01 25 0
Land Plants
Ananas comosus Eukaryota; Plants; GCA_001661175.1 Scaffold 524.07 LSRQ01 8448 23598
Land Plants
Ananas comosus Eukaryota; Plants; GCA_001540865.1 Chromosome 382.056 LODP01 3129 35775
Land Plants
Ananas comosus var. Eukaryota; Plants; GCA_902506285.1 Scaffold 513.235 CABWKS01 103 0
bracteatus Land Plants
Anastatica hierochuntica Eukaryota; Plants; GCA_900406275.1 Scaffold 542.343 OVAN01 72649 0
Land Plants
Andrographis paniculata Eukaryota; Plants; GCA_004354405.1 Chromosome 269.408 SMLO01 257 0
Land Plants
Apostasia shenzhenica Eukaryota; Plants; GCA_002786265.1 Scaffold 348.733 PEFY01 2985 21743
Land Plants
Aquilaria agallochum Eukaryota; Plants; GCA_000696445.1 Scaffold 726.71 JMHV01 27769 0
Land Plants
Aquilaria sinensis Eukaryota; Plants; GCA_005392925.1 Contig 699.794 SMDT01 3368 0
Land Plants
Aquilegia coerulea Eukaryota; Plants; GCA_002738505.1 Scaffold 301.98 NXFA01 970 41063
Land Plants
Arabidopsis halleri Eukaryota; Plants; GCA_003711535.1 Scaffold 164.574 RCNM01 40344 0
Land Plants
Arabidopsis halleri subsp. Eukaryota; Plants; GCA_900078215.1 Scaffold 196.243 FJVB01 2239 0
gemmifera Land Plants
Arabidopsis halleri subsp. Eukaryota; Plants; GCA_000523005.1 Scaffold 221.14 BASO01 282453 0
Arabidopsis halleri subsp. Eukaryota; Plants; GCA_003118655.1 Scaffold 413.881 BFAE01 344622 0
773

(continued)
774
Arabidopsis lyrata subsp. lyrata Eukaryota; Plants; GCA_000004255.1 Scaffold 206.823 ADBK01 696 39161
Land Plants
Arabidopsis lyrata subsp. Eukaryota; Plants; GCA_900205625.1 Scaffold 175.183 OANL01 1675 0
petraea Land Plants
Arabidopsis lyrata subsp. Eukaryota; Plants; GCA_000524985.1 Scaffold 202.972 BASP01 281536 0
petraea Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900303355.1 Contig 119.503 OMOL01 62 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_000835945.1 Contig 127.419 JSAD01 378 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900303345.1 Contig 119.75 OMOK01 78 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900243945.1 Contig 119.167 OFAM01 59 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900243935.1 Contig 119.203 OFEF01 40 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_001753755.2 Contig 244.583 MJMM01 411 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900243955.1 Contig 119.128 OFAN01 139 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_001742845.1 Scaffold 116.846 LXSY01 5197 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_000222345.1 Scaffold 98.0662 AFNB01 1740 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_000222325.1 Scaffold 96.5002 AFNA01 2143 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_000222365.1 Scaffold 96.2565 AFMZ01 1261 0
Land Plants
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Arabidopsis thaliana Eukaryota; Plants; GCA_000222385.1 Scaffold 96.694 AFNC01 2408 0

Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234075.1 Contig 0.195283 OCZF01 19 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234235.1 Contig 1.71971 OCYH01 133 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233735.1 Contig 1.87192 OCWZ01 156 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234125.1 Contig 2.02645 OCXO01 164 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234305.1 Contig 2.01361 OCYE01 163 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233785.1 Contig 2.68429 OCWV01 231 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234265.1 Contig 1.60963 OCYD01 141 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234185.1 Contig 2.04223 OCYB01 169 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233775.1 Contig 1.84553 OCWC01 154 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234295.1 Contig 1.88946 OCYF01 171 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234275.1 Contig 1.95856 OCYJ01 167 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233705.1 Contig 1.53762 OCVZ01 126 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234025.1 Contig 1.4141 OCXK01 116 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233685.1 Contig 1.72997 OCWA01 149 0
775
Land Plants
(continued)
776
Arabidopsis thaliana Eukaryota; Plants; GCA_900233725.1 Contig 2.27092 OCWQ01 190 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234165.1 Contig 2.13757 OCYN01 189 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234135.1 Contig 1.73315 OCXS01 143 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233965.1 Contig 1.78161 OCXD01 170 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234175.1 Contig 1.93182 OCYM01 173 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234115.1 Contig 1.8648 OCXN01 164 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233995.1 Contig 2.27278 OCXQ01 199 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234145.1 Contig 1.83575 OCXW01 156 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234285.1 Contig 2.15462 OCYA01 194 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234105.1 Contig 1.76999 OCXU01 156 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233755.1 Contig 1.9492 OCWR01 183 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233985.1 Contig 1.75614 OCXL01 156 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234215.1 Contig 1.86784 OCYC01 173 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234205.1 Contig 1.83401 OCYL01 182 0
Land Plants
A. Khan et al.
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Arabidopsis thaliana Eukaryota; Plants; GCA_900234065.1 Contig 1.56864 OCXH01 151 0

Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233765.1 Contig 1.97277 OCWY01 169 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233795.1 Contig 1.65792 OCXE01 153 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233745.1 Contig 1.86401 OCWJ01 172 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234155.1 Contig 1.93139 OCXX01 180 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234045.1 Contig 1.62901 OCXG01 158 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234195.1 Contig 1.93135 OCYG01 176 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233665.1 Contig 1.82675 OCWE01 191 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233875.1 Contig 1.72986 OCWP01 182 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233905.1 Contig 1.37218 OCWL01 132 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233635.1 Contig 1.68821 OCVY01 157 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233835.1 Contig 1.65938 OCWB01 159 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234085.1 Contig 2.05357 OCXM01 194 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234325.1 Contig 1.55025 OCYO01 153 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233645.1 Contig 1.34032 OCWG01 133 0
777
Land Plants
(continued)
778
Arabidopsis thaliana Eukaryota; Plants; GCA_900234245.1 Contig 1.90115 OCXY01 200 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234095.1 Contig 1.7997 OCXR01 173 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233865.1 Contig 1.33758 OCWN01 133 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233805.1 Contig 1.5272 OCWS01 146 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234035.1 Contig 2.18075 OCXI01 205 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234365.1 Contig 1.60312 OCYP01 153 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233825.1 Contig 1.4618 OCWH01 147 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233855.1 Contig 1.61604 OCXC01 153 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233695.1 Contig 1.80119 OCXF01 169 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234315.1 Contig 1.66717 OCYK01 179 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233915.1 Contig 1.36257 OCXB01 136 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234055.1 Contig 1.76317 OCXV01 182 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233885.1 Contig 1.38144 OCWT01 140 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233655.1 Contig 1.15189 OCWF01 124 0
Land Plants
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Arabidopsis thaliana Eukaryota; Plants; GCA_900234005.1 Contig 1.33529 OCXJ01 137 0

Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233815.1 Contig 1.71934 OCWO01 188 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234015.1 Contig 1.11018 OCXT01 124 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233675.1 Contig 1.03397 OCWK01 120 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233845.1 Contig 1.15 OCWM01 133 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233895.1 Contig 1.31957 OCWX01 158 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233715.1 Contig 1.14084 OCXA01 138 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233945.1 Contig 1.26394 OCWD01 151 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233925.1 Contig 2.03504 OCWI01 248 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233975.1 Contig 0.844674 OCXP01 119 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900233955.1 Contig 0.838703 OCWU01 109 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234225.1 Contig 0.32515 OCYI01 52 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900234255.1 Contig 0.847842 OCXZ01 146 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_902460285.1 Chromosome 120.338 CABPTM01 105 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_900660825.1 Chromosome 119.627 CAACVU01 109 0
779
Land Plants
(continued)
780
Arabidopsis thaliana Eukaryota; Plants; GCA_902460305.1 Chromosome 122.202 CABPTJ01 184 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_902460275.1 Chromosome 119.75 CABPTK01 102 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_902460295.1 Chromosome 120.29 CABPTI01 94 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_902460315.1 Chromosome 120.795 CABPTL01 142 0
Land Plants
Arabidopsis thaliana Eukaryota; Plants; GCA_001651475.1 Chromosome 118.891 LUHQ01 30 30837
Land Plants
Arabis alpina Eukaryota; Plants; GCA_000612745.1 Contig 171.788 CBTM01 37680 0
Land Plants
Arabis alpina Eukaryota; Plants; GCA_000733195.1 Chromosome 308.033 JNGA01 27779 23286
Land Plants
Arabis montbretiana Eukaryota; Plants; GCA_001484125.1 Contig 199.12 LNCH01 28775 0
Land Plants
Arabis nordmanniana Eukaryota; Plants; GCA_001484925.1 Scaffold 342.307 LNCG01 267228 0
Land Plants
Arachis duranensis Eukaryota; Plants; GCA_001687015.1 Scaffold 1075.96 MAMN01 20214 0
Land Plants
Arachis duranensis Eukaryota; Plants; GCA_000817695.2 Chromosome 1084.26 JQIN01 3189 52826
Land Plants
Arachis hypogaea Eukaryota; Plants; GCA_003086295.2 Chromosome 2557.07 PIVG01 385 100775
Land Plants
Arachis hypogaea Eukaryota; Plants; GCA_004170445.1 Chromosome 2551.68 SDMP01 29 101330
Land Plants
Arachis ipaensis Eukaryota; Plants; GCA_000816755.2 Chromosome 1353.5 JQIO01 997 57621
Land Plants
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Arachis monticola Eukaryota; Plants; GCA_003063285.2 Chromosome 2618.65 QBTX01 6909 0

Land Plants
Argania spinosa Eukaryota; Plants; GCA_003260245.1 Scaffold 670.097 QLOD01 75327 0
Land Plants
Aristotelia chilensis Eukaryota; Plants; GCA_008921755.1 Scaffold 96.3544 VEXP01 42602 0
Land Plants
Aristotelia chilensis Eukaryota; Plants; GCA_008126665.1 Scaffold 0.312713 VDCA01 93 0
Land Plants
Artemisia annua Eukaryota; Plants; GCA_003112345.1 Scaffold 1792.86 PKPP01 39400 213
Land Plants
Artocarpus camansi Eukaryota; Plants; GCA_002024485.1 Scaffold 631.308 LNSY01 396025 0
Land Plants
Asclepias syriaca Eukaryota; Plants; GCA_002018285.1 Scaffold 236.77 MSXX01 221855 0
Land Plants
Asparagus officinalis Eukaryota; Plants; GCA_001876935.1 Chromosome 1187.54 MPDI01 11792 36763
Land Plants
Atalantia buxifolia Eukaryota; Plants; GCA_002013935.1 Scaffold 315.806 MKYR01 25600 0
Land Plants
Aurinia saxatilis Eukaryota; Plants; GCA_900406295.1 Scaffold 316.42 OVAP01 76972 0
Land Plants
Avena sativa Eukaryota; Plants; GCA_002943605.1 Contig 67.3266 PKQH01 16667 0
Land Plants
Azadirachta indica Eukaryota; Plants; GCA_000439995.3 Contig 261.458 AMWY02 126142 0
Land Plants
Barbarea vulgaris Eukaryota; Plants; GCA_001920985.1 Scaffold 167.352 LXTM01 7810 0
Land Plants
Bassia scoparia Eukaryota; Plants; GCA_008642245.1 Scaffold 711.357 SNQN01 19671 0
Land Plants
Begonia fuchsioides Eukaryota; Plants; GCA_003255005.1 Scaffold 373.914 QAOC01 55006 0
781
Land Plants
(continued)
782
Berberis thunbergii Eukaryota; Plants; GCA_003290165.1 Contig 2240.74 QNQO01 11815 0
Land Plants
Beta patula Eukaryota; Plants; GCA_005862465.1 Scaffold 633.549 VASJ01 78458 0
Land Plants
Beta vulgaris subsp. maritima Eukaryota; Plants; GCA_005862445.2 Scaffold 608.27 VASK02 97415 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_002917755.1 Chromosome 540.534 PCNB01 40 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000510975.1 Chromosome 568.609 AYZY01 43635 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000510365.1 Scaffold 484.231 AYZT01 35771 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000510875.1 Scaffold 479.876 AYZX01 47405 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000510485.1 Scaffold 463.706 AYZW01 48733 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000510465.1 Scaffold 539.552 AYZU01 84234 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000397105.1 Scaffold 426.675 ARYA01 260142 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000729925.1 Contig 1.15347 JMBQ01 1287 0
Land Plants
Beta vulgaris subsp. vulgaris Eukaryota; Plants; GCA_000511025.2 Chromosome 566.55 AYZS02 40406 32874
Land Plants
Betula nana Eukaryota; Plants; GCA_000327005.1 Scaffold 564.011 CAOK01 551915 0
Land Plants
Betula pendula Eukaryota; Plants; GCA_900184695.1 Scaffold 435.915 FXXK01 5644 0
Land Plants
A. Khan et al.
32
Biscutella auriculata Eukaryota; Plants; GCA_900406285.1 Scaffold 384.77 OVAO01 150640 0

Land Plants
Biscutella laevigata subsp. Eukaryota; Plants; GCA_900406315.1 Scaffold 333.609 OVAU01 144912 0
laevigata Land Plants
Boechera puberula Eukaryota; Plants; GCA_900406335.1 Scaffold 182.592 OVAS01 123951 0
Land Plants
Boechera stricta Eukaryota; Plants; GCA_002079875.1 Scaffold 188.795 MLHT01 1944 0
Land Plants
Boehmeria nivea Eukaryota; Plants; GCA_002937015.1 Scaffold 344.617 PHNS01 12775 0
Land Plants
Boehmeria nivea Eukaryota; Plants; GCA_002806895.1 Scaffold 316.026 NHTU01 154955 0
Land Plants
Brachypodium distachyon Eukaryota; Plants; GCA_002892335.1 Contig 218.676 MXPZ01 60421 0
Land Plants
Brachypodium distachyon Eukaryota; Plants; GCA_002892295.1 Contig 218.015 MXQA01 65964 0
Land Plants
Brachypodium distachyon Eukaryota; Plants; GCA_001742125.1 Contig 214.716 LXJM01 68977 0
Land Plants
Brachypodium distachyon Eukaryota; Plants; GCA_000005505.4 Chromosome 271.299 ADDN03 15 37892
Land Plants
Brassica cretica Eukaryota; Plants; GCA_003260655.1 Contig 412.521 QGKV01 243461 0
Land Plants
Brassica cretica Eukaryota; Plants; GCA_003260635.1 Contig 208.354 QGKW01 100644 0
Land Plants
Brassica cretica Eukaryota; Plants; GCA_003260675.1 Contig 434.935 QGKX01 338759 0
Land Plants
Brassica cretica Eukaryota; Plants; GCA_003260695.1 Contig 400.212 QGKY01 396633 0
Land Plants
(continued)
783
784
Brassica juncea var. tumida Eukaryota; Plants; GCA_001687265.1 Chromosome 954.861 LFQT01 9746 0
Land Plants
Brassica napus Eukaryota; Plants; GCA_000751015.1 Scaffold 848.2 CCCW01 20899 61153
Land Plants
Brassica napus Eukaryota; Plants; GCA_000686985.2 Chromosome 976.191 JMKK02 1471 123467
Land Plants
Brassica nigra Eukaryota; Plants; GCA_001682895.1 Chromosome 402.145 LFLV01 2545 0
Land Plants
Brassica oleracea Eukaryota; Plants; GCA_900416815.2 Chromosome 554.977 OWNI02 129 0
Land Plants
Brassica oleracea var. capitata Eukaryota; Plants; GCA_000604025.1 Scaffold 514.431 AOIX01 1816 0
Land Plants
Brassica oleracea var. oleracea Eukaryota; Plants; GCA_000695525.1 Chromosome 488.954 JJMF01 32886 56687
Land Plants
Brassica rapa Eukaryota; Plants; GCA_900412535.2 Chromosome 401.927 OVXL02 304 0
Land Plants
Brassica rapa Eukaryota; Plants; GCA_000309985.1 Chromosome 284.129 AENI01 40432 52553
Land Plants
Brassica rapa Eukaryota; Plants; GCA_003434825.1 Chromosome 314.865 QMKI01 7071 43332
Land Plants
Brassica rapa subsp. pekinensis Eukaryota; Plants; GCA_008629595.1 Chromosome 234.688 VDME01 3421 0
Land Plants
Cajanus cajan Eukaryota; Plants; GCA_000230855.2 Contig 648.281 AFSP02 360028 0
Land Plants
Cajanus cajan Eukaryota; Plants; GCA_000340665.1 Chromosome 592.971 AGCT01 36536 41387
Land Plants
Calamus simplicifolius Eukaryota; Plants; GCA_900491605.1 Scaffold 1960.81 UESW01 5116 0
Land Plants
A. Khan et al.
32
Calotropis procera Eukaryota; Plants; GCA_004801955.1 Scaffold 209.215 LVCA01 20519 0

Land Plants
Camelina sativa Eukaryota; Plants; GCA_000496875.1 Scaffold 547.649 AUUT01 15937 0
Land Plants
Camelina sativa Eukaryota; Plants; GCA_000633955.1 Chromosome 641.356 JFZQ01 37212 107481
Land Plants
Camellia sinensis var. sinensis Eukaryota; Plants; GCA_004153795.1 Scaffold 3105.37 SDRB01 14028 76698
Land Plants
Cannabis sativa Eukaryota; Plants; GCA_001865755.1 Contig 585.824 MNPR01 11110 0
Land Plants
Cannabis sativa Eukaryota; Plants; GCA_002090435.1 Contig 512.174 MXBD01 18355 0
Land Plants
Cannabis sativa Eukaryota; Plants; GCA_003660325.2 Contig 1333.38 QVPT02 3372 0
Land Plants
Cannabis sativa Eukaryota; Plants; GCA_001509995.1 Scaffold 285.933 LKUB01 175088 0
Land Plants
Cannabis sativa Eukaryota; Plants; GCA_000230575.5 Chromosome 891.965 AGQN03 12836 0
Land Plants
Cannabis sativa Eukaryota; Plants; GCA_003417725.2 Chromosome 1009.67 QKVJ02 5303 0
Land Plants
Cannabis sativa Eukaryota; Plants; GCA_900626175.1 Chromosome 876.148 UZAU01 221 33677
Land Plants
Cannabis sativa subsp. indica Eukaryota; Plants; GCA_001510005.1 Contig 595.358 LKUA01 311039 0
Land Plants
Capsella bursa-pastoris Eukaryota; Plants; GCA_001974645.1 Scaffold 268.431 MPGU01 8186 0
Land Plants
Capsella rubella Eukaryota; Plants; GCA_000375325.1 Scaffold 133.064 ANNY01 773 34126
Land Plants
(continued)
785
786
Capsicum annuum Eukaryota; Plants; GCA_002878395.2 Chromosome 3212.12 NPHV01 81378 0
Land Plants
Capsicum annuum Eukaryota; Plants; GCA_000512255.2 Chromosome 3063.86 AYRZ02 35797 35845
Land Plants
Capsicum annuum Eukaryota; Plants; GCA_000710875.1 Chromosome 2935.88 ASJU01 6478 45410
Land Plants
Capsicum annuum var. Eukaryota; Plants; GCA_000950795.1 Chromosome 2768.13 ASJV01 16998 0
glabriusculum Land Plants
Capsicum baccatum Eukaryota; Plants; GCA_002271885.2 Chromosome 3215.61 MLFT02 23260 35853
Land Plants
Capsicum chinense Eukaryota; Plants; GCA_002271895.2 Chromosome 3070.91 MCIT02 87978 34974
Land Plants
Carica papaya Eukaryota; Plants; GCA_000150535.1 Scaffold 370.419 ABIM01 17766 26103
Land Plants
Carnegiea gigantea Eukaryota; Plants; GCA_002740515.1 Scaffold 980.351 NCQR01 57405 0
Land Plants
Carpinus fangiana Eukaryota; Plants; GCA_006937295.1 Chromosome 381.949 VIBQ01 4602 0
Land Plants
Carthamus tinctorius Eukaryota; Plants; GCA_001633085.1 Scaffold 661.938 LUCG01 463906 0
Land Plants
Caryocar brasiliense Eukaryota; Plants; GCA_004918865.1 Scaffold 212.173 STGP01 55248 0
Land Plants
Castanea mollissima Eukaryota; Plants; GCA_000763605.1 Scaffold 833.241 JRKL01 133589 0
Land Plants
Casuarina equisetifolia subsp. Eukaryota; Plants; GCA_003795335.1 Scaffold 301.458 RDRV01 2936 0
incana Land Plants
Casuarina glauca Eukaryota; Plants; GCA_003255045.1 Scaffold 282.811 QAOB01 39787 0
Land Plants
A. Khan et al.
32
Catharanthus roseus Eukaryota; Plants; GCA_000949345.1 Scaffold 522.654 JQHZ01 79302 0

Land Plants
Catharanthus roseus Eukaryota; Plants; GCA_001292525.1 Contig 0.11552 CCXB01 7 0
Land Plants
Catharanthus roseus Eukaryota; Plants; GCA_001292565.1 Contig 0.114931 CCXA01 32 0
Land Plants
Cenchrus americanus Eukaryota; Plants; GCA_002174835.2 Chromosome 1816.95 LKME02 52033 0
Land Plants
Cephalotus follicularis Eukaryota; Plants; GCA_001972305.1 Scaffold 1614.52 BDDD01 16307 36667
Land Plants
Cercis canadensis Eukaryota; Plants; GCA_003255065.1 Scaffold 329.325 QAOA01 8828 0
Land Plants
Chamaecrista fasciculata Eukaryota; Plants; GCA_003254925.1 Scaffold 429.103 QANZ01 56674 0
Land Plants
Chenopodium pallidicaule Eukaryota; Plants; GCA_001687005.1 Scaffold 337.011 MATR01 3013 0
Land Plants
Chenopodium quinoa Eukaryota; Plants; GCA_001683475.1 Scaffold 1333.55 LPWI01 3487 63173
Land Plants
Chenopodium quinoa Eukaryota; Plants; GCA_002732095.1 Scaffold 1336.74 NSDK01 3185 0
Land Plants
Chenopodium quinoa Eukaryota; Plants; GCA_001742885.1 Scaffold 1087.41 BDCQ01 24845 0
Land Plants
Chenopodium suecicum Eukaryota; Plants; GCA_001687025.1 Scaffold 536.949 MATQ01 11198 0
Land Plants
Chrysanthemum seticuspe Eukaryota; Plants; GCA_004359105.1 Scaffold 2721.84 BDUE01 354212 0
Land Plants
Cicer arietinum Eukaryota; Plants; GCA_002896005.2 Scaffold 653.867 PGTT02 13064 0
Land Plants
(continued)
787
788
Cicer arietinum Eukaryota; Plants; GCA_000347275.4 Chromosome 511.684 AHII03 30401 0
Land Plants
Cicer arietinum Eukaryota; Plants; GCA_000331145.1 Chromosome 530.894 ANPC01 7545 35754
Land Plants
Cicer echinospermum Eukaryota; Plants; GCA_002896215.2 Scaffold 657.414 PGTU02 17305 0
Land Plants
Cicer reticulatum Eukaryota; Plants; GCA_002896235.1 Contig 715.407 PGWS01 38802 0
Land Plants
Cicer reticulatum Eukaryota; Plants; GCA_003689015.2 Chromosome 416.904 QSLP02 3657 0
Land Plants
Cinnamomum micranthum Eukaryota; Plants; GCA_003546025.1 Scaffold 730.416 QPKB01 2150 26531
f. kanehirae Land Plants
Cissus quadrangularis Eukaryota; Plants; GCA_002878655.1 Scaffold 281.704 LLYR01 125206 0
Land Plants
Citrullus lanatus Eukaryota; Plants; GCA_000238415.2 Chromosome 365.45 AGCB02 113 0
Land Plants
Citrus cavaleriei Eukaryota; Plants; GCA_002013975.2 Scaffold 357.621 MKYP02 14916 0
Land Plants
Citrus clementina Eukaryota; Plants; GCA_000493195.1 Scaffold 301.365 AMZM01 1398 32586
Land Plants
Citrus hindsii Eukaryota; Plants; GCA_004802465.1 Contig 373.17 QWBT01 1331 0
Land Plants
Citrus maxima Eukaryota; Plants; GCA_002006925.1 Chromosome 345.757 MKYQ01 1612 0
Land Plants
Citrus medica Eukaryota; Plants; GCA_002013955.2 Scaffold 406.058 MKYO02 32732 0
Land Plants
Citrus reticulata Eukaryota; Plants; GCA_003258625.1 Scaffold 344.273 NIHA01 67725 0
Land Plants
A. Khan et al.
32
Citrus sinensis Eukaryota; Plants; GCA_000695605.1 Scaffold 319.225 JJOQ01 12573 51718
Land Plants
Citrus sinensis Eukaryota; Plants; GCA_000317415.1 Chromosome 327.83 AJPS01 4995 39056
Land Plants
Citrus unshiu Eukaryota; Plants; GCA_002897195.1 Scaffold 359.652 BDQV01 20876 37970
Land Plants
Citrus unshiu Eukaryota; Plants; GCA_001753815.1 Contig 1.1542 BDGO01 507 0
Land Plants
Citrus x paradisi x Citrus Eukaryota; Plants; GCA_001929425.1 Contig 265.534 AZHM01 238488 0
trifoliata Land Plants
Cochlearia officinalis Eukaryota; Plants; GCA_900406305.1 Scaffold 164.469 OVAT01 128459 0
Land Plants
Cocos nucifera Eukaryota; Plants; GCA_006176705.1 Scaffold 2102.42 QRFJ01 7998 0
Land Plants
Cocos nucifera Eukaryota; Plants; GCA_003604295.1 Scaffold 1839.17 PDMH01 59328 0
Land Plants
Cocos nucifera Eukaryota; Plants; GCA_008124465.1 Chromosome 2202.46 VOII01 113653 0
Land Plants
Codonopsis pilosula Eukaryota; Plants; GCA_004523855.1 Scaffold 937.71 SOPR01 1154 0
Land Plants
Coffea arabica Eukaryota; Plants; GCA_003713225.1 Chromosome 1094.45 RHJU01 2833 67222
Land Plants
Coffea canephora Eukaryota; Plants; GCA_900059795.1 Chromosome 568.612 CBUE02 13345 25574
Land Plants
Coffea eugenioides Eukaryota; Plants; GCA_003713205.1 Chromosome 699.904 RHJT01 3530 38150
Land Plants
Conringia planisiliqua Eukaryota; Plants; GCA_900108845.1 Scaffold 184.156 FNXX01 705 0
Land Plants
(continued)
789
790
Corchorus capsularis Eukaryota; Plants; GCA_001974805.1 Scaffold 317.178 AWWV01 16522 29356
Land Plants
Corchorus olitorius Eukaryota; Plants; GCA_001974825.1 Contig 334.912 AWUE01 24918 35704
Land Plants
Corchorus olitorius Eukaryota; Plants; GCA_002141455.1 Contig 377.377 LLWS01 52373 0
Land Plants
Crucihimalaya himalaica Eukaryota; Plants; GCA_004349715.1 Scaffold 234.721 SMJT01 582 0
Land Plants
Cucumis melo Eukaryota; Plants; GCA_000313045.1 Scaffold 374.928 CAJI01 31464 29798
Land Plants
Cucumis melo Eukaryota; Plants; GCA_902497455.1 Scaffold 357.857 CABVGI01 13 0
Land Plants
Cucumis melo var. makuwa Eukaryota; Plants; GCA_005549215.1 Scaffold 358.48 SSTE01 23444 38173
Land Plants
Cucumis melo var. makuwa Eukaryota; Plants; GCA_005549225.1 Scaffold 347.184 SSTD01 20255 36235
Land Plants
Cucumis sativus Eukaryota; Plants; GCA_001483825.2 Contig 342.654 LKUO02 8035 0
Land Plants
Cucumis sativus Eukaryota; Plants; GCA_000224045.1 Scaffold 323.986 ACYN01 13113 0
Land Plants
Cucumis sativus Eukaryota; Plants; GCA_000004075.2 Chromosome 195.669 ACHR02 190 25668
Land Plants
Cucurbita argyrosperma subsp. Eukaryota; Plants; GCA_004115005.1 Scaffold 230.034 SDJN01 938 0
argyrosperma Land Plants
Cucurbita maxima Eukaryota; Plants; GCA_002738345.1 Scaffold 271.413 NEWN01 8299 42777
Land Plants
Cucurbita moschata Eukaryota; Plants; GCA_002738365.1 Scaffold 269.943 NEWM01 3500 43715
Land Plants
A. Khan et al.
32
Cucurbita pepo subsp. pepo Eukaryota; Plants; GCA_002806865.2 Chromosome 261.355 NHTM01 25263 43466
Land Plants
Cuscuta australis Eukaryota; Plants; GCA_003260385.1 Contig 262.63 NQVE01 218 18157
Land Plants
Cuscuta campestris Eukaryota; Plants; GCA_900332095.1 Scaffold 476.792 OOIL01 6907 0
Land Plants
Cynara cardunculus var. Eukaryota; Plants; GCA_001531365.1 Chromosome 725.198 LEKV01 13588 38406
scolymus Land Plants
Dactylis glomerata Eukaryota; Plants; GCA_007115705.1 Scaffold 1781.32 QXEO01 2117 0
Land Plants
Dactylis glomerata Eukaryota; Plants; GCA_002892645.1 Scaffold 839.915 MVYT01 1072009 0
Land Plants
Datisca glomerata Eukaryota; Plants; GCA_003255025.1 Scaffold 688.404 QANY01 13864 0
Land Plants
Daucus carota subsp. sativus Eukaryota; Plants; GCA_001625215.1 Chromosome 421.539 LNRQ01 4826 44655
Land Plants
Dendrobium catenatum Eukaryota; Plants; GCA_001605985.2 Scaffold 1104.26 JSDN02 286090 34389
Land Plants
Dianthus caryophyllus Eukaryota; Plants; GCA_000512335.1 Scaffold 567.662 BAUD01 45088 0
Land Plants
Dichanthelium oligosanthes Eukaryota; Plants; GCA_001633215.2 Scaffold 589.166 LWDX02 17436 26468
Land Plants
Dioscorea alata Eukaryota; Plants; GCA_002904275.2 Scaffold 620.909 CZHE02 57706 0
Land Plants
Dioscorea rotundata Eukaryota; Plants; GCA_002260605.1 Scaffold 594.227 BBQW01 4723 0
Land Plants
Dioscorea rotundata Eukaryota; Plants; GCA_002260665.1 Scaffold 730.21 BDML01 615107 0
Land Plants
(continued)
791
792
Dioscorea rotundata Eukaryota; Plants; GCA_002260645.1 Scaffold 683.283 BDMK01 641416 0
Land Plants
Dioscorea rotundata Eukaryota; Plants; GCA_002240015.2 Chromosome 456.675 BDMI01 21 0
Land Plants
Dioscorea sansibarensis Eukaryota; Plants; GCA_900631875.1 Contig 0.128321 CABFPD01 6 0
Land Plants
Diospyros lotus Eukaryota; Plants; GCA_000774125.1 Scaffold 1.10419 JRBH01 796 0
Land Plants
Dorcoceras hygrometricum Eukaryota; Plants; GCA_001598015.1 Scaffold 1521.36 LVEL01 401752 47778
Land Plants
Drosera capensis Eukaryota; Plants; GCA_001925005.1 Scaffold 263.788 LIEC01 12713 0
Land Plants
Dryas drummondii Eukaryota; Plants; GCA_003254865.1 Scaffold 225.547 QANW01 13357 0
Land Plants
Durio zibethinus Eukaryota; Plants; GCA_002303985.1 Scaffold 715.23 NSDW01 677 63007
Land Plants
Echinochloa crus-galli Eukaryota; Plants; GCA_900205405.1 Scaffold 1486.61 OAMR01 4534 0
Land Plants
Echium plantagineum Eukaryota; Plants; GCA_003412495.2 Chromosome 349.028 QFAX02 809 0
Land Plants
Eichhornia paniculata Eukaryota; Plants; GCA_001647135.1 Scaffold 571.388 LTAE01 40286 0
Land Plants
Elaeis guineensis Eukaryota; Plants; GCA_001672495.1 Scaffold 499.029 JRVM01 218141 0
Land Plants
Elaeis guineensis Eukaryota; Plants; GCA_002146295.1 Contig 134.97 AXCU01 186862 0
Land Plants
Elaeis guineensis Eukaryota; Plants; GCA_000442705.1 Chromosome 1535.18 ASJS01 40349 43551
Land Plants
A. Khan et al.
32
Elaeis oleifera Eukaryota; Plants; GCA_000441515.1 Scaffold 1402.73 ASIR01 26756 0

Land Plants
Elaeis oleifera Eukaryota; Plants; GCA_002146275.1 Contig 60.0019 AXCH01 93897 0
Land Plants
Eleusine coracana subsp. Eukaryota; Plants; GCA_002180455.1 Scaffold 1195.99 LXGH01 525627 0
coracana Land Plants
Eleusine indica Eukaryota; Plants; GCA_003369855.1 Scaffold 492.27 QEPD01 24072 0
Land Plants
Embelia ribes Eukaryota; Plants; GCA_001753735.1 Scaffold 660.51 MKEJ01 107000 0
Land Plants
Ensete ventricosum Eukaryota; Plants; GCA_000818735.3 Scaffold 451.279 JTFG03 45742 58438
Land Plants
Ensete ventricosum Eukaryota; Plants; GCA_001884845.1 Scaffold 444.842 MKKT01 51525 58998
Land Plants
Ensete ventricosum Eukaryota; Plants; GCA_001884805.1 Contig 429.48 MKKS01 60129 56086
Land Plants
Ensete ventricosum Eukaryota; Plants; GCA_000331365.3 Scaffold 437.269 AMZH03 52691 55115
Land Plants
Eragrostis curvula Eukaryota; Plants; GCA_007726485.1 Chromosome 603.072 RWGY01 1143 55182
Land Plants
Eragrostis tef Eukaryota; Plants; GCA_000970635.1 Scaffold 607.318 LAPY01 13883 0
Land Plants
Erigeron canadensis Eukaryota; Plants; GCA_000775935.1 Contig 326.165 JSWR01 20075 0
Land Plants
Erucastrum elatum Eukaryota; Plants; GCA_900406325.1 Scaffold 362.114 OVBX01 80289 0
Land Plants
Erysimum cheiri Eukaryota; Plants; GCA_900406345.1 Scaffold 147.321 OVAQ01 37531 0
Land Plants
(continued)
793
794
Erysimum pusillum Eukaryota; Plants; GCA_900406355.1 Scaffold 184.064 OVBW01 47441 0
Land Plants
Erythranthe guttata Eukaryota; Plants; GCA_000504015.1 Scaffold 322.167 APLE01 2212 31861
Land Plants
Eschscholzia californica subsp. Eukaryota; Plants; GCA_002897215.1 Scaffold 489.065 BEHA01 53253 0
californica Land Plants
Eucalyptus camaldulensis Eukaryota; Plants; GCA_000260855.1 Contig 654.922 BADO01 274001 0
Land Plants
Eucalyptus grandis Eukaryota; Plants; GCA_000612305.1 Scaffold 691.43 AUSX01 4951 52554
Land Plants
Eucalyptus melliodora Eukaryota; Plants; GCA_004368105.1 Scaffold 643.228 SISH01 423 0
Land Plants
Eucalyptus pauciflora Eukaryota; Plants; GCA_007663325.1 Scaffold 594.528 VMYD01 415 0
Land Plants
Euclidium syriacum Eukaryota; Plants; GCA_900116095.1 Scaffold 229.211 FPAK01 160 0
Land Plants
Eugenia uniflora Eukaryota; Plants; GCA_004012085.1 Contig 3.15213 RQIG01 2601 0
Land Plants
Euphorbia esula Eukaryota; Plants; GCA_002919075.1 Scaffold 1124.89 PJAD01 1633094 0
Land Plants
Euphorbia esula Eukaryota; Plants; GCA_002918425.1 Scaffold 639.02 PJAE01 912031 0
Land Plants
Eutrema heterophyllum Eukaryota; Plants; GCA_002933915.1 Scaffold 348.971 PKMM01 57686 0
Land Plants
Eutrema salsugineum Eukaryota; Plants; GCA_000478725.1 Scaffold 243.11 ANOA01 638 33637
Land Plants
Eutrema salsugineum Eukaryota; Plants; GCA_000325905.2 Chromosome 231.893 AHIU01 2663 0
Land Plants
A. Khan et al.
32
Eutrema yunnanense Eukaryota; Plants; GCA_002933935.1 Scaffold 415.364 PKML01 78020 0

Land Plants
Fagopyrum esculentum Eukaryota; Plants; GCA_004303065.1 Scaffold 1087.7 QWEW01 14903 0
Land Plants
Fagopyrum esculentum Eukaryota; Plants; GCA_001661195.1 Scaffold 1177.69 BCYN01 387594 0
Land Plants
Fagopyrum tataricum Eukaryota; Plants; GCA_002928575.1 Scaffold 526.768 PKMW01 2564 0
Land Plants
Fagopyrum tataricum Eukaryota; Plants; GCA_002319775.1 Chromosome 505.883 NCTC01 7020 0
Land Plants
Fagus sylvatica Eukaryota; Plants; GCA_003347535.1 Scaffold 428.2 QCXR01 8673 0
Land Plants
Ficus carica Eukaryota; Plants; GCA_002002945.1 Scaffold 247.091 BDEM01 27995 0
Land Plants
Ficus erecta Eukaryota; Plants; GCA_008635985.1 Contig 595.835 BKCH01 2455 0
Land Plants
Foeniculum vulgare Eukaryota; Plants; GCA_003724115.1 Scaffold 1010.97 PHNY01 300377 0
Land Plants
Fragaria iinumae Eukaryota; Plants; GCA_000511975.1 Scaffold 199.628 BATU01 117822 0
Land Plants
Fragaria nipponica Eukaryota; Plants; GCA_000512025.1 Scaffold 206.415 BATV01 215024 0
Land Plants
Fragaria nubicola Eukaryota; Plants; GCA_000511995.1 Scaffold 203.686 BATW01 210780 0
Land Plants
Fragaria orientalis Eukaryota; Plants; GCA_000517285.1 Scaffold 214.184 BATX01 323163 0
Land Plants
Fragaria vesca subsp. vesca Eukaryota; Plants; GCA_000184155.1 Chromosome 214.373 AEMH01 3048 31387
Land Plants
(continued)
795
796
Fragaria x ananassa Eukaryota; Plants; GCA_000511835.1 Scaffold 697.762 BATT01 625966 0
Land Plants
Fragaria x ananassa Eukaryota; Plants; GCA_000511695.1 Scaffold 173.23 BATS01 211588 0
Land Plants
Fraxinus excelsior Eukaryota; Plants; GCA_900149125.1 Scaffold 867.455 FTPI01 89515 0
Land Plants
Gastrodiaelata f. glauca Eukaryota; Plants; GCA_002966915.1 Scaffold 1060.98 PVEL01 3768 0
Land Plants
Genlisea aurea Eukaryota; Plants; GCA_000441915.1 Scaffold 43.3578 AUSU01 10684 17685
Land Plants
Geum urbanum Eukaryota; Plants; GCA_900236755.1 Scaffold 1217.04 OEJZ01 170029 0
Land Plants
Glycine max Eukaryota; Plants; GCA_001269945.2 Contig 927.706 BBNX02 108601 0
Land Plants
Glycine max Eukaryota; Plants; GCA_003349995.1 Chromosome 1017.57 QKRT01 495 0
Land Plants
Glycine max Eukaryota; Plants; GCA_002905335.2 Chromosome 1016.28 PELE01 475 0
Land Plants
Glycine max Eukaryota; Plants; GCA_000004515.4 Chromosome 979.046 ACUP03 1579 71219
Land Plants
Glycine soja Eukaryota; Plants; GCA_000722935.2 Scaffold 863.568 AZNC01 33170 50399
Land Plants
Glycine soja Eukaryota; Plants; GCA_004193775.2 Chromosome 1013.77 QZWG01 1120 69277
Land Plants
Glycine soja Eukaryota; Plants; GCA_002907465.1 Chromosome 985.26 PGFP01 805 0
Land Plants
Glycine tomentella Eukaryota; Plants; GCA_007407185.1 Scaffold 1694.09 PYAF01 6353 0
Land Plants
A. Khan et al.
32
Gossypioides kirkii Eukaryota; Plants; GCA_002818315.1 Chromosome 528.715 PEQG01 745 0

Land Plants
Gossypium arboreum Eukaryota; Plants; GCA_000787975.1 Scaffold 1862.24 JRRC01 392831 33609
Land Plants
Gossypium arboreum Eukaryota; Plants; GCA_000612285.2 Chromosome 1694.6 AYOE01 75419 47568
Land Plants
Gossypium australe Eukaryota; Plants; GCA_005393395.2 Chromosome 1743.39 SMMG02 564 38281
Land Plants
Gossypium barbadense Eukaryota; Plants; GCA_002928715.1 Scaffold 1394.24 LAGA01 9269 40359
Land Plants
Gossypium barbadense Eukaryota; Plants; GCA_001856525.1 Scaffold 2566.74 AXCG01 29751 0
Land Plants
Gossypium barbadense Eukaryota; Plants; GCA_002926015.1 Scaffold 775.252 LAGB01 4265 36871
Land Plants
Gossypium barbadense Eukaryota; Plants; GCA_008761655.1 Chromosome 2195.8 VKDL01 4748 108363
Land Plants
Gossypium darwinii Eukaryota; Plants; GCA_007990325.1 Chromosome 2182.96 VKGI01 821 97407
Land Plants
Gossypium hirsutum Eukaryota; Plants; GCA_006980745.1 Chromosome 2287.87 VCQY01 599 0
Land Plants
Gossypium hirsutum Eukaryota; Plants; GCA_006980775.1 Chromosome 2308.22 VCQX01 2238 0
Land Plants
Gossypium hirsutum Eukaryota; Plants; GCA_000987745.1 Chromosome 2189.14 LBLM01 9148 90927
Land Plants
Gossypium mustelinum Eukaryota; Plants; GCA_007990455.1 Chromosome 2315.09 VKGF01 2146 106487
Land Plants
Gossypium raimondii Eukaryota; Plants; GCA_000331045.1 Scaffold 773.768 AMOP01 4699 0
Land Plants
(continued)
797
798
Gossypium raimondii Eukaryota; Plants; GCA_000327365.1 Chromosome 761.565 ALYE01 1034 59057
Land Plants
Gossypium thurberi Eukaryota; Plants; GCA_004027125.1 Chromosome 582.007 RCOT01 15297 0
Land Plants
Gossypium tomentosum Eukaryota; Plants; GCA_007990485.1 Chromosome 2193.56 VKGE01 749 112713
Land Plants
Handroanthus impetiginosus Eukaryota; Plants; GCA_002762385.1 Scaffold 503.289 NKXS01 13204 30271
Land Plants
Helianthus annuus Eukaryota; Plants; GCA_002127325.1 Chromosome 3027.84 MNCJ01 1528 73839
Land Plants
Heliophila coronopifolia Eukaryota; Plants; GCA_900406365.1 Scaffold 293.602 OVAW01 212649 0
Land Plants
Herrania umbratica Eukaryota; Plants; GCA_002168275.2 Scaffold 234.039 NHTG01 6074 27748
Land Plants
Hevea brasiliensis Eukaryota; Plants; GCA_001654055.1 Scaffold 1373.53 LVXX01 7453 58062
Land Plants
Hevea brasiliensis Eukaryota; Plants; GCA_002003025.1 Scaffold 1256.27 BDHL01 592579 0
Land Plants
Hevea brasiliensis Eukaryota; Plants; GCA_001907995.1 Scaffold 1550.51 MKXE01 189320 0
Land Plants
Hevea brasiliensis Eukaryota; Plants; GCA_000340545.1 Scaffold 1301.4 AJJZ01 1150326 0
Land Plants
Hibiscus syriacus Eukaryota; Plants; GCA_006381635.1 Scaffold 2573.67 VEPZ01 9646 0
Land Plants
Hibiscus syriacus Eukaryota; Plants; GCA_001696755.1 Scaffold 1748.25 MBGJ01 77488 0
Land Plants
Hordeum bulbosum Eukaryota; Plants; GCA_900070015.1 Scaffold 1294.87 CBQS01 2883554 0
Land Plants
A. Khan et al.
32
Hordeum pubiflorum Eukaryota; Plants; GCA_000582825.1 Scaffold 1425.27 CBMN01 1818420 0

Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_004114815.1 Scaffold 4006.12 SDOW01 1856 0
Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_900075435.2 Scaffold 9788.86 FJWB02 72295 0
Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_900067795.1 Contig 161.166 CEGI01 589134 0
Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_900067805.1 Contig 160.635 CEGJ01 598302 0
Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_900067785.1 Contig 316.11 CEGM01 1201146 0
Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_900067825.1 Contig 207.795 CEGK01 800872 0
Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_900067815.1 Contig 233.129 CEGL01 908607 0
Land Plants
Hordeum vulgare Eukaryota; Plants; GCA_000947855.1 Contig 98.056 CEGH01 391258 0
Land Plants
Hordeum vulgare subsp. Eukaryota; Plants; GCA_000326125.1 Scaffold 1779.49 CAJX01 2077901 0
vulgare Land Plants
Hordeum vulgare subsp. Eukaryota; Plants; GCA_001077415.1 Scaffold 1645.58 CBLZ01 2280908 0
vulgare Land Plants
Hordeum vulgare subsp. Eukaryota; Plants; GCA_000326085.1 Scaffold 1868.64 CAJW01 2670738 0
vulgare Land Plants
Hordeum vulgare subsp. Eukaryota; Plants; GCA_900002345.1 Scaffold 1825.17 CCJR01 2546226 0
vulgare Land Plants
Hordeum vulgare subsp. Eukaryota; Plants; GCA_002900805.1 Scaffold 2019.37 CAJV01 2742077 0
vulgare Land Plants
(continued)
799
800
Hordeum vulgare subsp. Eukaryota; Plants; GCA_902500625.1 Scaffold 4129.36 CABVVH01 8 0
vulgare Land Plants
Hordeum vulgare subsp. Eukaryota; Plants; GCA_000227425.1 Contig 28.016 BACC01 8583 0
vulgare Land Plants
Hordeum vulgare subsp. Eukaryota; Plants; GCA_002943585.1 Contig 57.9644 PKQG01 14158 0
vulgare Land Plants
Humulus lupulus var. Eukaryota; Plants; GCA_000830395.1 Scaffold 2049.21 BBPB01 132476 0
cordifolius Land Plants
Humulus lupulus var. lupulus Eukaryota; Plants; GCA_000831365.1 Scaffold 2049.21 BBPC01 132476 0
Land Plants
Iberis amara Eukaryota; Plants; GCA_900406375.1 Scaffold 361.245 OVAV01 191171 0
Land Plants
Iberis pinnata Eukaryota; Plants; GCA_900406425.1 Scaffold 686.283 OVBE01 141542 0
Land Plants
Ipomoea batatas Eukaryota; Plants; GCA_900092185.1 Contig 13.8613 FLTB01 41487 0
Land Plants
Ipomoea batatas Eukaryota; Plants; GCA_002525835.2 Chromosome 837.013 NXFB01 28461 0
Land Plants
Ipomoea nil Eukaryota; Plants; GCA_001879475.1 Scaffold 735.231 BDFN01 3418 51054
Land Plants
Ipomoea trifida Eukaryota; Plants; GCA_000978395.1 Scaffold 512.991 BBOG01 77400 0
Land Plants
Ipomoea trifida Eukaryota; Plants; GCA_000981105.1 Scaffold 712.155 BBOH01 181194 0
Land Plants
Ipomoea trifida Eukaryota; Plants; GCA_004706985.1 Chromosome 460.934 SMMV01 4236 0
Land Plants
Isatis lusitanica Eukaryota; Plants; GCA_900406415.1 Scaffold 203.919 OVBB01 61090 0
Land Plants
A. Khan et al.
32
Isatis tinctoria Eukaryota; Plants; GCA_900406385.1 Scaffold 244.177 OVBD01 106900 0

Land Plants
Jaltoma tasinuosa Eukaryota; Plants; GCA_003996215.1 Scaffold 1443.2 QJPP01 7667 0
Land Plants
Jatropha curcas Eukaryota; Plants; GCA_000696525.1 Scaffold 318.527 AFEW01 6024 32547
Land Plants
Jatropha curcas Eukaryota; Plants; GCA_004143595.1 Scaffold 265.767 QFZG01 2959 0
Land Plants
Jatropha curcas Eukaryota; Plants; GCA_000208675.2 Scaffold 297.661 BABX02 39277 0
Land Plants
Juglans cathayensis Eukaryota; Plants; GCA_003122765.1 Scaffold 600.151 QEOU01 19972 0
Land Plants
Juglans hindsii Eukaryota; Plants; GCA_003123825.1 Scaffold 611.109 QEOW01 73433 0
Land Plants
Juglans mandshurica Eukaryota; Plants; GCA_002916435.1 Scaffold 558.071 PKSJ01 13809 0
Land Plants
Juglans microcarpa Eukaryota; Plants; GCA_003123845.1 Scaffold 913.972 QEOX01 112570 0
Land Plants
Juglans microcarpa x Juglans Eukaryota; Plants; GCA_004785585.1 Chromosome 534.672 QKZY01 73 0
regia Land Plants
Juglans microcarpa x Juglans Eukaryota; Plants; GCA_004785595.1 Chromosome 527.896 QKZX01 154 0
regia Land Plants
Juglans nigra Eukaryota; Plants; GCA_003123865.1 Scaffold 620.767 QEOV01 90472 0
Land Plants
Juglans nigra Eukaryota; Plants; GCA_002916485.1 Scaffold 682.557 PKSI01 18575 0
Land Plants
Juglans regia Eukaryota; Plants; GCA_001411555.1 Scaffold 699.673 LIHL01 105803 55627
Land Plants
(continued)
801
802
Juglans regia Eukaryota; Plants; GCA_003122785.1 Scaffold 650.478 QEOZ01 4401 0
Land Plants
Juglans regia Eukaryota; Plants; GCA_002916465.1 Scaffold 634.748 PKSH01 25789 0
Land Plants
Juglans sigillata Eukaryota; Plants; GCA_003123805.1 Scaffold 648.117 QEOY01 134300 0
Land Plants
Kalanchoe fedtschenkoi Eukaryota; Plants; GCA_002312845.1 Scaffold 256.351 NQLW01 1324 0
Land Plants
Kernera saxatilis Eukaryota; Plants; GCA_900406395.1 Scaffold 143.969 OVAY01 18372 0
Land Plants
Kokia drynarioides Eukaryota; Plants; GCA_002814295.1 Scaffold 517.43 NTFQ01 15383 0
Land Plants
Lactuca sativa Eukaryota; Plants; GCA_002870075.1 Scaffold 2384.19 NBSK01 11453 45242
Land Plants
Lactuca sativa Eukaryota; Plants; GCA_900243165.1 Scaffold 2224.43 OFAD01 161898 0
Land Plants
Lactuca sativa Eukaryota; Plants; GCA_900198505.1 Scaffold 1975.25 FZNH01 138326 0
Land Plants
Lactuca sativa Eukaryota; Plants; GCA_000227445.1 Contig 1133.66 AFSA01 876110 0
Land Plants
Lagenaria siceraria Eukaryota; Plants; GCA_003268545.1 Scaffold 313.387 NHZF01 438 0
Land Plants
Lagenaria siceraria Eukaryota; Plants; GCA_000466325.1 Scaffold 176.727 ATBX01 305112 0
Land Plants
Lagenaria siceraria Eukaryota; Plants; GCA_002890555.2 Chromosome 297.879 MIMD02 27 0
Land Plants
Larix sibirica Eukaryota; Plants; GCA_004151065.1 Scaffold 12342.1 NWUY01 11325800 0
Land Plants
A. Khan et al.
32
Leavenworthiaalabamica Eukaryota; Plants; GCA_000411055.1 Scaffold 173.432 ASXC01 11715 0

Land Plants
Leersia perrieri Eukaryota; Plants; GCA_000325765.3 Chromosome 266.688 ALNV02 12 0
Land Plants
Lepidium africanum Eukaryota; Plants; GCA_900406405.1 Scaffold 225.262 OVAX01 17445 0
Land Plants
Lepidium aucheri Eukaryota; Plants; GCA_900406435.1 Scaffold 332.063 OVBA01 31191 0
Land Plants
Lindernia brevidens Eukaryota; Plants; GCA_004919715.1 Scaffold 266.105 SWDC01 158 0
Land Plants
Liriodendron chinense Eukaryota; Plants; GCA_003013855.2 Scaffold 1742.42 PVNU02 3710 0
Land Plants
Lolium perenne Eukaryota; Plants; GCA_001735685.1 Scaffold 481.479 MEHO01 666180 0
Land Plants
Lophocereus schottii Eukaryota; Plants; GCA_002740545.1 Scaffold 797.926 NCQV01 158704 0
Land Plants
Lotus japonicus Eukaryota; Plants; GCA_000181115.2 Contig 394.455 BABK02 44464 0
Land Plants
Lupinus angustifolius Eukaryota; Plants; GCA_000338175.1 Scaffold 523.298 AOCW01 71995 0
Land Plants
Lupinus angustifolius Eukaryota; Plants; GCA_001865875.1 Chromosome 609.203 MLAU01 14378 52821
Land Plants
Macadamia integrifolia Eukaryota; Plants; GCA_900631585.1 Scaffold 744.636 UZVR01 4098 0
Land Plants
Macadamia integrifolia Eukaryota; Plants; GCA_900087525.1 Scaffold 518.49 FLKO01 193493 0
Land Plants
Macleaya cordata Eukaryota; Plants; GCA_002174775.1 Scaffold 377.834 MVGT01 4547 21911
Land Plants
(continued)
803
804
Macropodium nivale Eukaryota; Plants; GCA_900406455.1 Scaffold 300.735 OVBF01 122604 0
Land Plants
Magnolia ashei Eukaryota; Plants; GCA_003571905.1 Scaffold 284.512 PCNC01 265493 0
Land Plants
Malus baccata Eukaryota; Plants; GCA_006547085.1 Scaffold 674.412 VIEB01 47473 45900
Land Plants
Malus domestica Eukaryota; Plants; GCA_004115385.1 Chromosome 660.463 RDQH01 343 42841
Land Plants
Malus domestica Eukaryota; Plants; GCA_000148765.2 Chromosome 1874.77 ACYM01 1667 0
Land Plants
Malus domestica Eukaryota; Plants; GCA_002114115.1 Chromosome 703.358 MJAX01 807 52039
Land Plants
Manihot esculenta Eukaryota; Plants; GCA_000737115.1 Scaffold 292.098 JPQF01 65771 0
Land Plants
Manihot esculenta Eukaryota; Plants; GCA_003957885.1 Scaffold 1276.89 RSFS01 4440 0
Land Plants
Manihot esculenta Eukaryota; Plants; GCA_003957995.1 Scaffold 1224.64 RSFT01 5398 0
Land Plants
Manihot esculenta Eukaryota; Plants; GCA_001659605.1 Chromosome 582.279 LTYI01 2020 43286
Land Plants
Manihot esculenta subsp. Eukaryota; Plants; GCA_000737105.1 Scaffold 390.836 JPQE01 54016 0
flabellifolia Land Plants
Marchantia inflexa Eukaryota; Plants; GCA_006177815.1 Scaffold 208.753 QLSQ01 41556 0
Land Plants
Marchantia polymorpha Eukaryota; Plants; GCA_003032435.1 Scaffold 225.761 PNPG01 2957 24674
Land Plants
Marchantia polymorpha subsp. Eukaryota; Plants; GCA_001641455.1 Scaffold 205.718 LVLJ01 4137 17956
ruderalis Land Plants
A. Khan et al.
32
Medicago truncatula Eukaryota; Plants; GCA_003473485.2 Chromosome 429.612 PSQE01 40 44450

Land Plants
Medicago truncatula Eukaryota; Plants; GCA_002024945.1 Scaffold 402.065 MWMB01 909 0
Land Plants
Medicago truncatula Eukaryota; Plants; GCA_002251925.1 Scaffold 427.447 MLKM01 2368 0
Land Plants
Medicago truncatula Eukaryota; Plants; GCA_002251935.1 Scaffold 394.059 MKZU01 1490 0
Land Plants
Medicago truncatula Eukaryota; Plants; GCA_002251955.1 Scaffold 426.024 MNAG01 2700 0
Land Plants
Medicago truncatula Eukaryota; Plants; GCA_000219495.2 Chromosome 412.924 APNO01 2187 41939
Land Plants
Mentha longifolia Eukaryota; Plants; GCA_001642375.1 Scaffold 353.287 LSBG01 190876 0
Land Plants
Metrosideros polymorpha var. Eukaryota; Plants; GCA_001662345.1 Scaffold 304.366 BCNH01 36376 0
glaberrima Land Plants
Mimosa pudica Eukaryota; Plants; GCA_003254945.1 Scaffold 557.202 QANV01 97892 0
Land Plants
Miscanthus sacchariflorus Eukaryota; Plants; GCA_002993905.1 Chromosome 2074.92 PUID01 137916 0
Land Plants
Momordica charantia Eukaryota; Plants; GCA_001995035.1 Scaffold 285.614 BDCS01 1052 28666
Land Plants
Momordica charantia Eukaryota; Plants; GCA_900491585.1 Scaffold 296.263 UESV01 3101 0
Land Plants
Monotropa hypopitys Eukaryota; Plants; GCA_002855965.1 Contig 2197.49 NMUG01 1259264 0
Land Plants
Morella rubra Eukaryota; Plants; GCA_003952965.1 Chromosome 313.02 RXIC01 500 0
Land Plants
(continued)
805
806
Morusn otabilis Eukaryota; Plants; GCA_000414095.2 Scaffold 320.379 ATGF01 31301 27648
Land Plants
Mucuna pruriens Eukaryota; Plants; GCA_003370565.1 Scaffold 397.042 QJKJ01 18487 56019
Land Plants
Musa acuminata subsp. Eukaryota; Plants; GCA_000313855.2 Chromosome 472.231 CAIC01 7512 47707
malaccensis Land Plants
Musa balbisiana Eukaryota; Plants; GCA_004837865.1 Chromosome 492.775 PYDT01 2590 33021
Land Plants
Musa itinerans Eukaryota; Plants; GCA_001649415.1 Scaffold 455.349 LVTN01 28415 0
Land Plants
Musa schizocarpa Eukaryota; Plants; GCA_900464855.1 Scaffold 525.283 UBIG01 194 0
Land Plants
Nasturtium officinale Eukaryota; Plants; GCA_900406445.1 Scaffold 216.122 OVAZ01 10793 0
Land Plants
Nelumbo nucifera Eukaryota; Plants; GCA_000365185.2 Scaffold 804.648 AQOG01 3603 38191
Land Plants
Nelumbo nucifera Eukaryota; Plants; GCA_000805495.1 Scaffold 790.339 APLB01 14895 0
Land Plants
Nelumbo nucifera Eukaryota; Plants; GCA_003033685.1 Chromosome 817.268 DLUB01 2341 0
Land Plants
Nelumbo nucifera Eukaryota; Plants; GCA_003033695.1 Chromosome 799.479 DLUA01 12643 0
Land Plants
Nicotiana attenuata Eukaryota; Plants; GCA_002018495.1 Scaffold 1827.78 MCOF01 951503 0
Land Plants
Nicotiana attenuata Eukaryota; Plants; GCA_001879085.1 Chromosome 2365.68 MJEQ01 37194 44491
Land Plants
Nicotiana benthamiana Eukaryota; Plants; GCA_000723945.1 Contig 61.9511 CBMM01 100480 0
Land Plants
A. Khan et al.
32
Nicotiana glauca Eukaryota; Plants; GCA_002930595.1 Scaffold 3222.83 PGPE01 514289 0

Land Plants
Nicotiana knightiana Eukaryota; Plants; GCA_005239525.1 Scaffold 2298.94 MDKJ01 160415 0
Land Plants
Nicotiana obtusifolia Eukaryota; Plants; GCA_002018475.1 Scaffold 1222.77 MCJB01 53128 0
Land Plants
Nicotiana otophora Eukaryota; Plants; GCA_000715115.1 Scaffold 2689.35 AWOL01 929607 0
Land Plants
Nicotiana paniculata Eukaryota; Plants; GCA_005239505.1 Scaffold 2190.56 MDKI01 181977 0
Land Plants
Nicotiana rustica Eukaryota; Plants; GCA_005239535.1 Scaffold 4231.29 MDKG01 337581 0
Land Plants
Nicotiana sylvestris Eukaryota; Plants; GCA_000393655.1 Scaffold 2221.99 ASAF01 253918 48160
Land Plants
Nicotiana tabacum Eukaryota; Plants; GCA_000715135.1 Scaffold 3643.47 AYMY01 168247 84255
Land Plants
Nicotiana tabacum Eukaryota; Plants; GCA_000715075.1 Scaffold 3732.64 AWOJ01 582565 0
Land Plants
Nicotiana tabacum Eukaryota; Plants; GCA_000715095.1 Scaffold 3735.82 AWOK01 643545 0
Land Plants
Nicotiana tabacum Eukaryota; Plants; GCA_002210045.1 Scaffold 4646.65 NCAA01 937112 0
Land Plants
Nicotiana tomentosiformis Eukaryota; Plants; GCA_000390325.2 Scaffold 1688.47 ASAG01 159548 48963
Land Plants
Nicotiana undulata Eukaryota; Plants; GCA_005239495.1 Scaffold 1914.3 MDKH01 117566 0
Land Plants
Nissolia schottii Eukaryota; Plants; GCA_003254905.1 Scaffold 466.099 QANU01 116213 0
Land Plants
(continued)
807
808
Noccaea caerulescens Eukaryota; Plants; GCA_900406465.1 Scaffold 140.792 OVBC01 19808 0
Land Plants
Noccaea goesingensis Eukaryota; Plants; GCA_900406475.1 Scaffold 150.323 OVBG01 183017 0
Land Plants
Nothapodytes nimmoniana Eukaryota; Plants; GCA_002091855.1 Contig 1.36527 BDGC01 2301 0
Land Plants
Nymphaea colorata Eukaryota; Plants; GCA_902499525.1 Scaffold 409.931 CABVML01 806 0
Land Plants
Nymphaea colorata Eukaryota; Plants; GCA_008831285.1 Chromosome 408.397 VYXN01 799 0
Land Plants
Nyssa sinensis Eukaryota; Plants; GCA_008638375.1 Chromosome 1001.45 VIRR01 654 36241
Land Plants
Ochetophila trinervis Eukaryota; Plants; GCA_003254975.1 Scaffold 309.116 QANX01 8237 0
Land Plants
Ocimum tenuiflorum Eukaryota; Plants; GCA_001278415.1 Contig 332.617 AYJT01 121993 0
Land Plants
Ocimum tenuiflorum Eukaryota; Plants; GCA_001748785.1 Contig 311.125 JQCZ01 230018 0
Land Plants
Odon tarrhena argentea Eukaryota; Plants; GCA_900406245.1 Scaffold 183.186 OVAF01 32097 0
Land Plants
Oenanthe javanica Eukaryota; Plants; GCA_008931105.1 Scaffold 1278.51 QRFB01 149923 0
Land Plants
Olea europaea subsp. europaea Eukaryota; Plants; GCA_900603015.1 Scaffold 1318.65 UWJE01 11038 0
Land Plants
Olea europaea var. sylvestris Eukaryota; Plants; GCA_002742605.1 Chromosome 1141.15 MSRW01 41226 58334
Land Plants
Oropetium thomaeum Eukaryota; Plants; GCA_001182835.1 Contig 243.175 LFJQ01 625 0
Land Plants
A. Khan et al.
32
Oryza barthii Eukaryota; Plants; GCA_002926215.1 Scaffold 295.586 PQXR01 46663 0

Land Plants
Oryza barthii Eukaryota; Plants; GCA_002926235.1 Scaffold 294.191 PQXQ01 52511 0
Land Plants
Oryza barthii Eukaryota; Plants; GCA_003020155.1 Scaffold 292.235 PTLQ01 66245 0
Land Plants
Oryza barthii Eukaryota; Plants; GCA_000182155.3 Chromosome 308.272 ABRL02 12 0
Land Plants
Oryza brachyantha Eukaryota; Plants; GCA_000710545.1 Chromosome 14.4404 JNWF01 1 0
Land Plants
Oryza brachyantha Eukaryota; Plants; GCA_000231095.2 Chromosome 259.908 AGAT01 2491 26803
Land Plants
Oryza glaberrima Eukaryota; Plants; GCA_000147395.2 Scaffold 303.295 ADWL01 25599 0
Land Plants
Oryza glumipatula Eukaryota; Plants; GCA_000576495.1 Chromosome 372.86 ALNU02 12 0
Land Plants
Oryza longistaminata Eukaryota; Plants; GCA_000789195.1 Scaffold 326.443 AMDW01 60198 0
Land Plants
Oryza longistaminata Eukaryota; Plants; GCA_001514335.2 Chromosome 362.064 LQBC01 11745 0
Land Plants
Oryza meridionalis Eukaryota; Plants; GCA_001551795.1 Contig 354.611 LONC01 3249 0
Land Plants
Oryza meridionalis Eukaryota; Plants; GCA_000338895.2 Chromosome 335.668 ALNW02 12 0
Land Plants
Oryza meyeriana var. granulata Eukaryota; Plants; GCA_005223365.1 Scaffold 736.649 SPHZ01 2389 0
Land Plants
Oryza meyeriana var. granulata Eukaryota; Plants; GCA_003991445.1 Contig 776.957 RYFJ01 4618 0
Land Plants
(continued)
809
810
Oryza meyeriana var. granulata Eukaryota; Plants; GCA_000325645.2 Scaffold 35.2457 ALNT01 1 0
Land Plants
Oryza minuta Eukaryota; Plants; GCA_000632695.1 Chromosome 45.1659 JJNN01 2 0
Land Plants
Oryza officinalis Eukaryota; Plants; GCA_008326285.1 Scaffold 584.134 BDMV01 91 0
Land Plants
Oryza officinalis Eukaryota; Plants; GCA_000717455.1 Chromosome 26.1885 JJMQ01 1 0
Land Plants
Oryza punctata Eukaryota; Plants; GCA_000710525.1 Chromosome 22.4654 JNWE01 1 0
Land Plants
Oryza punctata Eukaryota; Plants; GCA_000573905.1 Chromosome 393.817 AVCL01 12 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_000817225.1 Scaffold 339.177 CBQP01 3818 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_001551805.1 Contig 384.518 LONB01 2582 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609075.1 Contig 0.391959 UXAX01 64 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609175.1 Contig 0.439665 UXBI01 116 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609365.1 Contig 0.435203 UXBZ01 67 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609345.1 Contig 0.430311 UXBX01 112 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609235.1 Contig 0.431171 UXBN01 130 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609435.1 Contig 0.460371 UXCI01 141 0
Land Plants
A. Khan et al.
32
Oryza rufipogon Eukaryota; Plants; GCA_900609385.1 Contig 0.522775 UXCD01 399 0

Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609605.1 Contig 0.479941 UXCZ01 272 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609155.1 Contig 0.451183 UXBF01 185 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609195.1 Contig 0.444689 UXBK01 168 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609375.1 Contig 0.418576 UXCA01 72 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609635.1 Contig 0.437219 UXDC01 115 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609335.1 Contig 0.426264 UXBY01 80 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609565.1 Contig 0.416343 UXCV01 99 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609455.1 Contig 0.437726 UXCL01 172 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609145.1 Contig 0.4324 UXBJ01 128 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609205.1 Contig 0.429589 UXBM01 129 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609535.1 Contig 0.425584 UXCS01 176 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609445.1 Contig 0.428734 UXCJ01 87 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609595.1 Contig 0.445408 UXDA01 162 0
Land Plants
(continued)
811
812
Oryza rufipogon Eukaryota; Plants; GCA_900609645.1 Contig 0.448673 UXDD01 155 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609285.1 Contig 0.420471 UXBV01 143 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609165.1 Contig 0.426734 UXBG01 121 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609325.1 Contig 0.456272 UXCH01 231 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609355.1 Contig 0.427134 UXCB01 114 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609415.1 Contig 0.439065 UXCE01 125 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609575.1 Contig 0.426419 UXCW01 113 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609125.1 Contig 0.461807 UXBC01 223 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609475.1 Contig 0.435078 UXCN01 159 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609215.1 Contig 0.433062 UXBO01 157 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609395.1 Contig 0.399871 UXCF01 66 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609585.1 Contig 0.433896 UXCY01 137 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609555.1 Contig 0.423266 UXCT01 170 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609295.1 Contig 0.403327 UXBR01 109 0
Land Plants
A. Khan et al.
32
Oryza rufipogon Eukaryota; Plants; GCA_900609545.1 Contig 0.436432 UXCU01 221 0

Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609185.1 Contig 0.434658 UXBH01 155 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609525.1 Contig 0.44213 UXCQ01 233 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609105.1 Contig 0.42703 UXBD01 130 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609615.1 Contig 0.4457 UXCX01 188 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609505.1 Contig 0.49038 UXCR01 321 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609135.1 Contig 0.424383 UXBE01 108 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609495.1 Contig 0.429759 UXCM01 130 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609305.1 Contig 0.531837 UXBS01 488 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609625.1 Contig 0.442979 UXDB01 201 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609265.1 Contig 0.430376 UXBQ01 146 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609245.1 Contig 0.441818 UXBP01 94 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609405.1 Contig 0.466007 UXCC01 262 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609225.1 Contig 0.428872 UXBL01 138 0
Land Plants
(continued)
813
814
Oryza rufipogon Eukaryota; Plants; GCA_900609465.1 Contig 0.420063 UXCK01 121 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609255.1 Contig 0.551867 UXBT01 457 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609515.1 Contig 0.438855 UXCP01 218 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609085.1 Contig 0.467544 UXAY01 265 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609275.1 Contig 0.552388 UXBU01 559 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609095.1 Contig 0.4166 UXBA01 216 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609315.1 Contig 0.576561 UXBW01 564 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609485.1 Contig 0.512907 UXCO01 440 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609425.1 Contig 0.5568 UXCG01 519 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609115.1 Contig 0.416778 UXBB01 188 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_900609665.1 Contig 0.521122 UXDG01 471 0
Land Plants
Oryza rufipogon Eukaryota; Plants; GCA_000700045.1 Chromosome 12.7409 JNHC01 1 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_001648735.1 Scaffold 307.225 LVCG01 55637 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_001648745.1 Scaffold 295.39 LVCH01 64800 0
Land Plants
A. Khan et al.
32
Oryza sativa Eukaryota; Plants; GCA_900609875.1 Contig 0.418428 UXEA01 94 0

Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609755.1 Contig 0.412758 UXDO01 77 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900610885.1 Contig 0.415805 UXHM01 101 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609945.1 Contig 0.420498 UXEH01 97 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609745.1 Contig 0.412413 UXDL01 77 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609855.1 Contig 0.424341 UXDZ01 111 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609765.1 Contig 0.426436 UXDS01 105 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609895.1 Contig 0.420731 UXED01 102 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609705.1 Contig 0.413182 UXDI01 83 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609835.1 Contig 0.414506 UXDX01 86 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609655.1 Contig 0.4229 UXDE01 95 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609715.1 Contig 0.421534 UXDK01 109 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609735.1 Contig 0.412833 UXDM01 71 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609915.1 Contig 0.417059 UXEE01 85 0
Land Plants
(continued)
815
816
Oryza sativa Eukaryota; Plants; GCA_900609955.1 Contig 0.41464 UXEI01 83 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609885.1 Contig 0.415361 UXEC01 81 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900610895.1 Contig 0.414252 UXHO01 91 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609905.1 Contig 0.416009 UXEB01 104 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609865.1 Contig 0.4183 UXDY01 102 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609845.1 Contig 0.424074 UXDW01 114 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609685.1 Contig 0.415611 UXDH01 101 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609925.1 Contig 0.412137 UXEF01 98 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609695.1 Contig 0.414261 UXDJ01 76 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609775.1 Contig 0.411428 UXDP01 78 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609825.1 Contig 0.415893 UXDV01 83 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609725.1 Contig 0.413692 UXDN01 95 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609805.1 Contig 0.411297 UXDU01 88 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609675.1 Contig 0.413512 UXDF01 83 0
Land Plants
A. Khan et al.
32
Oryza sativa Eukaryota; Plants; GCA_900609935.1 Contig 0.415952 UXEG01 100 0

Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609815.1 Contig 0.405341 UXDT01 71 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609785.1 Contig 0.42347 UXDQ01 148 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_900609795.1 Contig 0.422231 UXDR01 125 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_003865215.1 Chromosome 395.354 PKRX01 127 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_004007595.1 Chromosome 377.604 RPSM01 615 0
Land Plants
Oryza sativa Eukaryota; Plants; GCA_004348155.2 Chromosome 415.393 QQAJ01 367 0
Land Plants
Oryza sativa aus subgroup Eukaryota; Plants; GCA_001952365.2 Chromosome 372.203 LWDA01 1312 0
Land Plants
Oryza sativa f. spontanea Eukaryota; Plants; GCA_006942195.1 Scaffold 377.675 QKSA01 350 0
Land Plants
Oryza sativa f. spontanea Eukaryota; Plants; GCA_000710535.2 Chromosome 19.4244 JNWG02 1 0
Land Plants
Oryza sativa f. spontanea Eukaryota; Plants; GCA_000576065.1 Chromosome 337.95 AWHD01 12 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_001623365.2 Chromosome 387.424 LNNK02 19 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_001623345.2 Chromosome 387.326 LNNJ02 20 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_001889745.1 Chromosome 389.088 MPPV01 66 0
Land Plants
(continued)
817
818
Oryza sativa Indica Group Eukaryota; Plants; GCA_001618795.1 Chromosome 386.486 LBBA01 8481 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_000725085.2 Chromosome 389.753 AZTA02 15907 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_001618785.1 Chromosome 398.762 LBAZ01 11486 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_000004655.2 Chromosome 426.337 AAAA02 10627 37358
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_003449045.1 Contig 388.772 QWGD01 410 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_001611195.1 Scaffold 351.225 LQHG01 39669 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_001611255.1 Scaffold 331.819 LQHF01 75421 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_001611235.1 Scaffold 352.227 LQHE01 116212 0
Land Plants
Oryza sativa Indica Group Eukaryota; Plants; GCA_006992885.1 Scaffold 281.326 SWLY01 91264 0
Land Plants
Oryza sativa Japonica Group Eukaryota; Plants; GCA_003449065.1 Contig 378.097 QWGC01 144 0
Land Plants
Oryza sativa Japonica Group Eukaryota; Plants; GCA_002573525.1 Contig 418.901 PDFQ01 588 0
Land Plants
Oryza sativa Japonica Group Eukaryota; Plants; GCA_003865235.1 Chromosome 379.626 PKRW01 115 0
Land Plants
Oryza sativa Japonica Group Eukaryota; Plants; GCA_000817635.1 Chromosome 337.74 JSUG01 1739 0
Land Plants
Oryza sativa Japonica Group Eukaryota; Plants; GCA_000817615.1 Chromosome 342.028 JSUF01 1160 0
Land Plants
A. Khan et al.
32
Oryza sativa Japonica Group Eukaryota; Plants; GCA_000149285.1 Chromosome 391.148 AACV01 7777 35394
Land Plants
Oryza sativa Japonica Group Eukaryota; Plants; GCA_000321445.1 Chromosome 382.627 BACJ01 12 0
Land Plants
Oryza sativa Japonica Group Eukaryota; Plants; GCA_000164945.1 Chromosome 382.151 BABO01 12 0
Land Plants
Pachycereus pringlei Eukaryota; Plants; GCA_002740445.1 Scaffold 629.656 NCQS01 171584 0
Land Plants
Panicum hallii Eukaryota; Plants; GCA_002211085.2 Chromosome 535.889 NCQW02 1027 37612
Land Plants
Panicum hallii var. hallii Eukaryota; Plants; GCA_003061485.1 Chromosome 487.474 QAVV01 144 42523
Land Plants
Panicum miliaceum Eukaryota; Plants; GCA_002895445.2 Chromosome 848.352 PPDP02 466 0
Land Plants
Panicum miliaceum Eukaryota; Plants; GCA_003046395.2 Chromosome 854.793 PQIB02 1306 55964
Land Plants
Papaver somniferum Eukaryota; Plants; GCA_003573695.1 Chromosome 2715.53 PUWZ01 34381 84179
Land Plants
Parasponia andersonii Eukaryota; Plants; GCA_002914805.1 Scaffold 475.834 JXTB01 2732 37227
Land Plants
Passiflora edulis Eukaryota; Plants; GCA_002156105.1 Scaffold 165.657 MUZT01 234012 0
Land Plants
Penstemon barbatus Eukaryota; Plants; GCA_003313485.1 Contig 696.306 QOIQ01 18827 0
Land Plants
Penstemon centranthifolius Eukaryota; Plants; GCA_000737435.1 Contig 4.47159 JPFH01 6761 0
Land Plants
Penstemon cyananthus Eukaryota; Plants; GCA_000281005.1 Contig 4.62226 AKKG01 9712 0
Land Plants
(continued)
819
820
Penstemon davidsonii Eukaryota; Plants; GCA_000280985.1 Contig 2.37523 AKKI01 4880 0
Land Plants
Penstemon dissectus Eukaryota; Plants; GCA_000280965.1 Contig 2.62809 AKKH01 5361 0
Land Plants
Penstemon fruticosus Eukaryota; Plants; GCA_000281025.1 Contig 2.31904 AKKJ01 4770 0
Land Plants
Penstemon grinnellii Eukaryota; Plants; GCA_000737425.1 Contig 3.66352 JPFI01 5523 0
Land Plants
Pereskia humboldtii Eukaryota; Plants; GCA_002740485.1 Scaffold 414.047 NCQU01 126352 0
Land Plants
Perilla citriodora Eukaryota; Plants; GCA_004303085.1 Scaffold 618.797 SDAM01 29924 0
Land Plants
Persea americana Eukaryota; Plants; GCA_008087245.1 Contig 912.698 SDSS01 8135 0
Land Plants
Persea americana Eukaryota; Plants; GCA_002908915.1 Contig 446.756 NXHZ01 5000 0
Land Plants
Persea americana var. Eukaryota; Plants; GCA_008033785.1 Scaffold 820.369 SDXN01 43777 0
drymifolia Land Plants
Phalaenopsis aphrodite Eukaryota; Plants; GCA_003013225.1 Scaffold 1025.1 NEWO01 13732 0
Land Plants
Phalaenopsis equestris Eukaryota; Plants; GCA_001263595.1 Scaffold 1064.2 APLD01 89584 29894
Land Plants
Phalaenopsis hybrid cultivar Eukaryota; Plants; GCA_002079205.1 Scaffold 2687.66 JXCR01 149149 0
Land Plants
Phaseolus coccineus subsp. Eukaryota; Plants; GCA_003122825.1 Scaffold 371.086 QBDZ01 192921 0
coccineus Land Plants
Phaseolus vulgaris Eukaryota; Plants; GCA_001517995.1 Chromosome 549.748 LPQZ01 68335 0
Land Plants
A. Khan et al.
32
Phaseolus vulgaris Eukaryota; Plants; GCA_000499845.1 Chromosome 521.077 ANNZ01 708 32720
Land Plants
Phoenix dactylifera Eukaryota; Plants; GCA_000413155.1 Scaffold 556.481 ATBV01 80317 40634
Land Plants
Phoenix dactylifera Eukaryota; Plants; GCA_000181215.2 Scaffold 381.563 ACYX02 57277 0
Land Plants
Phoenix dactylifera Eukaryota; Plants; GCA_007821505.1 Scaffold 454.367 PEFZ01 252335 0
Land Plants
Physaria acutifolia Eukaryota; Plants; GCA_900406485.1 Scaffold 199.442 OVBH01 276837 0
Land Plants
Physaria fendleri Eukaryota; Plants; GCA_900406525.1 Scaffold 331.342 OVBV01 99932 0
Land Plants
Physaria ovalifolia Eukaryota; Plants; GCA_900406505.1 Scaffold 290.267 OVBY01 335399 0
Land Plants
Physcomitrella patens Eukaryota; Plants; GCA_000002425.2 Chromosome 472.081 ABEU02 359 48022
Land Plants
Picea abies Eukaryota; Plants; GCA_900067695.1 Scaffold 11961.4 CBVK01 11340369 0
Land Plants
Picea abies var. abies Eukaryota; Plants; GCA_900491625.1 Scaffold 42.7831 UETF01 41150 0
Land Plants
Picea glauca Eukaryota; Plants; GCA_000411955.5 Scaffold 24633.1 ALWZ04 3033322 6445
Land Plants
Picea glauca Eukaryota; Plants; GCA_000966675.1 Scaffold 26936.2 JZKD01 3353683 0
Land Plants
Picea glauca Eukaryota; Plants; GCA_001687225.1 Contig 258.272 LDPM01 222034 0
Land Plants
Pinus lambertiana Eukaryota; Plants; GCA_001447015.2 Scaffold 27602.7 LMTP01 4253097 0
Land Plants
(continued)
821
822
Pinus sylvestris Eukaryota; Plants; GCA_900143225.1 Contig 0.985624 FRDG01 224 0
Land Plants
Pinus taeda Eukaryota; Plants; GCA_000404065.3 Scaffold 22103.6 APFE03 1760464 0
Land Plants
Pistacia vera Eukaryota; Plants; GCA_008641045.1 Scaffold 671.28 VTWI01 1865 82
Land Plants
Pisum sativum Eukaryota; Plants; GCA_003013575.1 Scaffold 4275.93 PUCA01 5449423 0
Land Plants
Platycodon grandiflorus Eukaryota; Plants; GCA_004681165.1 Scaffold 680.178 SPEA01 4816 0
Land Plants
Pleurozium schreberi Eukaryota; Plants; GCA_006891605.1 Contig 220.032 VACF01 2689 0
Land Plants
Pogostemon cablin Eukaryota; Plants; GCA_003675935.1 Scaffold 1916.69 QKXD01 41698 0
Land Plants
Populus alba Eukaryota; Plants; GCA_005239225.1 Contig 416.961 RCHU01 1287 32959
Land Plants
Populus euphratica Eukaryota; Plants; GCA_000495115.1 Scaffold 496.033 AOFL01 9615 49760
Land Plants
Populus simonii Eukaryota; Plants; GCA_007827005.2 Chromosome 441.407 VJNQ02 686 0
Land Plants
Populus trichocarpa Eukaryota; Plants; GCA_000002775.3 Chromosome 434.29 AARH03 1694 51717
Land Plants
Primula veris Eukaryota; Plants; GCA_000788445.1 Scaffold 309.693 JTKG01 8756 0
Land Plants
Primula vulgaris Eukaryota; Plants; GCA_001077355.1 Scaffold 1.50478 CDJJ02 229 0
Land Plants
Primula vulgaris Eukaryota; Plants; GCA_001403715.1 Scaffold 1.50478 CYSU01 229 0
Land Plants
A. Khan et al.
32
Prosopis alba Eukaryota; Plants; GCA_004799145.1 Contig 707.162 SMJV01 6087 57572
Land Plants
Prunus avium Eukaryota; Plants; GCA_002207925.1 Scaffold 272.362 BDGV01 10148 35009
Land Plants
Prunus avium Eukaryota; Plants; GCA_003946875.1 Contig 287.192 QXJJ01 1540 0
Land Plants
Prunus dulcis Eukaryota; Plants; GCA_902201215.1 Chromosome 227.599 CABIKO01 691 32556
Land Plants
Prunus mume Eukaryota; Plants; GCA_000346735.1 Chromosome 234.03 AOHF01 8626 29705
Land Plants
Prunus persica Eukaryota; Plants; GCA_000218175.1 Scaffold 214.225 AEJG01 30834 0
Land Plants
Prunus persica Eukaryota; Plants; GCA_000218215.1 Scaffold 207.185 AEKV01 43890 0
Land Plants
Prunus persica Eukaryota; Plants; GCA_000218195.1 Scaffold 211.308 AEKW01 35219 0
Land Plants
Prunus persica Eukaryota; Plants; GCA_000346465.2 Chromosome 227.569 AKXU02 192 32595
Land Plants
Prunus yedoensis Eukaryota; Plants; GCA_005406145.1 Contig 690.106 BJCG01 4571 0
Land Plants
Prunus yedoensis var. nudiflora Eukaryota; Plants; GCA_002966975.2 Scaffold 319.21 PJQY01 4016 41294
Land Plants
Prunus yedoensis var. nudiflora Eukaryota; Plants; GCA_900382725.1 Scaffold 319.21 OSDV01 4016 0
Land Plants
Pseudotsuga menziesii Eukaryota; Plants; GCA_001517045.1 Scaffold 14673.2 LPNX01 1236665 0
Land Plants
Pseudoturritis turrita Eukaryota; Plants; GCA_900406555.1 Contig 321.563 OVBL01 1111 0
Land Plants
(continued)
823
824
Pseudoturritis turrita Eukaryota; Plants; GCA_900406515.1 Scaffold 263.513 OVBK01 25008 0
Land Plants
Psidium guajava Eukaryota; Plants; GCA_002914565.1 Contig 386.852 NTGF01 4728 0
Land Plants
Pterocarya stenoptera Eukaryota; Plants; GCA_003123785.1 Scaffold 955.601 QEOT01 124315 0
Land Plants
Punica granatum Eukaryota; Plants; GCA_002864125.1 Scaffold 274.043 MTJX01 2117 0
Land Plants
Punica granatum Eukaryota; Plants; GCA_002201585.1 Scaffold 296.383 MTKT01 17405 29127
Land Plants
Punica granatum Eukaryota; Plants; GCA_002837095.1 Scaffold 380.178 PGOL01 45308 50476
Land Plants
Punica granatum Eukaryota; Plants; GCA_007655135.2 Chromosome 320.336 MABG02 473 0
Land Plants
Purshia tridentata Eukaryota; Plants; GCA_003254885.1 Scaffold 175.971 QANT01 9353 0
Land Plants
Pyrus betulifolia Eukaryota; Plants; GCA_007844245.1 Chromosome 532.747 VDML01 139 0
Land Plants
Pyrus x bretschneideri Eukaryota; Plants; GCA_000315295.1 Scaffold 508.551 AJSU01 2182 47086
Land Plants
Quercus lobata Eukaryota; Plants; GCA_001633185.2 Chromosome 846.07 LRBV02 2010 53228
Land Plants
Quercus robur Eukaryota; Plants; GCA_900291515.1 Scaffold 814.336 OLKR01 550 0
Land Plants
Quercus robur Eukaryota; Plants; GCA_003013145.1 Scaffold 719.602 PVWZ01 84416 0
Land Plants
Quercus suber Eukaryota; Plants; GCA_002906115.1 Scaffold 953.299 PKMF01 23344 59614
Land Plants
A. Khan et al.
32
Quillaja saponaria Eukaryota; Plants; GCA_003338715.1 Contig 248.908 PVLG01 48349 0

Land Plants
Raddia distichophylla Eukaryota; Plants; GCA_005191435.1 Scaffold 580.833 SPJY01 38257 0
Land Plants
Raparia bulbosa Eukaryota; Plants; GCA_900406535.1 Scaffold 152.784 OVBJ01 13874 0
Land Plants
Raphanus raphanistrum subsp. Eukaryota; Plants; GCA_000769845.1 Contig 253.834 JRQH01 64732 0
raphanistrum Land Plants
Raphanus sativus Eukaryota; Plants; GCA_000801105.2 Scaffold 426.614 JRUI02 10676 61216
Land Plants
Raphanus sativus Eukaryota; Plants; GCA_000715565.1 Scaffold 402.328 BAUK01 76592 0
Land Plants
Raphanus sativus Eukaryota; Plants; GCA_001047155.1 Scaffold 383.105 BAOO01 40123 0
Land Plants
Raphanus sativus Eukaryota; Plants; GCA_002197605.1 Chromosome 382.79 JSDR01 44239 0
Land Plants
Rhamnella rubrinervis Eukaryota; Plants; GCA_007844105.1 Chromosome 245.336 VOIH01 133 0
Land Plants
Rhazya stricta Eukaryota; Plants; GCA_001752375.1 Scaffold 274.354 MEJB01 979 0
Land Plants
Rhizophora apiculata Eukaryota; Plants; GCA_900174605.1 Scaffold 232.055 FWPW01 142 0
Land Plants
Rhizophora apiculata Eukaryota; Plants; GCA_900004065.1 Scaffold 232.431 CELW01 45996 0
Land Plants
Rhodamnia argentea Eukaryota; Plants; GCA_900635035.1 Scaffold 414.816 CAAAGQ01 15781 42570
Land Plants
Rhodoleia championii Eukaryota; Plants; GCA_008932045.1 Contig 105.726 VMOD01 278658 0
Land Plants
(continued)
825
826
Ricinus communis Eukaryota; Plants; GCA_000151685.2 Scaffold 350.622 AASG02 25763 28584
Land Plants
Rosa chinensis Eukaryota; Plants; GCA_002994745.1 Chromosome 513.854 PDCK01 45 45097
Land Plants
Rosa lucieae Eukaryota; Plants; GCA_006954505.1 Scaffold 786.105 RQIQ01 500476 0
Land Plants
Rosa multiflora Eukaryota; Plants; GCA_002564525.1 Scaffold 739.638 BDJD01 83189 0
Land Plants
Rosa x damascena Eukaryota; Plants; GCA_001662545.1 Scaffold 711.72 LYNE01 307872 0
Land Plants
Ruellia speciosa Eukaryota; Plants; GCA_001909325.1 Contig 740.036 MAYD01 794288 0
Land Plants
Saccharum hybrid cultivar Eukaryota; Plants; GCA_900465005.1 Scaffold 530.66 UBIK01 5708 0
Land Plants
Saccharum hybrid cultivar Eukaryota; Plants; GCA_008692665.1 Scaffold 4014.93 QPEU01 398353 0
SP80-3280 Land Plants
Saccharum hybrid cultivar Eukaryota; Plants; GCA_002018215.1 Contig 1169.95 JXQF01 199028 0
Saccharum hybrid cultivar Eukaryota; Plants; GCA_009173535.1 Scaffold 49.3852 PYBL01 461 0
Saccharum spontaneum Eukaryota; Plants; GCA_900500655.1 Contig 3924.19 UINE01 75981 0
Land Plants
Saccharum spontaneum Eukaryota; Plants; GCA_003544955.1 Chromosome 3133.29 QVOL01 15303 0
Land Plants
Salix brachista Eukaryota; Plants; GCA_009078335.1 Chromosome 339.588 VDCV01 30 30209
Land Plants
Salvia splendens Eukaryota; Plants; GCA_004379255.1 Scaffold 809.16 PNBA01 1525 53354
Land Plants
A. Khan et al.
32
Santalum album Eukaryota; Plants; GCA_002911635.1 Contig 196.101 NXEK01 180 0

Land Plants
Santalum album Eukaryota; Plants; GCA_002925775.1 Scaffold 220.961 LOCJ01 12821 0
Land Plants
Schrenkiella parvula Eukaryota; Plants; GCA_000218505.1 Chromosome 137.073 AFAN01 1463 0
Land Plants
Scutellaria baicalensis Eukaryota; Plants; GCA_005771605.1 Chromosome 386.674 VALJ01 114 0
Land Plants
Secale cereale Eukaryota; Plants; GCA_900079665.1 Scaffold 1684.93 FKKI01 1581707 0
Land Plants
Secale cereale Eukaryota; Plants; GCA_900002355.1 Scaffold 1684.93 CCJQ01 1581707 0
Land Plants
Sedum album Eukaryota; Plants; GCA_006409495.1 Contig 302.251 QZGG01 6038 0
Land Plants
Selaginella kraussiana Eukaryota; Plants; GCA_001021135.1 Scaffold 114.503 LDJE01 105914 0
Land Plants
Selaginella moellendorffii Eukaryota; Plants; GCA_000143415.2 Scaffold 212.315 ADFJ01 757 45247
Land Plants
Selaginella tamariscina Eukaryota; Plants; GCA_003024785.1 Scaffold 300.729 PUQB01 1391 0
Land Plants
Sequoia sempervirens Eukaryota; Plants; GCA_007258455.1 Scaffold 26537.2 VDFB01 517852 0
Land Plants
Sequoia dendrongiganteum Eukaryota; Plants; GCA_007115665.1 Scaffold 8122.13 VCHN01 39798 0
Land Plants
Sesamum indicum Eukaryota; Plants; GCA_001692995.1 Scaffold 210.758 MBSK01 5868 0
Land Plants
Sesamum indicum Eukaryota; Plants; GCA_003268515.1 Scaffold 242.679 LUAT01 48805 0
Land Plants
(continued)
827
828
Sesamum indicum Eukaryota; Plants; GCA_000975565.1 Scaffold 340.464 JPLX01 76023 0
Land Plants
Sesamum indicum Eukaryota; Plants; GCA_000512975.1 Chromosome 275.059 APMJ01 16369 35410
Land Plants
Setaria italica Eukaryota; Plants; GCA_001652605.1 Chromosome 477.542 LWRS01 2689 0
Land Plants
Setaria italica Eukaryota; Plants; GCA_000263155.2 Chromosome 405.868 AGNK02 337 35844
Land Plants
Setaria viridis Eukaryota; Plants; GCA_005286985.1 Chromosome 395.732 SNSE01 75 52459
Land Plants
Silene latifolia Eukaryota; Plants; GCA_003260165.1 Scaffold 1185.09 QBIE01 319506 0
Land Plants
Silene latifolia Eukaryota; Plants; GCA_900095335.1 Contig 36.0486 FMHP01 46178 0
Land Plants
Silene latifolia subsp. alba Eukaryota; Plants; GCA_001412135.1 Scaffold 665.279 LHUT01 307720 0
Land Plants
Silphium perfoliatum Eukaryota; Plants; GCA_900538075.1 Contig 121.712 UXAI01 1197534 0
Land Plants
Silybum marianum Eukaryota; Plants; GCA_001541825.1 Contig 1477.57 LMWD01 258575 0
Land Plants
Sisymbrium altissimum Eukaryota; Plants; GCA_900406495.1 Scaffold 178.647 OVBI01 14597 0
Land Plants
Sisymbrium irio Eukaryota; Plants; GCA_000411075.1 Scaffold 245.55 ASZH01 21357 0
Land Plants
Solanum americanum Eukaryota; Plants; GCA_900188915.1 Contig 9.01369 FYFB01 837 0
Land Plants
Solanum americanum Eukaryota; Plants; GCA_900188785.1 Contig 7.74921 FYHF01 1085 0
Land Plants
A. Khan et al.
32
Solanum americanum Eukaryota; Plants; GCA_900188895.1 Contig 7.74473 FYHB01 1085 0

Land Plants
Solanum americanum Eukaryota; Plants; GCA_900188885.1 Contig 7.6066 FYHD01 1085 0
Land Plants
Solanum americanum Eukaryota; Plants; GCA_900198685.1 Contig 8.308 FZPQ01 1460 0
Land Plants
Solanum americanum Eukaryota; Plants; GCA_900188835.1 Contig 9.83576 FYHH01 1483 0
Land Plants
Solanum arcanum Eukaryota; Plants; GCA_000612985.1 Contig 665.187 CBYQ01 46594 0
Land Plants
Solanum chilense Eukaryota; Plants; GCA_006013705.1 Scaffold 913.881 RXGB01 81304 19
Land Plants
Solanum commersonii Eukaryota; Plants; GCA_001239805.1 Scaffold 729.603 JXZD01 63664 0
Land Plants
Solanum habrochaites Eukaryota; Plants; GCA_000577655.1 Contig 724.285 CBYS01 42990 0
Land Plants
Solanum lycopersicum Eukaryota; Plants; GCA_000181095.1 Scaffold 540.589 BABP01 100783 0
Land Plants
Solanum lycopersicum Eukaryota; Plants; GCA_000325825.1 Scaffold 0.575198 AFYB01 195 0
Land Plants
Solanum lycopersicum Eukaryota; Plants; GCA_000188115.3 Chromosome 828.349 AEKE03 3150 37660
Land Plants
Solanum melongena Eukaryota; Plants; GCA_000787875.1 Scaffold 833.081 BAUE01 33873 0
Land Plants
Solanum pennellii Eukaryota; Plants; GCA_000577875.1 Contig 720.458 CBYR01 57205 0
Land Plants
Solanum pennellii Eukaryota; Plants; GCA_000820945.1 Contig 720.458 CCXL01 57205 0
Land Plants
(continued)
829
830
Solanum pimpinellifolium Eukaryota; Plants; GCA_003660305.1 Scaffold 748.694 NRDK01 48863 0
Land Plants
Solanum pimpinellifolium Eukaryota; Plants; GCA_000230315.1 Contig 688.247 AGFK01 309180 0
Land Plants
Solanum tuberosum Eukaryota; Plants; GCA_000226075.1 Scaffold 705.934 AEWC01 14854 37966
Land Plants
Solanum tuberosum Eukaryota; Plants; GCA_900004685.1 Contig 90.4582 CVMJ01 39446 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185145.1 Scaffold 730.142 FYAA01 224100 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185165.1 Scaffold 667.086 FXZQ01 2343 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185285.1 Contig 659.291 FXZO01 2461 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185275.1 Scaffold 662.264 FXZP01 1331 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185325.1 Scaffold 659.429 FXZS01 1377 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185305.1 Scaffold 716.55 FXZW01 4814 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185245.1 Contig 715.934 FXZV01 5571 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185335.1 Scaffold 749.835 FXZU01 5164 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185175.1 Scaffold 764.063 FXZT01 7840 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185215.1 Contig 722.285 FXZR01 8138 0
Land Plants
A. Khan et al.
32
Solanum verrucosum Eukaryota; Plants; GCA_900185185.1 Scaffold 751.585 FYAB01 321725 0

Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185195.1 Scaffold 740.946 FYAC01 170623 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185295.1 Scaffold 728.86 FYAD01 228374 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185265.1 Scaffold 729.31 FYAF01 224108 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185155.1 Scaffold 690.417 FXZY01 22476 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185205.1 Scaffold 688.737 FXZX01 22492 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185315.1 Scaffold 710.407 FYAG01 246082 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185225.1 Scaffold 730.903 FXZZ01 182474 0
Land Plants
Solanum verrucosum Eukaryota; Plants; GCA_900185235.1 Scaffold 759.168 FYAE01 569872 0
Land Plants
Sorghum bicolor Eukaryota; Plants; GCA_003482435.1 Scaffold 666.155 QWKM01 308 0
Land Plants
Sorghum bicolor Eukaryota; Plants; GCA_008000285.1 Contig 374.252 VOIB01 2657 0
Land Plants
Sorghum bicolor Eukaryota; Plants; GCA_000236765.2 Contig 0.015475 AHAO01 16 16
Land Plants
Sorghum bicolor Eukaryota; Plants; GCA_000236725.2 Contig 0.018494 AHAQ01 20 22
Land Plants
Sorghum bicolor Eukaryota; Plants; GCA_000236745.2 Contig 0.021299 AHAP01 35 35
Land Plants
(continued)
831
832
Sorghum bicolor Eukaryota; Plants; GCA_000003195.3 Chromosome 709.345 ABXC03 869 39248
Land Plants
Spatholobus suberectus Eukaryota; Plants; GCA_004329165.1 Chromosome 798.47 QUWT01 816 31106
Land Plants
Spinacia oleracea Eukaryota; Plants; GCA_002007265.1 Scaffold 869.946 LZYP01 78263 32794
Land Plants
Spinacia oleracea Eukaryota; Plants; GCA_000510995.2 Scaffold 493.772 AYZV02 103502 23522
Land Plants
Spirodela polyrhiza Eukaryota; Plants; GCA_900492545.1 Scaffold 138.592 UIDA01 20 0
Land Plants
Spirodela polyrhiza Eukaryota; Plants; GCA_008360905.1 Scaffold 138.536 SWLF01 134 0
Land Plants
Spirodela polyrhiza Eukaryota; Plants; GCA_900536055.1 Scaffold 142.661 UNPA01 2585 0
Land Plants
Spirodela polyrhiza Eukaryota; Plants; GCA_000504445.1 Contig 132.009 ATDW01 16051 0
Land Plants
Sporobolus alterniflorus Eukaryota; Plants; GCA_008808055.1 Contig 365.573 VSTD01 52184 0
Land Plants
Stenocereus thurberi Eukaryota; Plants; GCA_002740465.1 Scaffold 853.348 NCQT01 159477 0
Land Plants
Striga asiatica Eukaryota; Plants; GCA_008636005.1 Scaffold 471.563 BKCP01 13846 33426
Land Plants
Syzygium oleosum Eukaryota; Plants; GCA_900635055.1 Scaffold 431.291 CAAAGS01 19039 38158
Land Plants
Tarenaya hassleriana Eukaryota; Plants; GCA_000463585.1 Scaffold 249.93 AOUI01 12249 41094
Land Plants
Theobroma cacao Eukaryota; Plants; GCA_000403535.1 Chromosome 345.994 ALXC01 814 44186
Land Plants
A. Khan et al.
32
Theobroma cacao Eukaryota; Plants; GCA_000208745.2 Chromosome 324.88 FLSQ01 431 30854
Land Plants
Thlaspi arvense Eukaryota; Plants; GCA_000956625.1 Scaffold 343.012 AZNP01 6768 0
Land Plants
Trema orientale Eukaryota; Plants; GCA_002914845.1 Scaffold 387.958 JXTC01 2756 35849
Land Plants
Trichopus zeylanicus subsp. Eukaryota; Plants; GCA_005019695.1 Scaffold 713.407 RXID01 22601 0
travancoricus Land Plants
Trifolium medium Eukaryota; Plants; GCA_003490085.1 Scaffold 492.653 LXQA01 1471389 0
Land Plants
Trifolium pratense Eukaryota; Plants; GCA_900292005.1 Chromosome 351.622 OMTE01 38479 0
Land Plants
Trifolium pratense Eukaryota; Plants; GCA_900079335.1 Chromosome 345.991 FKJA01 39051 0
Land Plants
Trifolium pratense Eukaryota; Plants; GCA_000583005.2 Contig 304.972 ASHM01 267372 63850
Land Plants
Trifolium subterraneum Eukaryota; Plants; GCA_001742945.1 Scaffold 471.834 BCLP01 27424 42059
Land Plants
Trifolium subterraneum Eukaryota; Plants; GCA_002003065.1 Contig 392.71 BBPR01 968279 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_002220415.2 Contig 15344.7 NMPL02 279439 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_900241085.1 Scaffold 13916.9 OETA01 519179 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_900067645.1 Scaffold 13427.4 FAOM01 735943 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_900000045.1 Scaffold 9134.02 CCYC01 6870110 0
Land Plants
(continued)
833
834
Triticum aestivum Eukaryota; Plants; GCA_900067735.1 Scaffold 10058.1 CBTL01 11673940 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_001077335.1 Scaffold 58.5022 CBUC01 1450 250
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_002158495.1 Scaffold 567.21 MOLT01 10339 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_000818885.1 Scaffold 65.1111 JROL01 15144 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_002999095.1 Scaffold 574.295 PKRY01 9044 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_900236235.1 Scaffold 452.948 OEIT01 23433 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_002780475.1 Contig 240.199 NTGG01 127921 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_002780545.1 Contig 609.488 NTGH01 523543 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_002780565.1 Contig 564.3 NTGI01 565551 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_900235945.1 Scaffold 839.076 OEIJ01 767884 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_900235935.1 Scaffold 804.001 ODGO01 749802 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_001485685.1 Contig 44.4015 FAOV01 50000 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_000334095.1 Contig 3800.33 CALP01 5321847 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_000188135.1 Contig 159.087 AEOM01 311945 0
Land Plants
A. Khan et al.
32
Triticum aestivum Eukaryota; Plants; GCA_000334135.1 Contig 437.106 CALO01 945079 0

Land Plants
Triticum aestivum Eukaryota; Plants; GCA_001889245.1 Contig 2.42226 LOLC01 9834 0
Land Plants
Triticum aestivum Eukaryota; Plants; GCA_001889205.1 Contig 0.942499 LOLD01 3746 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323645.1 Scaffold 392.684 OOGV01 252227 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323695.1 Scaffold 552.916 OOHG01 360242 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323615.1 Scaffold 477.083 OOGX01 350258 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323635.1 Scaffold 434.779 OOGY01 363434 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323595.1 Scaffold 431.061 OOHA01 427274 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323585.1 Scaffold 288.316 OOGW01 327364 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323625.1 Scaffold 518.951 OOHB01 592030 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323565.1 Scaffold 353.054 OOGZ01 455525 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323665.1 Scaffold 441.177 OOHC01 572876 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323605.1 Scaffold 497.42 OOHD01 649826 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323575.1 Scaffold 585.067 OOHE01 730770 0
Land Plants
(continued)
835
836
Triticum dicoccoides Eukaryota; Plants; GCA_900323655.1 Scaffold 624.303 OOHF01 905302 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323675.1 Scaffold 388.595 OOHH01 638342 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900323685.1 Scaffold 700.417 OOHP01 1181661 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_002162155.2 Chromosome 10677.9 LSYQ02 148396 0
Land Plants
Triticum dicoccoides Eukaryota; Plants; GCA_900184675.1 Chromosome 10495 FXXJ01 149145 0
Land Plants
Triticum urartu Eukaryota; Plants; GCA_000347455.1 Scaffold 3747.05 AOTI01 499221 24169
Land Plants
Triticum urartu Eukaryota; Plants; GCA_003073215.1 Chromosome 4851.9 MKGO01 10284 0
Land Plants
Turritis glabra Eukaryota; Plants; GCA_900406565.1 Contig 171.13 OVBN01 250 0
Land Plants
Turritis glabra Eukaryota; Plants; GCA_900406545.1 Scaffold 157.63 OVBM01 6441 0
Land Plants
Urochloa ruziziensis Eukaryota; Plants; GCA_003016355.1 Scaffold 732.531 PVZT01 102577 0
Land Plants
Utricularia gibba Eukaryota; Plants; GCA_002189035.1 Chromosome 100.689 NEEC01 518 0
Land Plants
Vaccinium macrocarpon Eukaryota; Plants; GCA_000775335.2 Scaffold 414.622 JOTO01 200203 0
Land Plants
Vachellia collinsii Eukaryota; Plants; GCA_006871305.1 Scaffold 461.065 QFDD01 122260 0
Land Plants
Vanilla planifolia Eukaryota; Plants; GCA_004338375.1 Scaffold 2203.64 SDXO01 794534 0
Land Plants
A. Khan et al.
32
Vicia faba Eukaryota; Plants; GCA_001375635.1 Contig 80.3627 CSVX01 74659 0

Land Plants
Vigna angularis Eukaryota; Plants; GCA_001190045.1 Chromosome 467.301 JZJH01 37727 37769
Land Plants
Vigna angularis var. angularis Eukaryota; Plants; GCA_000465365.1 Scaffold 291.824 AUGG01 14501 0
Land Plants
Vigna angularis var. angularis Eukaryota; Plants; GCA_001723775.1 Chromosome 444.439 JRFV01 3387 0
Land Plants
Vigna radiata Eukaryota; Plants; GCA_000180895.1 Contig 10.1012 BABL01 46645 0
Land Plants
Vigna radiata var. radiata Eukaryota; Plants; GCA_001584445.1 Scaffold 454.907 LJIH01 2418 0
Land Plants
Vigna radiata var. radiata Eukaryota; Plants; GCA_000741045.2 Chromosome 463.638 JJMO01 2499 42284
Land Plants
Vigna unguiculata Eukaryota; Plants; GCA_004118075.1 Chromosome 519.067 NBOW01 682 41173
Land Plants
Vigna unguiculata subsp. Eukaryota; Plants; GCA_001687525.1 Scaffold 695.046 MATU01 224035 0
unguiculata Land Plants
Viola pubescens var. Eukaryota; Plants; GCA_002752925.1 Scaffold 318.366 NBIL01 157716 0
scabriuscula Land Plants
Vitis aestivalis Eukaryota; Plants; GCA_001562795.1 Contig 432.755 LOML01 756125 0
Land Plants
Vitis cinerea x Vitis riparia Eukaryota; Plants; GCA_001282645.1 Scaffold 539.624 CCJE01 210444 0
Land Plants
Vitis riparia Eukaryota; Plants; GCA_004353265.1 Chromosome 500.106 SJAQ01 174 0
Land Plants
Vitis vinifera Eukaryota; Plants; GCA_002923105.1 Scaffold 427.211 BDSR01 21 0
Land Plants
(continued)
837
838
Vitis vinifera Eukaryota; Plants; GCA_002922885.1 Scaffold 427.171 BDSO01 21 0
Land Plants
Vitis vinifera Eukaryota; Plants; GCA_002923015.1 Scaffold 427.04 BDSQ01 21 0
Land Plants
Vitis vinifera Eukaryota; Plants; GCA_002923165.1 Scaffold 426.616 BDSS01 21 0
Land Plants
Vitis vinifera Eukaryota; Plants; GCA_004011995.1 Contig 868.043 QGNW01 2737 112320
Land Plants
Vitis vinifera Eukaryota; Plants; GCA_000003745.2 Chromosome 486.197 CAAP03 2061 41208
Land Plants
Vitis x labruscana x Vitis Eukaryota; Plants; GCA_008326845.1 Scaffold 490.143 BKBX01 8696 0
vinifera Land Plants
Xanthoceras sorbifolium Eukaryota; Plants; GCA_003430845.1 Chromosome 504.383 QUWJ01 2297 0
Land Plants
Xerophyta viscosa Eukaryota; Plants; GCA_002076135.1 Scaffold 295.462 MJHO01 896 0
Land Plants
Zea mays Eukaryota; Plants; GCA_000223545.1 Scaffold 177.051 AECO01 196697 0
Land Plants
Zea mays Eukaryota; Plants; GCA_000275765.1 Contig 1.33507 AHID01 1844 0
Land Plants
Zea mays Eukaryota; Plants; GCA_003185045.1 Chromosome 2182.61 NCVQ01 2203 46530
Land Plants
Zea mays Eukaryota; Plants; GCA_003704525.1 Chromosome 2198.5 RAQR01 797 0
Land Plants
Zea mays Eukaryota; Plants; GCA_003709335.1 Chromosome 2288.19 RAQT01 972 0
Land Plants
Zea mays Eukaryota; Plants; GCA_000005005.6 Chromosome 2135.08 LPUQ01 598 58411
Land Plants
A. Khan et al.
32
Zea mays subsp. mays Eukaryota; Plants; GCA_002813505.1 Scaffold 2041.55 LMUZ01 48268 0
Land Plants
Zea mays subsp. mays Eukaryota; Plants; GCA_001990705.1 Chromosome 2392.8 MTTB01 62610 0
Land Plants
Zea mays subsp. mays Eukaryota; Plants; GCA_001984235.2 Chromosome 2455.26 MTTA01 60567 0
Land Plants
Zea mays subsp. mays Eukaryota; Plants; GCA_001644905.2 Chromosome 2133.88 LWRW02 191 0
Land Plants
Zea mays subsp. mays Eukaryota; Plants; GCA_002682915.2 Chromosome 2197.97 NWUM01 3538 0
Land Plants
Zea mays subsp. mays Eukaryota; Plants; GCA_002237485.1 Chromosome 2155.82 NKIA01 43301 0
Land Plants
Zea mays subsp. mexicana Eukaryota; Plants; GCA_002813485.1 Scaffold 1204.28 LMVA01 107418 0
Land Plants
Zizania latifolia Eukaryota; Plants; GCA_000418225.1 Scaffold 603.989 ASSH01 4522 0
Land Plants
Ziziphus jujuba Eukaryota; Plants; GCA_001835785.1 Scaffold 351.097 LPXJ01 36119 0
Land Plants
Ziziphus jujuba Eukaryota; Plants; GCA_000826755.1 Chromosome 437.754 JREP01 5897 43574
Land Plants
Zostera marina Eukaryota; Plants; GCA_001185155.1 Scaffold 203.914 LFYR01 2228 20648
Land Plants
Zoysia japonica Eukaryota; Plants; GCA_001602275.1 Scaffold 334.384 BCLF01 11786 0
Land Plants
Zoysia matrella Eukaryota; Plants; GCA_001602295.1 Scaffold 563.439 BCLG01 13609 0
Land Plants
Zoysia pacifica Eukaryota; Plants; GCA_001602315.1 Scaffold 397.01 BCLH01 11428 0
Land Plants
839
840 A. Khan et al.
32.6 Conclusion
The applications of bioinformatics to plant pathology have been pivotal role in

understanding of host and pathogen evolution and molecular interactions
between host and pathogen. Availability of next-generation sequencing data
of candidate model organisms of all kingdom through high-throughput tech-
nology is convenient to deal with biological systems and understand the
biological sequence–structure–function correlation using in-silico biology
tools, technology and databases. Genome annotation, assembly, bioproject,
biosample submission, sequence data submission, retrieval of data, data analy-
sis, variation analysis, conserved domain analysis, gene identification, regu-
latory elements analysis, gene expression analysis, structure prediction,
structure visualization, structure analysis, structure classification, molecular
modeling, epitope identification and mapping using 3D, drug designing, active
site analysis and molecular docking, etc. play an important role to achieve
biological function and understand the sequence–structure–function relation-
ship. These all in-silico biology techniques will be further helpful in genomics-
assisted crop improvement and development of designer crops with high yield
and super quality.
References
Alioto T (2012) Gene prediction. Methods Mol Biol 855:175–201
Aljanabi S (2001) Genomics and plant breeding. Biotechnol Annu Rev 7:195–238
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J
Mol Biol 215:403–410
Batley J, Edwards D (2016) The application of genomics and bioinformatics to accelerate crop
improvement in a changing climate. Curr Opin Plant Biol 30:78–81
Bawono P, Dijkstra M, Pirovano W, Feenstra A, Abeln S, Heringa J (2017) Multiple sequence
alignment. Methods Mol Biol 1525:167–189
Berman HM (2008) The protein data bank: a historical perspective. Acta Crystallogr A 64
(Pt 1):88–95
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE
(2000) The protein data bank. Nucleic Acids Res 28(1):235–242
Bolger ME, Arsova B, Usadel B (2018) Plant genome and transcriptome annotations: from
misconceptions to simple solutions. Brief Bioinform 19:437–449
Bolser D, Staines DM, Pritchard E, Kersey P (2016) Ensembl plants: integrating tools for
visualizing, mining, and analyzing plant genomics data. Methods Mol Biol 1374:115–140
Bolser DM, Staines DM, Perry E, Kersey PJ (2017) Ensembl plants: integrating tools for
visualizing, mining, and analyzing plant genomic data. Methods Mol Biol 1533:1–31
Cavasotto CN, Phatak SS (2009) Homology modeling in drug discovery: current trends and
applications. Drug Discov Today 14:676–683
Chen YP, Chen F (2008) Using bioinformatics techniques for gene identification in drug discovery
and development. Curr Drug Metab 9:567–573
Chen J, Anderson JB, DeWeese-Scott C, Fedorova ND, Geer LY, He S, Hurwitz DI, Jackson JD,
Jacobs AR, Lanczycki CJ, Liebert CA, Liu C, Madej T, Marchler-Bauer A, Marchler GH,
Mazumder R, Nikolskaya AN, Rao BS, Panchenko AR, Shoemaker BA, Simonyan V, Song JS,
Thiessen PA, Vasudevan S, Wang Y, Yamashita RA, Yin JJ, Bryant SH (2003) MMDB:
Entrez’s 3D-structure database. Nucleic Acids Res 31:474–477
Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple
sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500
Clough E, Barrett T (2016) The gene expression omnibus database. Methods Mol Biol
1418:93–110
De Las Rivas J, Fontanillo C (2010) Protein-protein interactions essentials: key concepts to building
and analyzing interactome networks. PLoS Comput Biol 6(6):e1000807
de Ruyck J, Brysbaert G, Blossey R, Lensink MF (2016) Molecular docking as a popular tool in
drug design, an in silico travel. Adv Appl Bioinforma Chem 9:1–11
Demuth JP, Hahn MW (2009) The life and death of gene families. BioEssays 31:29–39
Dong Q, Schlueter SD, Brendel V (2004) PlantGDB, plant genome database and analysis tools.
Nucleic Acids Res 32(Database issue):D354–D359
Duvick J, Fu A, Muppirala U, Sabharwal M, Wilkerson MD, Lawrence CJ, Lushbough C, Brendel
V (2008) PlantGDB: a resource for comparative plant genomics. Nucleic Acids Res 36(Data-
base issue):D959–D965
Eisenberg D, Lüthy R, Bowie JU (1997) VERIFY3D: assessment of protein models with three-
dimensional profiles. Methods Enzymol 277:396–404
Ferreira LG, Dos Santos RN, Oliva G, Andricopulo AD (2015) Molecular docking and structure-
based drug design strategies. Molecules 20:13384–13421
França TC (2015) Homology modeling: an important tool for the drug discovery. J Biomol Struct
Dyn 33(8):1780–1793
Galperin MY, Fernández-Suárez XM (2012) The 2012 nucleic acids research database issue and the
online molecular biology database collection. Nucleic Acids Res 40(Database issue):D1–D8
Gebhardt C, Schmidt R, Schneider K (2005) Plant genome analysis: the state of the art. Int Rev
Cytol 247:223–284
Gonçalves JP, Madeira SC, Oliveira AL (2009) BiGGEsTS: integrated environment for biclustering
analysis of time series gene expression data. BMC Res Notes 2:124
Goodsell DS, Morris GM, Olson AJ (1996) Automated docking of flexible ligands: applications of
AutoDock. J Mol Recognit 9:1–5
Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U,
Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics.
Nucleic Acids Res 40(Database issue):D1178–D1186
Gschwend DA, Good AC, Kuntz ID (1996) Molecular docking towards drug discovery. J Mol
Recognit 9:175–186
Guedes IA, de Magalhães CS, Dardenne LE (2014) Receptor-ligand molecular docking. Biophys
Rev 6:75–87
Hardison RC (2003) Comparative genomics. PLoS Biol 1:E58
Higgins DG, Thompson JD, Gibson TJ (1996) Using CLUSTAL for multiple sequence alignments.
Methods Enzymol 266:383–402
Jones G, Willett P (1995) Docking small-molecule ligands into active sites. Curr Opin Biotechnol
6:652–656
King GJ (2004) Bioinformatics: harvesting information for plant and crop science. Semin Cell Dev
Biol 15:721–731
Kodama Y, Mashima J, Kaminuma E, Gojobori T, Ogasawara O, Takagi T, Okubo K, Nakamura Y
(2012) The DNA Data Bank of Japan launches a new resource, the DDBJ Omics Archive of
functional genomics experiments. Nucleic Acids Res 40(Database issue):D38–D42
Kozakov D, Hall DR, Xia B, Porter KA, Padhorny D, Yueh C, Beglov D, Vajda S (2017) The
ClusPro web server for protein-protein docking. Nat Protoc 12:255–278
Krieger E, Vriend G (2014) YASARA view – molecular graphics for all devices – from
smartphones to workstations. Bioinformatics 30:2981–2982
Krieger E, Nabuurs SB, Vriend G (2003) Homology modeling. Methods Biochem Anal
44:509–523
842 A. Khan et al.
Kroemer RT (2007) Structure-based drug design: docking and scoring. Curr Protein Pept Sci
8:312–328
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA (2009)
Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–1645
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics
analysis across computing platforms. Mol Biol Evol 35(6):1547–1549
Kuntal BK, Aparoy P, Reddanna P (2010) Easy modeller: a graphical interface to MODELLER.
BMC Res Notes 3:226
Laskowski RA, Hutchinson EG, Michie AD, Wallace AC, Jones ML, Thornton JM (1997)
PDBsum: a web-based database of summaries and analyses of all PDB structures. Trends
Biochem Sci 22(12):488–490. PubMed PMID: 9433130
Laskowski RA, Jabłońska J, Pravda L, Vařeková RS, Thornton JM (2017) PDBsum: structural
summaries of PDB entries. Protein Sci 27:129–134
Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS,
Richardson DC (2003) Structure validation by Calpha geometry: phi, psi and Cbeta deviation.
Proteins 50:437–450
Lybrand TP (1995) Ligand-protein docking and rational drug design. Curr Opin Struct Biol
5:224–228
Lyons E, Freeling M (2008) How to usefully compare homologous plant genes and chromosomes
as DNA sequences. Plant J 53:661–673
Macindoe G, Mavridis L, Venkatraman V, Devignes MD, Ritchie DW (2010) HexServer: an
FFT-based protein docking server powered by graphics processors. Nucleic Acids Res 38
(Web Server issue):W445–W449
Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC,
Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019.
Nucleic Acids Res 47(W1):W636–W641
Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC,
Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z,
Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH (2017) CDD/SPARCLE: functional
classification of proteins via subfamily domain architectures. Nucleic Acids Res 45:D200–D203
Martinez M (2013) From plant genomes to protein families: computational tools. Comput Struct
Biotechnol J 8:e201307001
McClure MA, Vasi TK, Fitch WM (1994) Comparative analysis of multiple protein-sequence
alignment methods. Mol Biol Evol 11:571–592
Meng XY, Zhang HX, Mezei M, Cui M (2011) Molecular docking: a powerful approach for
structure-based drug discovery. Curr Comput Aided Drug Des 7:146–157
Metsalu T, Vilo J (2015) ClustVis: a web tool for visualizing clustering of multivariate data using
principal component analysis and heatmap. Nucleic Acids Res 43:W566–W570
Mitchell AL, Attwood TK, Babbitt PC, Blum M, Bork P, Bridge A, Brown SD, Chang HY,
El-Gebali S, Fraser MI, Gough J, Haft DR, Huang H, Letunic I, Lopez R, Luciani A,
Madeira F, Marchler-Bauer A, Mi H, Natale DA, Necci M, Nuka G, Orengo C, Pandurangan
AP, Paysan-Lafosse T, Pesseat S, Potter SC, Qureshi MA, Rawlings ND, Redaschi N,
Richardson LJ, Rivoire C, Salazar GA, Sangrador-Vegas A, Sigrist CJA, Sillitoe I, Sutton
GG, Thanki N, Thomas PD, Tosatto SCE, Yong SY, Finn RD (2019) InterPro in 2019:
improving coverage, classification and access to protein sequence annotations. Nucleic Acids
Res 47:D351–D360
Mochida K, Shinozaki K (2010) Genomics and bioinformatics resources for crop improvement.
Plant Cell Physiol 51:497–523
Moody G (2004) Digital code of life: how bioinformatics is revolutionizing science, medicine, and
business. Wiley, Hoboken. ISBN 978-0-471-32788-2
Morris GM, Lim-Wilby M (2008) Molecular docking. Methods Mol Biol 443:365–382
Mount DW (2007) Using the basic local alignment search tool (BLAST). CSH Protoc 2007:pdb.
top17
NCBI Resource Coordinators (2016) Database resources of the national center for biotechnology
information. Nucleic Acids Res 44:D7–D19
Ong Q, Nguyen P, Thao NP, Le L (2016) Bioinformatics approach in plant genomic research. Curr
Genomics 17:368–378
Pagadala NS, Syed K, Tuszynski J (2017) Software for molecular docking: a review. Biophys Rev
9:91–102
Pirovano W, Heringa J (2008) Multiple sequence alignment. Methods Mol Biol 452:143–161.
https://doi.org/10.1007/978-1-60327-159-2_7. Review. PubMed PMID: 18566763
Rambaut A (2012) FigTree v1. 4.0. University of Oxford, Oxford. http://tree.bio.ed.ac.uk/software/
figtree/
Sayers EW, Agarwala R, Bolton EE, Brister JR, Canese K, Clark K, Connor R, Fiorini N, Funk K,
Hefferon T, Holmes JB, Kim S, Kimchi A, Kitts PA, Lathrop S, Lu Z, Madden TL, Marchler-
Bauer A, Phan L, Schneider VA, Schoch CL, Pruitt KD, Ostell J (2019) Database resources of
the national center for biotechnology information. Nucleic Acids Res 47:D23–D28
Schneidman-Duhovny D, Inbar Y, Nussinov R, Wolfson HJ (2005) PatchDock and SymmDock:
servers for rigid and symmetric docking. Nucleic Acids Res 33(Web Server issue):W363–W367
Seo J, Gordish-Dressman H, Hoffman EP (2006) An interactive power analysis tool for microarray
hypothesis testing and generation. Bioinformatics 22(7):808–814
Sievers F, Higgins DG (2014) Clustal Omega, accurate alignment of very large numbers of
sequences. Methods Mol Biol 1079:105–116
Sigrist CJ, Cerutti L, Hulo N, Gattiker A, Falquet L, Pagni M, Bairoch A, Bucher P (2002)
PROSITE: a documented database using patterns and profiles as motif descriptors. Brief
Bioinform 3(3):265–274
Sousa SF, Fernandes PA, Ramos MJ (2006) Protein-ligand docking: current status and future
challenges. Proteins 65:15–26
Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva
NT, Morris JH, Bork P, Jensen LJ, Mering CV (2019) STRING v11: protein-protein association
networks with increased coverage, supporting functional discovery in genome-wide experimen-
tal datasets. Nucleic Acids Res 47:D607–D613
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and
ClustalX. Curr Protoc Bioinformatics. Chapter 2:Unit 2.3
Tippmann HF (2004) Analysis for free: comparing programs for sequence analysis. Brief Bioinform
5(1):82–87
Trosset JY, Cavé C (2019) In silico drug-target profiling. Methods Mol Biol 1953:89–103
UniProt Consortium T (2018) UniProt: the universal protein knowledgebase. Nucleic Acids Res
46:2699
Wang Z, Chen Y, Li Y (2004) A brief review of computational gene prediction methods. Genomics
Proteomics Bioinformatics 2:216–221
Wheeler DL, Smith-White B, Chetvernin V, Resenchuk S, Dombrowski SM, Pechous SW,
Tatusova T, Ostell J (2005) Plant genome resources at the national center for biotechnology
information. Plant Physiol 138:1280–1288
Xiang Z (2006) Advances in homology protein structure modeling. Curr Protein Pept Sci
7:217–227. Review. PubMed PMID: 16787261; PubMed Central PMCID: PMC1839925
Xu D, Xu Y, Uberbacher EC (2000) Computational tools for protein modeling. Curr Protein Pept
Sci 1:1–21
Zhao H, Caflisch A (2015) Molecular dynamics in drug design. Eur J Med Chem 91:4–14
844 A. Khan et al.
URLs
http://bioinformatics.cau.edu.cn/PMRD/
http://circos.ca/
http://hex.loria.fr/
http://mordred.bioc.cam.ac.uk/~rapper/rampage.php
http://tree.bio.ed.ac.uk/software/figtree/
http://www.cs.umd.edu/hcil/hce/
http://www.mbio.ncsu.edu/BioEdit/bioedit.html
http://www.plantgdb.org/
https://biit.cs.ut.ee/clustvis/
https://bioinfo3d.cs.tau.ac.il/PatchDock/
https://blast.ncbi.nlm.nih.gov/Blast.cgi
https://en.wikipedia.org/wiki/Category:Bacterial_plant_pathogens_and_diseases
https://en.wikipedia.org/wiki/Category:Fungal_plant_pathogens_and_diseases
https://en.wikipedia.org/wiki/Category:Lists_of_plant_diseases
https://en.wikipedia.org/wiki/Category:Viral_plant_pathogens_and_diseases
https://phytozome.jgi.doe.gov/pz/portal.html
https://plants.ensembl.org/index.html
https://prosite.expasy.org/
https://servicesn.mbi.ucla.edu/Verify3D/
https://www.ddbj.nig.ac.jp/index-e.html
https://www.ebi.ac.uk/interpro/search/sequence/
https://www.ebi.ac.uk/Tools/msa/clustalo/
https://www.embl.org/
https://www.megasoftware.net/index.html
https://www.ncbi.nlm.nih.gov/
https://www.ncbi.nlm.nih.gov/geo/
https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml
https://www.ncbi.nlm.nih.gov/Structure/MMDB/mmdb.shtml
https://www.plantcyc.org/
https://www.rcsb.org/
https://www.uniprot.org/

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Krishna

Emerging Trends in Plant

Birinchi Kumar Sarma

ISBN 978-981-15-6274-7 ISBN 978-981-15-6275-4 (eBook)

# Springer Nature Singapore Pte Ltd. 2021

Executive Secretary, APAARI Ravi Khetarpal

Pantnagar, Uttarakhand, India Krishna P. Singh

1 Emerging Plant Diseases: Research Status and Challenges . . . . . . . 1

11 New-Generation Fungicides for Sustainable Production and

22 Concept of Effectors and Receptors in Improving Plant

About the Editors

Dr. Krishna P. Singh is a Professor in the Department of Plant Pathology, G. B.

Dr. Shamarao Jahagirdar is a Professor in the Department of Plant Pathology,

Dr. Birinchi Kumar Sarma is a Professor in the Department of Mycology and

S. Abeysinghe Department of Botany, University of Ruhuna, Matara, Sri Lanka

V. Celia Chalam Division of Plant Quarantine, ICAR-National Bureau of Plant

N. Jhansirani Department of Plant Pathology, College of Horticulture, Bengaluru,

Sunita Mahapatra Department of Plant Pathology, Bidhan Chandra Krishi

Laxmi Rawat Plant Pathology Division, College of Forestry, Tehri, India

Saurabh Singh Department of Environment & Sustainable Development, Institute

Plant diseases result in signiﬁcant crop destruction thereby inadequate food

# Springer Nature Singapore Pte Ltd. 2021 1

on increasing the fundamental understanding of host-pathogen interactions to detect

Fig. 1.1 Multitunnel

Fig. 1.2 Aerial image of

1.2 Emerging Plant Diseases and Their Research Status

Our knowledge of global crop losses due to plant-pathogen infestations is very

Fig. 1.5 Disease triangle

1.3 Overview of Disease Detection Methods and Their Ability

According to plant pathologists, serological and PCR-based methods are the

1.3.1 Molecular Methods for Disease Detection

1.3.2 Serological Assays

In the ELISA-based method of plant disease detection, the microbial protein

1.3.3 Spectroscopic and Imaging Techniques

Recent research developments focus on automated nondestructive methods of plant

1.3.4 Other Innovative Detection Methods

1.3.5 Remote Sensing Method

1.4 Challenges of Plant Disease Management

Remote sensing Biophotonics

Climate change · Emerging plant disease · Pathogen · Environment

# Springer Nature Singapore Pte Ltd. 2021 19

2.2 Historical Impact of Emerging Diseases

Emergence of new disease or a new strain of pathogen in a non-native place is a

2.3 Impact of Changing Climatic Components on Plant Disease

geographical distribution, increased overwintering, changes in population growth

2.3.2 Carbon Dioxide Gas (CO2)

2.3.3 Light and Ultraviolet (UV)

2.3.4 Ozone Gas

2.3.5 Rainfall and Humidity

is generally needed for fungal spore germination, multiplication and penetration of

2.3.6 Effect of Soil Moisture

2.3.7 Effect of Wind

2.4 Emergence of New Diseases and Pathogens Due to Climate

Climate change may cause favorable environment to the emergence of new

2.5 Possible Causes of Emergence of Plant Pathogens

laccase protein produced by C. gloeosporioides. The inﬂuence of geography is

2.6 Actions for Mitigating Emergence of New Pathogens

Climate change is a nature’s phenomenon which cannot be regulated or managed by

Phytosanitary programs include surveillance at ports, airports, and borders to

Climate change is an important phenomenon that affects agricultural production

Carnicer J, Coll M, Ninyerola M, Pons X, Sánchez G, Peñuelas J (2011) Widespread crown

M. R. Khan (*) · I. Ahamad · M. H. Shah

# Springer Nature Singapore Pte Ltd. 2021 33

Trichoderma spp. or Pseudomonas ﬂuorescens as a general treatment may prove