Management of Fungal Plant Pathogens

frans QNAP

Among the most damaging fungal pathogens of wheat,Fusarium graminearum, main causal agent of Fusarium head blight (FHB), reduces significantly yield worldwide, and affects grains quality by mycotoxins contamination. During the past years, numbers of FHB resistance QTL with moderate effects against this pathogen have been identified in very genetically diverse wheat collections. However the lack of diagnostic markers as well as detrimental linkage drag associated with some of these QTL (QTL governing the yield and grain quality) limit breeding for resistance. Understanding the molecular basis of FHB susceptibility can provide alternative tools to improve the control of this disease in fields. To decipher the molecular crosstalk involved during compatible interaction between F. graminearum and its host, and to gain information about susceptibility host factors, we analyzed a time course infection of the susceptible French wheat cultivar Recital by F. graminearum through transcriptomic...

India has the largest pulse producing area of about 24 million ha with production of grains only 14.8 million tones per annum. The productivity of pulses staggers around 600 kg/ha well below the world average productivity of 846 kg/ha over last 15-20 years. The low productivity of the pulse in India is caused by the effect of several biotic constraints, of which diseases caused by viral pathogens are most important. Further, the variation in production of pulses in India is common as pulses are cultivated mainly under rainfed conditions. The over increasing population of the country and low productivity of pulses has resulted depletion of per capita availability to about 40 gm in recent year. Thus, it is necessitated to import pulses from neighboring countries. Therefore, it is urgent need to overcome the constrains of pulse production, so that, our country is able to produce about 27 million tones pulses by 2015 to meet the demand of our citizens. Mungbean and urdbean are mainly cultivated in many tropical and sub-tropical continents like Pakistan, Bangladesh, Thailand, Philippines, China, Vietnam, Indonesia and Burma. The major pulses grown in this country are chick pea (Cicer arietinum), pigeon pea (Cajanus cajan), mungbean (Vigna radiata), urdbean (V. mungo), lentil (Lens culinaris), peas (Pisum sativum), French bean (Phaseolus vulgaris) and cowpea (P. unguiculata). More than 100 viruses have been reported to causes diseases in the pulse crops. In present chapter viral disease in two important pulse, mungbean and urdbean will be discussed.

Management of Fungal Plant Pathogens This page intentionally left blank Management of Fungal Plant Pathogens Edited by Arun Arya Professor and Head, Department of Botany and Coordinator Environment Science Programme, Faculty of Science The Maharaja Sayajirao University of Baroda, Vadodara, India and Analía Edith Perelló Assistant Professor and Research Scientist, CONICET - CIDEFI, and Coordinator MSc Vegetal Protection Programme, Plant Pathology, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Provincia de Buenos Aires, Argentina CABI is a trading name of CAB International CABI Head Ofﬁce Nosworthy Way Wallingford Oxfordshire OX10 8DE UK CABI North American Ofﬁce 875 Massachusetts Avenue 7th Floor Cambridge, MA 02139 USA Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 E-mail: cabi@cabi.org Website: www.cabi.org Tel: +1 617 395 4056 Fax: +1 617 354 6875 E-mail: cabi-nao@cabi.org © CAB International 2010. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK Library of Congress Cataloging-in-Publication Data Management of fungal plant pathogens / edited by Arun Arya, Analía Edith Perelló. p. cm. Includes bibliographical references and index. ISBN 978-1-84593-603-7 (alk. paper) 1. Fungal diseases of plants. 2. Phytopathogenic fungi–Control. 3. Plant-pathogen relationships. I. Arya, Arun. II. Perelló, Analía Edith. III. title. SB733.M36 2010 632'.4–dc22 2009023395 ISBN-13: 978 1 84593 603 7 Typeset by AMA Dataset, Preston, UK. Printed and bound in the UK by the MPG Books Group. Contents Contributors Preface viii xi PART I: BOTANICALS IN FUNGAL PEST MANAGEMENT 1 Recent Advances in the Management of Fungal Pathogens of Fruit Crops Arun Arya 2 Botanicals in Agricultural Pest Management Ashok Kumar, Priyanka Singh and N.K. Dubey 3 Deleterious Effects of Fungi on Postharvest Crops and Their Management Strategies A.O. Ogaraku 28 Exploitation of Botanicals in the Management of Phytopathogenic and Storage Fungi Pramila Tripathi and A.K. Shukla 36 Use of Plant Extracts as Natural Fungicides in the Management of Seedborne Diseases Gustavo Dal Bello and Marina Sisterna 51 4 5 3 14 PART II: DISEASE CONTROL THROUGH RESISTANCE 6 Resistance to Septoria Leaf Blotch in Wheat María R. Simón 69 7 Barley and Wheat Resistance Genes for Fusarium Head Blight S.A. Stenglein and W.J. Rogers 78 v vi 8 Contents Sustainable Management of Rice Blast (Magnaporthe grisea (Hebert) Barr): 50 Years of Research Progress in Molecular Biology S. Nandy, N. Mandal, P.K. Bhowmik, M.A. Khan and S.K. Basu 92 PART III: BIOLOGICAL CONTROL MECHANISMS 9 10 11 Postharvest Technology – Yeast as Biocontrol Agents: Progress, Problems and Prospects Neeta Sharma and Pallavi Awasthi 109 Biological Control of Plant Diseases: An Overview and the Trichoderma System as Biocontrol Agents Abhishek Tripathi, Neeta Sharma and Nidhi Tripathi 121 Physiological Specialization of Ustilaginales (Smut) of Genera Bromus, Zea and Triticum in Argentina Marta M. Astiz Gassó and María del C. Molina 138 PART IV: ENDOPHYTES IN PLANT DISEASE CONTROL 12 13 14 15 Status and Progress of Research in Endophytes from Agricultural Crops in Argentina Silvina Larrán and Cecilia Mónaco 149 Effect of Tillage Systems on the Arbuscular Mycorrhizal Fungi Propagule Bank in Soils Santiago Schalamuk and Marta N. Cabello 162 Mechanism of Action in Arbuscular Mycorrhizal Symbionts to Control Fungal Diseases Arun Arya, Chitra Arya and Renu Misra 171 Role of Fungal Endophytes in Plant Protection S.K. Gond, V.C. Verma, A. Mishra, A. Kumar and R.N. Kharwar 183 PART V: MANAGING FUNGAL PATHOGENS CAUSING LEAF DAMAGE 16 The Rust Fungi: Systematics, Diseases and Their Management M.S. Patil and Anjali Patil 201 17 Etiology, Epidemiology and Management of Fungal Diseases of Sugarcane Ayman M.H. Esh 217 18 New and Emerging Fungal Pathogens Associated with Leaf Blight Symptoms on Wheat (Triticum aestivum) in Argentina Analía Edith Perelló 231 Diseases of Fenugreek (Trigonella foenum-graecum L.) and Their Control Measures, with Special Emphasis on Fungal Diseases S.N. Acharya, J.E. Thomas, R. Prasad and S.K. Basu 245 19 Contents vii 20 Fungal Diseases of Oilseed Crops and Their Management S.S. Adiver and Kumari 263 21 Occurrence of Pyrenophora tritici-repentis Causing Tan Spot in Argentina M.V. Moreno and A.E. Perelló 275 22 Epidemiological Studies on Septoria Leaf Blotch of Wheat in Argentina Cristina A. Cordo 291 PART VI: ALTERNATIVE CONTROL STRATEGIES 23 24 25 Review of Thecaphora amaranthicola M. Piepenbr., Causal Agent of Smut on Amaranthus mantegazzianus Pass. M.C.I. Noelting, M.C. Sandoval, M.M.A. Gassó and M.C. Molina 311 Population Biology and Management Strategies of Phytophthora sojae Causing Phytophthora Root and Stem Rots of Soybean Shuzhen Zhang and Allen G. Xue 318 Management of Fungal Pathogens – A Prerequisite for Maintenance of Seed Quality During Storage Anuja Gupta 329 26 Controlling Root and Butt Rot Diseases in Alpine European Forests Paolo Gonthier 345 27 Some Important Fungal Diseases and Their Impact on Wheat Production Aakash Goyal and Rajib Prasad 362 Index The colour plate section can be found following page 50. 375 Contributors Acharya, S.N., Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada T1J 4B1 Adiver, S.S., Oilseeds Scheme, Main Agricultural Research Station, University of Agricultural Sciences, Dharwad 580 005, Karnataka, India (shivaputra_adiver@rediffmail.com) Arya, Arun, Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, India (aryaarunarya@rediffmail.com) Arya, Chitra, Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, India (caarya@yahoo.co.in) Astiz Gassó, Marta M., Instituto Fitotécnico Santa Catalina (IFSC), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, CC 4, 1836 Llavallol, Buenos Aires, Argentina (astizgasso@yahoo.com.ar) Awasthi, Pallavi, Mycology and Plant Pathology Division, Department of Botany, University of Lucknow, Lucknow 226007, India Basu, S.K., Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4 (saikat.basu@uleth.ca) Bhowmik, P.K., Bioproducts and Bioprocesses, Lethbridge Research Center, Agriculture and Agri-Food Canada, Lethbridge, AB Canada T1J 4B1 Cabello, Marta N., Comisión de Investigaciones Cientíﬁcas de la Provincia de Buenos Aires (CICBA) – Instituto de Botánica Spegazzini, Calle 53 N° 577, 1900 La Plata, Argentina (mcabello@museo.fcnym.unlp.edu.ar) Cordo, Cristina A., Comisión de Investigaciones Cientíﬁcas de la Provincia de Buenos Aires, Centro de Investigaciones de Fitopatología (CIDEFI) – Facultad de Ciencias Agrarias y Forestales, 60 y 119, (1900) La Plata, Argentina (cristcordo@hotmail.com) Dal Bello, Gustavo, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata, Argentina (dalbello@speedy.com.ar) Dubey, N.K., Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India (nkdubey2@rediffmail.com) Esh, Ayman M.H., Biotechnology and Tissue Culture Laboratories, Sugar Crops Research Institute, Agricultural Research Center, Giza, Egypt (aymanesh@gmail.com) Gond, S.K., Mycopathology and Microbial Technology Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India viii Contributors ix Gonthier, Paolo, Department of Exploitation and Protection of Agricultural and Forestry Resources (DIVAPRA), Plant and Forest Pathology, University of Torino, Via L. da Vinci, 44, I-10095 Grugliasco (TO), Italy (paolo.gonthier@unito.it) Goyal, Aakash, Agriculture and Agri-Food Canada, Lethbridge Research Center, Lethbridge AB-T1J4B1, Canada (akgroyal@gmail.com) Gupta, Anuja, Indian Agricultural Research Institute, Regional Station, Karnal – 132 001, Haryana, India (agupta_2005@yahoo.com) Khan, M.A., Department of Weed Science, NWFP Agricultural University, Peshawar, NWFP, Pakistan 25130 Kharwar, R.N., Mycopathology and Microbial Technology Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India (rnkharwar@yahoo.com) Kumar, Ashok, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India Kumari, Oilseeds Scheme, Main Agricultural Research Station, University of Agricultural Sciences, Dharwad 580 005, Karnataka, India Larrán, Silvina, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata, Argentina Mandal N., Bidhan Chandra Krishi Vishavidalay, Nadia, WB, India 741252 Mishra, A., Mycopathology and Microbial Technology Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India Misra, Renu, Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, India Molina, María del C., Consejo de Investigaciones Cientíﬁcas y Técnicas (CONICET), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, CC 4, 1836 Llavallol, Buenos Aires, Argentina Mónaco, Cecilia, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata, Argentina (cecilia.monaco7@gmail.com) Moreno, M.V., CONICET – Facultad de Agronomía de Azul, Universidad Nacional del Centro de la Provincia de Buenos Aires, República de Italia No. 780, Azul CP 7300, Buenos Aires, Argentina (morevir@yahoo.com.ar) Nandy, S., Bioproducts and Bioprocesses, Lethbridge Research Center, Agriculture and Agri-Food Canada, Lethbridge, AB Canada T1J 4B1 Noelting, M.C.I., Instituto Fitotécnico de Santa Catalina, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Garibaldi 3400, Llavallol 1836 CC 4 Buenos Aires, Argentina (mcnoelting@hotmail.com) Ogaraku, A.O., Plant Science and Biotechnology Unit, Department of Biological Sciences, Nasarawa State University, PMB 1022, Kefﬁ, Nasarawa State, Nigeria (ogara006@yahoo. com) Patil, Anjali, Department of Botany, Rajaram College, Kolhapur 416004 (M.S.), India (dhirajanj@gmail.com) Patil, M.S., Department of Botany, Shivaji University, Kolhapur (M.S.), India Perelló, Analía Edith, CIDEFI (Centro de Investigaciones de Fitopatología) – CONICET (Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas), Facultad de Ciencias Agrarias y Forestales de la Universidad Nacional de La Plata, La Plata, Provincia de Buenos Aires, Argentina (anaperello@yahoo.com.ar) Prasad, Rajib, Agriculture and Agri-Food Canada, Lethbridge Research Center, Lethbridge AB-T1J4B1, Canada Rogers, W.J., Laboratorio de Biología Funcional y Biotecnología (BIOLAB), Facultad de Agronomía, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNICEN), x Contributors Av. República de Italia # 780 (CC 47), (7300) Azul, Buenos Aires, Argentina; FIBA – Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET), Argentina Sandoval, M.C., Facultad de Ciencias Agrarias, UNLZ, Ruta 4 Km 2 Llavallol, Buenos Aires, Argentina Schalamuk, Santiago, CONICET – Centro de Investigaciones de Fitopatología (CIDEFI) y Cerealicultura, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata, Argentina (sschala@yahoo.com.ar) Sharma, Neeta, Mycology and Plant Pathology Division, Department of Botany, University of Lucknow, Lucknow 226007, India (dr_neeta_sharma2003@yahoo.com) Shukla, A.K., Department of Botany, Rajiv Gandhi University, Rono Hills, Itanagar 791 112, India Singh, Priyanka, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India Simón, María R., Cerealicultura, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata, Argentina (mrsimon@agro.unlp. edu.ar) Sisterna, Marina, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata, Argentina (mnsisterna@infovia.com.ar) Stenglein, S.A., Laboratorio de Biología Funcional y Biotecnología (BIOLAB), Facultad de Agronomía, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNICEN), Av. República de Italia # 780 (CC 47), (7300) Azul, Buenos Aires, Argentina; FIBA – Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET), Argentina (stenglein@ faa.com.unicen.edu.ar) Thomas, J.E., Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4 Tripathi, Abhishek, Department of Bioscience and Biotechnology, Banasthali University, PO Banasthali Vidyapith, 304022 Rajasthan, India (abhitri77@yahoo.com) Tripathi, Nidhi, Department of Bioscience and Biotechnology, Banasthali University, PO Banasthali Vidyapith, 304022 Rajasthan, India Tripathi, Pramila, Department of Botany, D.A.V.-P.G. College, Kanpur 208001 (U.P.), India (pramilatripathi_bhu@rediffmail.com) Verma, V.C., Mycopathology and Microbial Technology Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India Xue, Allen G., Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa, Ontario, Canada, K1A 0C6 (axue@agr.gc.ca) Zhang, Shuzhen, Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese Education Ministry, Northeast Agricultural University, Harbin, Heilongjiang, China, 150030 Preface Oldest life forms have been reported from the North Pole Dome area of Western Australia, which dates back 3556 million years. Non-septate mycelium remains of Eomycetopsis robusta were recovered from late Precambrian chert of Australia. Having appeared ﬁrst on planet Earth, microbes have immense potential to inﬂuence all other life forms. Plant diseases have caused epidemics and have had a profound inﬂuence on wars, famine and the changing economy. Microbes including fungi need no introduction to common man; they are progressive, ever changing and evolving in their own way, so they are capable of adapting to every condition of life. The French biochemist, Louis Pasteur, once said, ‘The role of the inﬁnitely small is inﬁnitely large.’ Potentially immortal fungi spread their tentacles in 1845, when potato late blight fungus caused havoc in Ireland. Soon after, Plasmopara viticola threatened the wine industry in France. First reported in 1819 in Sweden, apple scab disease caused by Venturia inaequalis threatened apple cultivation in the Kashmir Valley in India in 1973. Panama disease of banana, wilt diseases of pigeon pea, castor and guava and smut and rust of cereals are some other serious fungal diseases. The chance discovery of Bordeaux mixture by P.A. Millardet in France paved the way to the chemical control of plant diseases. Phytopathologists are confronted by a volley of challenges in the wake of a resurgence of new diseases and the obligation to fulﬁl international trade agreements. We have to protect the environment and at the same time ensure the safety and security of farmers in the ﬁeld by making a concentrated effort to minimize crop losses due to fungi and other microbes. This book provides an overview of our current knowledge of some plant–pathogen interactions in economically important crops, emphasizing the importance of pathogenic fungi on fruits, cereals, postharvest crops and the establishment of plant diseases and drawing together fundamental new information on their management strategies based on conventional and eco-friendly methods, with an emphasis on the use of microorganisms and various biotechnological aspects of agriculture, which could lead to sustainability in modern agriculture. The book examines the role of microbes in growth promotion, as bioprotectors and bioremediators, and presents practical strategies for using microbes in sustainable agriculture. In addition, the use of botanicals vis-à-vis chemical pesticides has also been reviewed. Contributions on new research ﬁelds such as mycorrhizae and endophytes have been xi xii Preface included. The book also examines in different chapters host–pathogen interactions in the light of the new tools and techniques of molecular biology and genetics. Dr Arya expresses his deep sense of indebtedness and admiration to the late Dr S.N. Bhargava and to Professor Bihari Lal, ex Head of the Department of Botany, University of Allahabad, who taught him his ﬁrst lessons in plant pathology at the University of Allahabad. He is grateful to his father, the late Shri O.P. Arya, for inspiring him to write about the management of plant diseases and pests, which has proved most useful to plant growers. He honours his grandfather, Baba Shankaranand, who fed him with sweet mangoes during his childhood and who motivated him to love plants and to learn how to nurture them and research into new and improved varieties. We are grateful to the entire staff of our institutions and the cooperation and collaborative efforts of the plant pathology experts of Argentina (Universidad Nacional de La Plata, Universidad Nacional de Lomas de Zamora, Universidad Nacional del Centro) and India (Botany Department, The Maharaja Sayajirao University of Baroda), who made this book possible. We thank all those who have contributed their valuable articles to this volume and are sure that the present work, which consists of 27 different chapters written by learned experts in the ﬁeld, will be immensely useful to postgraduate students, researchers, academics, progressive farmers and practising horticulturists, as well as those involved in the various agro-industries. We are hopeful that the available knowledge in the ﬁeld, newer technologies and disease-resistant varieties will be used in different parts of the world and that ultimately the plant disease scenario will change. All appreciations and good wishes are extended to the members of the CABI team, particularly Ms. Sarah Mellor, for helpful discussions and skilled assistance in the reviewing of the manuscripts, and also for helping us in various ways to accomplish this project satisfactorily in the stipulated time. And also for the cooperation and collaborative effort of the Plant Pathology experts that made this book possible. Arun Arya Analia Edith Perelló Part I Botanicals in Fungal Pest Management This page intentionally left blank 1 Recent Advances in the Management of Fungal Pathogens of Fruit Crops Arun Arya Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India Abstract Fruits constitute a rich source of sugars, vitamins, minerals and medicinally important compounds like ﬂavonoids, which prevent cancer and cardiovascular diseases. These are eaten as a dessert or processed into jams, jellies, ice creams and drinks; grapes are dried to make raisins. The science of protecting fruit crops began with the discovery of Bordeaux mixture by P.A. Millardet in France. But still we have yet to ﬁnd many new techniques and fungicide formulations to control diseases; such as bunch rot of grapes (Botrytis cinerea), apple scab (Venturia inaequalis), wilt of guava (Fusarium solani), Panama wilt of banana (F. cubense), mango malformation (F. moniliforme), blue mould of citrus (Penicillium citrinum) and anthracnose of papaya (Colletotrichum papayae), etc. Losses from postharvest fruit diseases range from 1 to 20% in the USA and from 10 to 40% in India. The pathogens have developed resistance against various fungicides and the postharvest phase is minimized. Alternative strategies like the use of biocontrol methods and the application of botanicals have been tried. A large number of plants are screened for the presence of effective secondary metabolites. Integrated pest management, using improved cultural practices (pruning methods to control Botrytis bunch rot in grapes), the use of solarization (in strawberries), the application of growth hormone (NAA in the case of mango malformation), along with minimum dosage of fungicides, are recommended to control various fruit diseases. The world fruit market is expanding; we are more concerned about human nutrition now, but at the same time serious enough to protect the environment from pollution. The economics of a success story will have to revolve around the use of various cutting-edge technologies and, at the same time, the use of simpler and more effective methods acceptable to fruit growers. Biotechnologists have tried to enhance the activity of biocontrol agents; at the same time, efforts are being made for genetic transformation involving molecular breeding. This technology involves intimate knowledge of the gene, regulatory components and gene functional environment (i.e. the domain where the gene is located). Once an understanding of the molecular basis of genes involved in resistance has been achieved, we will be able to isolate the alleles of those genes and their inclusion will lead to transformed, diseasefree plants. Introduction Fruits constitute an important component of our daily diet. The use of dates, ﬁg, mango and grapes is mentioned in ancient texts. Taken either as a dessert or processed, the nutritional value of fruits depends chieﬂy on the quality and concentration of sugars, vitamins and other essential minerals. Plants suffer with a number of diseases and pests  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 3 4 A. Arya during their growth phase. Fungi not only blemish, disﬁgure or cause rot to a number of fruits but also reduce their market value (Arya, 2004). Realizing the importance of postharvest diseases, Stevens and Stevens (1952) mentioned, ‘of all losses caused by plant disease those that occur after harvest are the most costly, whether measured in monetary terms or in man hours’. India is the second most important fruit producing country of the world. It produces the highest quantity of mango, while the productivity of grapes in India at 56 t/ha is a world record. The export of mango increased in the nineties from 25,000 to 44,000 t (a 25% share of world trade) (Neelam, 1993). Fruit production is 49 Mt (Arya, 2004). The fruit growing industry has developed a lot, overcoming the hurdles of biotic and abiotic stresses. The industry needs a comprehensive strategy to face the challenges and opportunities of a global economy. Lewis (1985) stated that ‘a few key discoveries have led to a breakthrough in our understanding of the biological genome and our ability to alter it, which may equal in signiﬁcance the development of nuclear energy in the Physical Sciences.’ Fungal Infection of Fruits and Fruit Trees Brown rot of citrus fruit is caused in the orchard by Phytophthora citrophthora. Many fungi that penetrate the host in the ﬁeld cause quiescent infection. The grey mould in strawberries is caused by B. cinerea. The Botrytis spores with which the strawberry ﬁeld is ﬁlled during bloom can germinate in a drop of water on the petal or other parts of the ﬂower and later penetrate the senescenced parts of the ﬂower into the edge of the receptacle of the strawberry, where they develop a dormant mycelium. During ripening and storage, as the resistance of the fruit to the pathogen decreases, the preliminary mycelium enters an active stage and decay develops (Powelson, 1960; Jarvis, 1962). Postharvest pathogens can be divided, according to the timing of their penetration of the host, into those that penetrate the fruits while still in the ﬁeld, but develop in their tissues only after harvest, during storage or marketing, and those that initiate penetration during or after harvest. Symptoms in stylar end rot of guava caused by Phomopsis psidii become more prominent during storage (Arya, 1983). Verhoeff (1974) describes how quiescent infection is established in young fruits: 1. Shortage of adequate substances in young fruits. 2. The incapability of the pathogen to produce cell wall degrading enzymes in the young fruit. 3. The presence of antifungal compounds. 4. The accumulation of phytoalexins (Swinburne, 1983). The ﬁrst theory claims that young unripe fruit does not provide the pathogen with the nutrition and energy required for its development. The artiﬁcial increase of the sugar level in apple was achieved by the use of a chemical such as 2,4-dinitrophenol on the fruit. It accelerated the decay caused by Botryosphaeria ribis (Sitterly and Shay, 1960). It has been found that antifungal compounds become toxic in the presence of sugars. The second theory suggests that the unripe fruit does not supply the pathogen with compounds that induce activity in cell wall degrading pectolytic enzymes. The third and fourth theories point to a relation between the formation of antifungal compounds in the young tissues and the creation of quiescent infections. Chemicals such as 3,4-dihydroxy benzaldehyde have proven fungistatic activity in the green banana fruit. In unripe avocado fruit, a link has been established between the presence of a diene and monoene antifungal compounds in the fruit rind and the quiescent infection of C. gloeosporioides in such a fruit. The reduction in the concentration of the diene probably results from lipoxygenase enzymatic activity that increases as ripening progresses and the fruit softens (Prusky et al., 1982, 1985). The dormant state of Alternaria alternata in young mango fruits has been attributed to the presence of two antifungal resorcinols in the unripe fruit rind (Droby et al., 1986). Recent Advances in the Management of Fungal Pathogens of Fruit Crops Recent Advances in the Management of Fungal Pathogens Cultural practices Initial infection of most temperate fruits is carried from the orchard; therefore, preharvest cultural practices, if adopted, considerably reduce postharvest diseases during transit and storage. Strict orchard hygiene and maintenance of tree vigour is recommended to reduce losses from Botryosphaeria rot of apple. Pezicula malicorticis and Nectria galligena infection in apple start from cankered portions. The removal of dead and senile plant parts and canker portions helps to reduce the incidence of many postharvest diseases. The incidence of many rots may also be reduced if the rotted fruits are frequently collected and dumped in a deep trench and later covered with a thick layer of soil to prevent the dissemination of their spores. If such rotted fruits are destroyed by burning some distance away from the orchard, this also helps to reduce the incidence of many rots in temperate fruits. Proper pruning can prevent Botrytis rot of grapes (Philips et al., 1990). The inﬂuence of N, P, K, Ca and Mg nutrients on storage rots of apple and pear has been studied extensively (Sharples, 1980). Susceptibility to Gloeosporium rot was correlated negatively with fruit Ca, but correlated positively with K/Ca ratios. Higher doses of nitrogen increase the incidence of G. album (Montgomery and Wilkinson, 1962). Calcium sprays to control bitter pit in apples also confer resistance to P. expansum. Fumigation Safe fumigating agents that disappear after a short time, such as the use of ozone and sulphur dioxide and acetic acid, can be recommended to reduce dependence on conventional fungicides. Ozone application to grapes (0.1 mg/g grapes) during 20 min exposure reduced decay caused by Rhizopus stolonifer and prolonged shelf life. This treatment was as effective as sulphur dioxide 5 (Sarig et al., 1996). The tolerance of grapes to sulphur dioxide is unique among fresh fruits. It eradicates most of the postharvest pathogens. However, the beneﬁts of sulphur dioxide disappear after a short period of time. Hence, sodium bisulphate in packing cases reacts with the moisture in the air in grape containers. This treatment is used exclusively for the long distance transportation of grapes (Hedberg, 1977). Fumigation with acetic acid is effective in controlling M. fructicola, R. stolonifer and Alternaria species on peaches, nectarines, apricot and cherries (Sholberg and Gaunce, 1996). Relatively few fumigation treatments have been developed for pome and stone fruits. Heat treatments Heat treatments may be applied by hot water dips or hot vapour exposure. Hot water is useful in controlling fungal infections, while exposure to hot vapour controls insects. Postharvest decay of strawberries caused by B. cinerea and R. stolonifer has been controlled by exposing the fruits to humid air at 44°C for 40–60 min (Couey and Follstad, 1966). Akamine and Arisumi (1953) have reported hot water treatments for fruit rot of papaya (48°C for 20 min). Two methods have been suggested: one involves a shortterm heat treatment above 40°C (usually 44–55°C) for a few minutes to 1 h and in the other, the fruits are exposed to 38–46°C but for a longer duration (12 h to 4 days) (Fallik et al., 1996). The LD50 temperature for sporangiospores of R. stolonifer exposed to hot water for 4 min was 49°C, whereas that for germinating spores was only 39°C (Eckert and Sommer, 1967). Ionizing radiation and UV illumination Ionizing radiation may harm the genetic material of the living cell directly, leading to mutagenesis and eventually to cell death. Most studies are carried out with Co60 gamma rays. It has been seen that multicellular conidia of Alternaria and Stemphylium or 6 A. Arya bicellular spores such as Cladosporium and Diplodia are more resistant to gamma radiation than the unicellular spores of other fungal species (Sommer et al., 1964). Since radiation can penetrate fruit tissues, it has a therapeutic effect. Plant tissues can produce phytoalexins (defence chemicals) in response to radiation effect. Low doses of UV-C light (wavelength 190–280 nm) can induce resistance in a wide range of fruit and vegetables (Barkai-Golan, 2001). UV light has a germicidal effect and, at the same time, it induces activity of PAL and peroxidase enzymes (Droby et al., 1993). Another treatment for extending the postharvest life of apple, pear and plum is by coating the skin with a product called ‘Prolong’, a mixture of sucrose esters of fatty acids and polysaccharide (Banks and Harper, 1981). It alters the permeability of fruits to gases in such a way that oxygen permeability is reduced considerably, while carbon dioxide permeability is little affected. This coating had little effect on grapes and strawberries. Chemically impregnated wrappers Various strains of antagonist must be compared for effectiveness in controlling fruit decay and for phenotypic characteristics that are useful in determining their commercial potential; for example, the differentiation criteria for decay control on apple includes the biological control efﬁcacy of the strains, spectrum of activity (pathogens to be tested, cultivar range, fruit maturity stages), ability to colonize wounded and sound fruit surfaces under various conditions, utilization of substrates occurring in fruits, or growth at cold storage temperatures and at 37°C. In addition, these antagonists must meet strict regulations for safety as they are being applied to consumable commodities, i.e. fruits. Thus, in developing biocontrol systems for postharvest disease management of fruits, the key requirements for successful commercialization of an antagonist must be well deﬁned and strain searches should continue until adequate strains are found that meet all the safety requirements. Wrapping grape clusters in tissue paper impregnated with sodium orthophenyl butyrate and sodium metabisulphate reduces postharvest decay. Volatile fungal inhibitors also provide effective control of grapes against A. niger and P. canescens (Sharma and Vir, 1984). Potassium iodide wraps provide effective control of G. roseum on apples (Sharma and Kaul, 1988). Development of Botryodiplodia rot of apples was retarded by wrapping them in papers dipped in culture ﬁltrate of Streptomyces thermoﬂavus (Gupta and Gupta, 1983). Fruit skin coatings Skin coatings can improve the keeping quality of fruits by decreasing water loss and retarding ripening and rotting by various pathogens. Coating is generally done with oils, waxes and colloidal solutions of carboxymethyl cellulose. Apples coated with mustard oil, parafﬁn and castor oil checked the infection of a large number of pathogens (Sumbali and Mehrotra, 1980; Kaul and Munjal, 1982; Sharma and Kaul, 1988). Application of hydrogenated groundnut oil provided effective control of Alternaria rot of apple (Tak et al., 1985). Skin coating with neem oil completely checked blue mould rot in apples (Kerni et al., 1983). Search for the antagonists: criteria of selection Enhancement in biocontrol activity of antagonists Postharvest environments are better deﬁned than ﬁeld conditions, wherein abiotic and biotic factors can be determined with relative ease and manipulated to the antagonist’s advantage, although the mechanism(s) of biocontrol have not yet been fully explained Recent Advances in the Management of Fungal Pathogens of Fruit Crops and, to date, there have been only a few attempts to exploit these mechanisms to improve postharvest biocontrol (Janisiewiez et al., 1992). The reports available on the mechanism of the biocontrol of postharvested commodities suggest that competition for nutrients and space plays a major role in most cases (Wisniewski et al., 1991; Calvente et al., 1999). In most of the systems where microbial communities are involved, interactions are density dependent and often more than one type of interaction occurs at a speciﬁc time which is dependent on the growth phase of different microorganisms, population density and species diversity. Basically, three different types of interactions, namely competition for nutrients, competition for space and inhibition by secondary metabolites, have been observed in preharvest sprays of B. subtilis to control C. gloeosporioides on avocado (Korsten et al., 1997). The main approaches used to improve biological control in postharvest systems are: (i) manipulation of the environment; (ii) use of mixed cultures of antagonists; (iii) physiological and genetic manipulation of antagonists; (iv) combining ﬁeld and postharvest applications; (v) manipulation of formulations; and (vi) integration with other methods. In the case of the development of BioSave, the effectiveness of the antagonist, a saprophytic strain of P. syringae L-59-66, in reducing blue mould and grey mould decay on apples and pears in a laboratory setting was demonstrated to EcoScience Corp (Orlando, Florida, USA). The commercial setting of the test, the involvement of industry in conducting those tests and the encouraging results were the key factors in obtaining a commitment to develop the antagonist for commercial use. EcoScience Corp then investigated the potential for registration and formulation of the antagonist before making this commitment. Mass production by fermentation and the biomass yield of P. syringae strain L-59-66 was determined before scale-up experiments (Janisiewiez, 1998). Extensive technical support and quality control have been instrumental in the success of this product. Similar support and testing need to be conducted for the development 7 of many more biocontrol agents for postharvest fruit rots. Biocontrol: an integrated approach Recently there has been an increased interest in enhancing the efﬁcacy of biocontrol agents by adding some synthetic chemicals like calcium chloride or nitrogenous compounds or sugar analogues. For example, a mixture of Cryptococcus laurentis and thiabendazole has been observed to reduce 95% of P. expansum infection in pear (Sugar et al., 1994). Enhancement of biocontrol activity of antagonists by the addition of nitrogenous (L-asparagine, and L-proline) and carbohydrate (2-deoxy-D-glucose) compound has been reported in apple and pear fruit (Janisiewiez, 1994). Similarly, a combination of 2-deoxy-D-glucose and Candida saitoana is reported to be useful in reducing postharvest diseases (Wilson and El-Ghaouth, 1997). Recently, a bioactive coating having a combination of C. saitoana and 0.2% glycolchitosan has been found more effective in controlling rot development caused by B. cinerea, P. digitatum and P. expansum in several cultivars of apples, oranges and lemon (El-Ghaouth et al., 2000a,b). The same group of researchers showed that the application of C. saitoana with 0.2% 2-deoxy-D-glucose, before inoculation of pathogens, was more effective in controlling the decay of apple, orange and lemon caused by B. cinerea, P. expansum and P. digitatum than either C. saitoana or 0.2% 2-deoxy-D-glucose alone. For the postharvest treatment of fruits, stock of biocontrol agent is usually made in lyophilized cultures, agar slant or spore suspensions and is maintained at low temperature and at the same osmotic concentration in culture medium (Churchill, 1982). Botanicals as Antifungal Agents in Postharvest Disease Control of Fruits Fruits and vegetables have a number of constituents and inducible volatile aromatic and ﬂavour compounds (Tripathi, 2007). 8 A. Arya These aromatic and ﬂavour components are produced generally by fruits during ripening and provide resistance to the fruits at the postharvest stage. The ﬂavour compounds are secondary metabolites having unique properties of volatility and low water solubility. As potential fungicides, their natural occurrence as part of the diet, their ephemeral nature and their biodegradability suggest low toxic residue problems. Such compounds could be extracted and applied to other harvested perishables. Some of the volatile aromatic components, namely acetaldehyde, six carbon (C6) aldehydes, benzaldehyde, hexenel and hexanal, are of signiﬁcant importance. Vapours of acetaldehyde have been used to control B. cinerea (Prasad and Stadelbacher, 1973). Avissar and Pesis (1991) reported acetaldehyde to be active against B. cinerea and R. stolonifer causing rot to strawberry fruits. The effect of trans-2-hexenel on the control of blue mould disease (P. expansum) in the reduction of patulin content and on fruit quality improvement of ‘Conference’ pears was evaluated and greater reduction of decay was obtained by treatment at 12.5 µl/l at 20°C for 24 or 48 h after inoculation (Neri et al., 2006). Jasmonates are naturally occurring plant growth regulators that are widely distributed in the plant kingdom and are known to regulate various aspects of plant development and responses to environmental stresses. The antifungal activity of six glucosinolates has been tested on several postharvest pathogens, namely B. cinerea, R. stolonifer, Monilinia laxa, Mucor piriformis and P. expansum, both in vitro and in vivo (Mari et al., 1996). Fumigation of apples with acetaldehyde, a natural volatile compound produced by various plant organs, inhibits P. expansum development in the fruit (Stadelbacher and Prasad, 1974), while fumigation of strawberries with acetaldehyde considerably reduces decay caused by R. stolonifer and B. cinerea. Evaluation of 15 volatile odour compounds, released from raspberries and strawberries during ripening, for their ability to inhibit postharvest decay fungi showed that 5 of them inhibited the growth of A. alternata, B. cinerea and C. gloeosporioides directly on the fruit at 0.4 µl ml (Vaughn et al., 1993). Among the ﬁve compounds, benzaldehyde was the most toxic to the fungi. Plant extracts Fungitoxic activity of plant extracts can be tested by the poisoned food technique (Grover and Moore, 1962). Tripathi (2005) tested 24 taxa belonging to 12 different families for their antifungal activity against P. italicum. Most of the plants showed either poor or moderate (50–100%) activity. Leaf extracts of seven plants, namely Acacia nilotica (ethyl alcohol), Citrus aurantifolia (ethyl acetate), Murraya koenigii (ethyl acetate), Nerium indicum (ethyl acetate), Ocimum gratissimum (benzene, ethyl acetate), O. sanctum (petroleum ether), Prunus persica (ethyl acetate) and bark extract of A. farnesiana and A. nilotica (ethyl acetate extract) showed 100% activity against test fungus. The leaves of Achyranthes aspera and Hyptia suaveolens showed poor activity. Arya (1988) tried leaf extracts of Aegle marmelos, O. sanctum, Azadirachta indica, Crataeva nurvala, Ephedra foliata (shoot), Eucalyptus occidentalis, Lawsonia inermis and Strichnos nux vomica in three different concentrations on two fruit rot pathogens, P. psidii and P. viticola. Extracts obtained from Ephedra and Eucalyptus were most effective at 25% concentration in the case of P. viticola, while a higher concentration (75%) leaf extract of ‘neem’ (A. indica) was most effective, causing 82.3% spore inhibition. Tulsi caused 76.4% inhibition. The fungicidal nature of ‘neem’ and ‘tulsi’ was reported earlier by Pandey et al. (1983) against Pestalotia psidii. Essential oils Volatile oils are sweet-smelling lipids synthesized and stored in various plant parts. These oils are essentially mixtures of two classes of terpenoids, i.e. the monoterpenes and the sesquiterpenes, the former predominating in Recent Advances in the Management of Fungal Pathogens of Fruit Crops most cases. Among the 49 essential oils tested, those of palmrosa (Cymbopogon martini) and red thyme (Thymus zygis) showed the greatest inhibitory effect on B. cinerea spore germination at the lowest concentration. The next best inhibitors were essential oils of clove buds (Eugenia caryophyllata) and cinnamon leaf (Cinnamomum zeylanicum). The most frequently occurring constituents in essential oils showing high antifungal activity were: D-limonene, cineole, a-pinene, b-pinene, b-myrcene and camphor. The fungicidal activity of the individual components, singly and in combination, is being studied (Wilson et al., 1997). Essential oil derived from another species of Thymus, T. capitatus, reduced the development of B. cinerea markedly in inoculated mandarin fruits when applied as a vapour. Scanning electron microscopic observations indicated a direct damaging effect of the thyme oil on fungal hyphae (Arras and Piga, 1994). Gel and latex Gel derived from Aloe vera has been found to have antifungal activity against four common postharvest pathogens, P. digitatum, P. expansum, B. cinerea and A. alternata. The natural gel suppressed both germination and mycelial growth. Latex present in some fruits is another natural fungicide which is effective against diseases of banana, papaya and other fruits (Adikaram et al., 1996). Papaya latex contains proteases, glucosidases, chitinases and lipases, while a cysteinrich protein, hevien, was isolated from the latex of rubber tree (Hevea brasiliensis). It showed a strong antifungal activity in vitro against B. cinerea and species of Fusarium and Trichoderma (van Parijs et al., 1991). Use of Plantibodies for Disease Control Drawing a clue from the potential antibodies in combating human diseases, plant scientists are now geared to extend this remarkable technology to plant disease control. 9 Antibodies are produced in response to invasion of an antigen. The remarkable potential of recombinant DNA technology has made it possible for plants to express antibodies against pathogen proteins, which in turn enable them to defend against the target pathogen. The expression of pathogenspeciﬁc antibody in plants is termed ‘plantibody’ (Smith, 1996; Gibbs, 1997). The plantibodies produced in the cell cytosol are expected to interact with their targets, rendering them inactive (Zhang and Wu, 1998). Induced Resistance Induced resistance is a new concept proposed by the American phytopathologist, Joseph Kuc (1995). According to Kuc, resistance in plant tissues can be enhanced by modulating their natural defence mechanisms. Various physical, chemical and biological elicitors can enhance resistance in plants. Use of chitosan, a deacetylated derivative of chitin, and salicylic acid can be made to offer a possible alternative to synthetic pesticides. ASM (acibenzolar-s-methyle) is the ﬁrst commercially available product that activates a systemic acquired resistance (SAR) in plants like other biological inducers. Host Defence Through Gene Silencing Scientists working on Eutypa dieback disease of grapevine in Switzerland (2008) found the involvement of glutathion-stransferase in the detoxiﬁcation of toxins, of the jasmonic acid signalling path way, and of several effector genes underlying a more general response where the toxins could be recognized as an elicitor for the trunk pathogens. Grapevines were tested for inﬁltration of double standard RNA into leaves for easy testing of genes. dsRNA were functional in Puccinia striiformis to suppress recognition by host plants (Newton, 2002). Genes that encode for post-transcriptional gene silencing have been characterized in plants and fungi (Dalmay et al., 2000). 10 A. Arya A variety of gene silencing phenomena that have been discovered are: (i) the duplicated DNA sequence is inactivated by mutation in the meiotic phase, a process known as repeat induced mutation (RIP) (Selker et al., 1987); (ii) the duplicated DNA sequence during the meiotic phase is inactivated by methylation, methylation induced premeiotically (MIP) (Goyon and Faugeron, 1989); (iii) multiple copies of transgenes in the vegetative phase are irreversely inactivated and silencing is called ‘quelling’ (Romano and Macino, 1992); and (iv) silencing is maintained even in the absence of transgenes (van West et al., 1999) or another process called MSUD (Shiu et al., 2001). Disease-resistant Transgenic Plants Newly developed techniques in plant breeding such as restriction fragment length polymorphism techniques and gene transfer methods can be used to develop these cultivars. In contrast to conventional breeding, this later technology allows the transfer of traits from one species into the genomes of plants of other species with the preservation of the intrinsic properties of the acceptor plant (Cornelissen and Melchers, 1993). A transgenic plant contains, within its genome, a foreign DNA that has been introduced artiﬁcially via genetic engineering. The creation of such plants involves the introduction of genes for resistance from unrelated plant species. Desirable target genes are isolated from plant viruses, bacteria, fungi or other plants and introduced in the plants. Genes have been transferred by scientists in India from Amaranthus to potato for improving protein quality and quantity, and from mangroves to annual crops for imparting tolerance to salinity. Powell et al. (1994) reported that transgenic tomato fruits expressing the gene of fungal PG-inhibiting glycoproteins of plants were more resistant to B. cinerea than the control fruits. Scientists have tried to prevent ethylene production by plant tissue using an antisense gene. The fruits would not ripen here until treated exogenously with ethylene. PR protein genes appear to be a very potential source for candidate genes providing fungal resistance. These proteins may play a direct role in defence by attacking and degrading pathogen cell wall components. The ﬁrst speciﬁc fungal-resistant gene, Hm1, has been isolated from maize, conferring resistance to race 1 of the fungus Helminthosporium carbonum (Johal and Briggs, 1992). After fungal-resistance genes have been isolated, they can be transferred to provide resistance to a speciﬁc race of fungal pathogens. Woloshuk et al. (1991) identiﬁed in tobacco a salt stress-inducible vacuolar protein with an inhibitory effect on the growth of P. infestans in vitro. It was suggested that this protein, described as Osmotin, inhibited growth by interfering with the fungal membrane, hence disturbing cellular function. 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Dubey Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, India Abstract The overzealous and indiscriminate use of most of the synthetic fungicides has created different types of environmental and toxicological problems. The ultimate aim of recent research in this area has been the development of alternative control strategies to reduce dependency on synthetic fungicides. Recently, in different parts of the world, attention has been paid to the exploitation of higher plant products as novel chemotherapeutants in plant protection because of their non-phytotoxicity, systemicity and easy biodegradability. The exploitation of natural products to control fungal infestation and prolong storage life of food commodities has received more attention. Biologically active natural products have the potential to replace synthetic fungicides. Currently, different plant products have been formulated for large-scale application as botanical pesticides in the eco-friendly management of plant pests and are being used as alternatives to synthetic pesticides in crop protection. This chapter deals with the current status and future prospects of botanical pesticides in eco-friendly management of different plant pests. Introduction The constant growth of the world’s population requires substantial resources for the production of food. One of the greatest challenges of the world is to produce enough food for the growing population. Production as well as protection of food commodities is necessary to nourish the ever-growing population. The situation is particularly critical in developing countries, where the rate of net food production is slowing down in relation to population growth. The world food situation is aggravated by the fact that in spite of the use of all available means of plant protection, a major proportion of the yearly production of food commodities of 14 the world is destroyed by various pests, including bacteria, fungi, viruses, insects, rodents, nematodes, etc. Losses at times are so severe as to lead to famine in large areas of the world that are densely populated. Considerable attention has been given to losses in the ﬁeld caused by different pests, but research into postharvest losses of food commodities is still required. So, priority should be given to postharvest studies, particularly in hot and humid tropical climates where at least half of the foodstuffs may be lost between harvest and consumption. Considerable postharvest losses of food commodities are brought about due to fungi, insects and rodents. International agencies that monitor world food resources have acknowledged  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Botanicals in Agricultural Pest Management that one of the most feasible options for meeting future food needs is the reduction of postharvest losses (Tripathi and Dubey, 2004). Fungi are signiﬁcant destroyers of foodstuffs during storage, rendering them unﬁt for human consumption by retarding their nutritive value. Many agricultural commodities are vulnerable to attack by a group of fungi that are able to produce toxic metabolites called mycotoxins. Production of mycotoxins by several fungi has added a new dimension to the gravity of the problem. Fungal toxins are low molecular weight chemical compounds which are not detected by the body’s antigens. Their effect is more often chronic rather than acute; hence, they produce no obvious symptoms. Thus, mycotoxins are insidious poisons (Pitt, 2002). Cereals and grains are major mycotoxin vectors because they are consumed by both humans and animals. According to FAO estimates, 25% of the world food crops are affected by mycotoxins each year. These toxins can develop during production, harvesting, or storage of grains, nuts and other crops. Mycotoxins are among the most potent mutagenic and carcinogenic substances known. They pose chronic health risks: prolonged exposure through diet has been linked to cancer and kidney, liver and immune system disease (Srivastava et al., 2008). Among mycotoxins, aﬂatoxins chieﬂy produced by strains of Aspergillus ﬂavus are the most dangerous and about 4.5 billion people in underdeveloped countries are at risk of chronic exposure to aﬂatoxicosis through contaminated foods (Williams et al., 2004; Srivastava et al., 2008). In most of the developing countries, total permissible aﬂatoxin content in food has been set around 20 ppb (Mishra and Das, 2003). Aﬂatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive agents, produced as secondary metabolites by the fungus Aspergillus, A. parasiticus and A. nomius on a variety of food products. In addition, aﬂatoxin inhibits seed germination, seedling growth, root elongation, chlorophyll and carotenoid synthesis, as well as protein, nucleic acid and some enzyme synthesis in seeds. Climatic conditions in India are most conducive to mould invasion and elaboration 15 of mycotoxins. Unseasonal rains and ﬂash ﬂoods are very common in India, which enhances the moisture content of the grains, making them more vulnerable to fungal attack (Srivastava, 1987). Fungi can grow on simple and complex food products and produce various metabolites (Khosravi et al., 2007). Up to now, more than 100,000 fungal species are considered as natural contaminants of agricultural and food products (Kacaniova, 2003). The quality and safety of food is of importance so that markets are not compromised by the sale of low quality or unsafe food. Control of Fungal Infestation During Storage Attempts to control postharvest diseases have been carried out by different physical and chemical treatments. Physical methods Several techniques are used for the preservation of food and feeds. Drying, freezedrying, cold storage, modiﬁed atmosphere storage and heat treatments are all physical methods of food preservation (Farkas, 2001) (Table 2.1). Cold storage Low temperature inhibits the germination of spore/conidia and pathogenicity signiﬁcantly (Tian, 2001). It reduces the metabolic activities of various microbes associated with foodstuffs, which would be helpful in enhancing the shelf life of edibles. However, cold storage has its limitations, such as unavailability in most developing countries and an inability to check psychrophilic microorganisms. Heat treatment High temperature plays a signiﬁcant role in controlling the metabolic activities of organisms because it affects the enzymatic activities in all organisms adversely (Lagunas and Castaigne, 2008; Moatsou et al., 2008). Heat treatment can check microbial 16 A. Kumar et al. Table 2.1. Some physical and chemical methods used in the prevention of fungal contamination and mycotoxin production. Methods Fungi/mycotoxins References Physical: Sunlight Solar irradiation Electric light UV light UV-C radiation Infrared light γ Radiation α Radiation Autoclaving Cooking Roasting Dry heat Low temperature/ refrigeration Aspergillus ﬂavus/aﬂatoxin A. parasiticus/aﬂatoxin B1 A. ﬂavus/aﬂatoxin B1 A. ﬂavus/aﬂatoxin Colletotrichum gloeosporioides Penicillium citrinum Cryptococcus neoformans C. neoformans All types of moulds Food-spoiling moulds Food-spoiling moulds Fusarium graminearum Some soil fungi Shantha and Sreenivasamurthy (1977) Samarajeewa et al. (1985) Chourasia and Roy (1991) Shantha and Sreenivasamurthy (1977) Cia et al. (2007) Qing et al. (2002) Dadachova et al. (2004) Martinez et al. (2006) Coomes et al. (1966) Rehana and Basappa (1990) Ogunsanwo et al. (2004) Clear et al. (2002) Janna et al. (2005) A. ﬂavus/aﬂatoxin A. ﬂavus/aﬂatoxin C. lupini C. lupini C. lupini A. carbonarius/ochratoxin Penicillium sp., Trichoderma sp. F. graminearum/ZEN Sclerophoma pityophila C. gloeosporioides Aspergillus spp. Aspergillus spp. Aspergillus spp. Aspergillus spp. A. ﬂavus A. ﬂavus A. ﬂavus A. ﬂavus A. ﬂavus Sreenivasamurthy et al. (1967) Shantha et al. (1986) Thomas et al. (2008) Thomas et al. (2008) Thomas et al. (2008) Medina et al. (2007) Magarey et al. (1997) D’Mello et al. (1998) Olender et al. (2008) Rehman et al. (2008) Satish et al. (2008) Satish et al. (2008) Satish et al. (2008) Satish et al. (2008) Kumar et al. (2008) Kumar et al. (2008) Kumar et al. (2008) Kumar et al. (2008) Kumar et al. (2008) Chemical: H2O2 Na-hypochlorite Azoxystrobin Chlorothalonil Copper oxychloride Carbendazim Mancozeb Maneb Nitroimidazole Organotin Blitox Captan Dithane M-45 Thiram SAAF Bavistin Wettasul-80 Ceresan Diphenylamine growth efﬁciently but the technique is not suitable for long-term storage. Radiation Sun drying of food commodities (grains and pulses) before storage is preferable in most underdeveloped countries but the technique is unsuitable in the case of vegetable crops. High-energy radiation like γ rays (Petushkova et al., 1988), UV rays (Oteiza et al., 2005), infrared (Qing et al., 2002), etc., is also efﬁcient in checking microbial growth and proliferation, as well as mycotoxin production. The irradiation of food commodities during storage is unattainable in developing countries. Chemical methods In order to minimize the losses caused by moulds in the ﬁeld and also during storage, Botanicals in Agricultural Pest Management many synthetic fungicides have been introduced (Table 2.1). The discovery of Bordeaux mixture is signiﬁcant in the history of the chemical control of plant diseases. In the past few decades, various synthetic chemicals have played a signiﬁcant role in the management of such losses. Several chemical additives also function as preservatives, even though the exact mechanisms or targets are often not known (Davidson, 2001. The organic acids, acetic, lactic, propionic, sorbic and benzoic acids, are used as food preservatives (Brul and Coote, 1999). Both sorbic and benzoic acid have a broad spectrum of activity (Nielsen and De Boer, 2000; Davidson, 2001). Benzoic acid and sodium benzoate are used primarily as antifungal agents (Davidson, 2001). Recently, some technology like TiO2 photocatalytic ozonation has been found to be efﬁcient in controlling postharvest spoilage of kiwifruit (Hur et al., 2005). The indiscriminate application of synthetic chemicals as antimicrobials has contributed greatly to the management of losses caused by fungi, but these chemicals have led to a number of ecological and health problems due to their residual toxicity (Kneževi and Serdar, 2008), carcinogenicity, teratogenicity, hormonal imbalance, spermatotoxicity, etc. (Pandey, 2003; Kumar et al., 2007). History also shows that overzealous use of synthetic pesticides has led to numerous problems unforeseen at the time of their introduction. Different types of ecological problems have been reported from time to time by these xenobiotics, such as acute and chronic poisoning of applicators, farm workers, and even consumers, extensive groundwater contamination, resistance development in pests (Wilson et al., 1997), effect on non-target organisms (Wiktelius et al., 1999), ozone layer depletion by methyl bromide (Lee et al., 2001), etc. Biocontrol Agents in Pest Management Considerable attention has also been given to the potential of biological control of 17 postharvest diseases of fruits, vegetables and other edibles as a viable alternative to the use of present day synthetic fungicides (Wilson et al., 1999; Pang et al., 2002). Microbial antagonists have been reported to protect a variety of harvested perishable commodities against a number of postharvest pathogens (Wisniewski et al., 2001). However, decreasing efﬁcacy and lack of consistency when applied as stand-alone treatments under commercial conditions (Droby et al., 2001) are limiting their use. Hence, these drawbacks in alternative methods have increased interest in developing further alternative control methods, particularly those which are environmentally sound and biodegradable. Botanicals as Fungitoxicants Recently, in different parts of the world, attention has been drawn towards the exploitation of higher plant products as novel chemotherapeutants in plant protection. Because of non-phytotoxicity, systemicity, easy biodegradability and the stimulatory nature of host metabolism, plant products possess the potential to be of value in pest management (Mishra and Dubey, 1994). Higher plants contain a wide spectrum of secondary metabolites such as phenols, ﬂavonoids, quinones, tannins, essential oils, alkaloids, saponins and sterols. Such plant-derived chemicals may be exploited for their different biological properties (Tripathi et al., 2004). Terrestrial plants produce a spectrum of natural products, namely terpenoids, phenolics and alkaloids. Many of these are thought to have an ecological function for the plants producing them, serving to defend the plants from herbivores and pathogens (Isman and Akhtar, 2007). Such defensive chemistry is thought to be extremely widespread among the plant kingdom. The body of scientiﬁc literature documenting the bioactivity of plant derivatives to different pests continues to expand; yet only a handful of botanicals are currently used in agriculture in the industrialized world. In the context of agricultural pest 18 A. Kumar et al. management, botanical pesticides are well suited for use in industrialized countries and can play a much greater role in the postharvest protection of food commodities in developing countries (Isman, 2006). Among the different plant products, the application of essential oils is a very attractive method for controlling postharvest losses (Table 2.2). Production of essential oils by plants is believed to be predominantly a defence mechanism against pathogens and pests (Oxenham, 2003). Essential oils and their components are gaining increasing interest because of their relatively safe status, wide acceptance by consumers and their exploitation for potential multi-purpose use (Sawamura, 2000; Ormancey et al., 2001; Feng and Zheng, 2007). The problem of the development of resistant strains of fungi and other organisms may be solved by the use of essential oils of higher plants as fumigants in the management of storage pests because of synergism between different components of the oils (Varma and Dubey, 1999; Dubey et al., 2006). The antifungal activity of essential oils is well documented and characterized with their bioactivity in vapour phase. The pesticidal activities of essential oils are due to the presence of some aroma compounds. Fumigation with such aroma compounds greatly reduces postharvest decay without causing any toxicity (Chu et al., 2001; Liu et al., 2002). Recently, some monoterpenes isolated from essential oils exhibited fungicidal activity and have been shown to inhibit fungal rotting of vegetables without altering taste and quality (Hartmans et al., 1995; Oosterhaven, 1995). The fungitoxic properties of essential oils from higher plants are well documented but little attention has been paid towards the bioactivity of essential oil constituents. The fungitoxic activity of some essential oil components is listed in Table 2.3 and Fig. 2.1. However, more work on the bioactivity of plant products including essential oil and constituents in in vitro and in vivo conditions is required. The literature is also silent on the mode of action of the essential oils and components when used as postharvest fungitoxicants. Conclusions Plants are a virtually untapped reservoir of different valuable chemicals that can be used directly or as templates for the formulation of pesticides. Numerous factors have increased the interest of the pesticide industry and the pesticide market in this source of natural products as pesticides. Pesticides based on plant essential oils or their constituents have demonstrated their efﬁcacy against a range of fungal pests responsible for pre- and postharvest diseases, as well as mycotoxin production. Encouraging results on the use of natural products to control postharvest fungal spoilage indicate that we should be able to develop natural pesticides that could be as effective as synthetic fungicides and presumably safer for man and the environment. Biological compounds, because of their natural origin, are comparatively biodegradable and most of them are almost non-residual in nature (Beye, 1978). During recent years, products of some pesticidal plants have received global attention for the protection of several food commodities because of their antimicrobial properties (Kumar et al., 2007). Such plant products have been formulated for largescale application as botanical pesticides, which are used as alternatives to synthetic pesticides in crop protection. A consolidated and continuous search of natural products may yield safer alternative control measures comparable to azadirachtin and pyrethroids, which are being used in different parts of the world as ideal natural fungicides. The number of options that must be considered in the discovery and development of a natural product as a pesticide is larger than for a synthetic pesticide. However, current advances in plant chemistry and biotechnology, combined with increasing need and environmental pressure, are greatly increasing the interest in plant products as pesticides. Products from higher plants are a safe and economical option in the management of agricultural pests and will be in high demand in the global pesticide market. Botanicals in Agricultural Pest Management Table 2.2. 19 Efﬁcacy of some higher plant products in checking fungal growth and mycotoxin production. Plants Products Hypericum linarioides EO/PEE/ 6 Fusarium spp. ME/ChlE EO Colletotrichum gloeosporioides, Rhizoctonia solani, F. oxysporum EO/ME/ F. oxysporum, C. capsici, HexE Botrytis cinerea EO 17 pathogenic fungi EO Trametes versicolor, Lenzites betulina, Laetiporus sulphureus EO Candida albicans EO Aspergillus niger, A. parasiticus EO F. oxysporum, Cladosporium herbarum, A. ﬂavus EO C. albicans Cakir et al. (2005) AqE/EO Omidbeygi et al. (2007); Aldred et al. (2008); Reddy et al. (2008) Reddy et al. (2008) Reddy et al. (2008) Tatsadjieu et al. (2009) Viuda-Martos et al. (2008) Deba et al. (2008) Omidbeygi et al. (2007); Abyaneh et al. (2008); Dikbas et al. (2008) Rasooli et al. (2006) Rasooli and Abyaneh (2004) Atanda et al. (2007) Matan and Matan (2008) Pinto et al. (2007) Calocedrus macrolepis Silene armeria Origanum acutidens Cinnamomum osmophloeum Thymus numidicus Lantana camara O. glandulosum Tarchonanthus camphoratus Syzygium aromaticum Curcuma longa Allium sativum Lippia rugosa Citrus sp. AqE AqE EO EO Bidens pilosa Satureja hortensis EO/AqE EO/ME T. eriocalyx T. x-porlock Ocimum basilicum Pimpinella anisum Salvia ofﬁcinalis EO EO EO EO EO T. vulgaris Cympopogon citratus EO EO Fungi/mycotoxins A. ﬂavus/aﬂatoxin B1, A. ﬂavus, Penicillium verrucosum/ochratoxin A A. ﬂavus/aﬂatoxin B1 A. ﬂavus/aﬂatoxin B1 A. ﬂavus/aﬂatoxin B1 A. ﬂavus, P. chrysogenum, P. verrucosum Corticium rolfsii, F. solani A. ﬂavus, A. parasiticus/aﬂatoxin A. niger A. parasiticus/aﬂatoxin A. parasiticus/aﬂatoxin A. niger, P. chrysogenum C. albicans, Trichophyton rubrum, A. ﬂavus A. ﬂavus/aﬂatoxin B1 B. cinerea, C. herbarum, A. niger A. parasiticus/aﬂatoxin A. parasiticus/aﬂatoxin R. solani, T. mentagrophytes Rosmarinus ofﬁcinalis EO Trachyspermum copticum EO Cordia curassavica EO/HexE/ ChlE/ME Sesuvium portulacastrum EO A. niger, A. ﬂavus, P. notatum Calamintha ofﬁcinalis EO B. cinerea Olea europaea AE/ME Alternaria alternata, A. ﬂavus, F. oxysporum Citrus sinensis EO A. niger Azadirachta indica AqE P. citrinum/Citrinin Agave asperrima ME/AqE A. ﬂavus, A. parasiticus/ aﬂatoxin B1 Adenocalymma alliaceum AqE A. ﬂavus/aﬂatoxin B1 Lupinus albus AqE A. ﬂavus/aﬂatoxin B1 References Chang et al. (2008) Bajpai et al. (2008) Kordali et al. (2008) Cheng et al. (2006) Giordani et al. (2008) Deena and Thoppil (2000) Bendahou et al. (2008) Matasyoh et al. (2007) Kumar et al. (2008) Tzortzakis and Economakis (2007) Rasooli et al. (2008) Rasooli et al. (2008) Hernandez et al. (2007) Magwa et al. (2006) Bouchra et al. (2003) Korukluoglu et al. (2008) Sharma and Tripathi (2008) Aparecida et al. (2008) Sánchez et al. (2005) Shukla et al. (2008) Mahmoud (1999) Note: EO, essential oil; ME, methanolic extract; AqE, aqueous extract; PEE, petroleum ether extract; AE, acetone extract; ChlE, chloroformic extract; HexE, hexane extract. 20 A. Kumar et al. Table 2.3. Efﬁcacy of some essential oil components in checking fungal growth. Compounds of plant origin Ajoene Allicin Myrcene Limonene r-Cymene a-Pinene Fungi References Aspergillus niger, Candida albicans, Saccharomyces cerevisiae C. albicans Rhizoctonia solani, Fusarium oxysporum Colletotrichum gloeosporioides, Botryosphaeria parva, F. verticillioides Fusarium sp. C. albicans, S. cerevisiae, A. niger Yoshida et al. (1987) Naganawa et al. (1996) Ankri and Mirelman (1999) Chang et al. (2008) Regnier et al. (2008) Dambolena et al. (2008) Kordali et al. (2008) Yousefzadi et al. (2008) Sonboli et al. (2006) Chang et al. (2008) Moleyar and Narasimham (1986) Da Silva et al. (2008) Cheng et al. (2006) Pitarokili et al. (2003) Regnier et al. (2008) Duru et al. (2004) Dambolena et al. (2008) Shaﬁ et al. (2004) Zhang et al. (2006) Zhang et al. (2006) Moleyar and Narasimham (1986) Barra et al. (2007) Dambolena et al. (2008) Moleyar and Narasimham (1986) Serrano et al. (2005) Braga et al. (2008) Kordali et al. (2008) Dambolena et al. (2008) Cheng et al. (2006) Gayoso et al. (2005) Shaﬁ et al. (2004) Regnier et al. (2008) Lee (2007) Lee et al. (2004) Agarwal et al. (2001) Agarwal et al. (2001) Meepagala et al. (2003) Meepagala et al. (2003) Kordali et al. (2008) Romero et al. (2007) Chang et al. (2008) Chang et al. (2008) Caryophyllene Citral R. solani, F. oxysporum A. niger, F. oxysporum, Penicillium digitatum, C. albicans Cinnamaldehyde Camphor Carvone Pulegone Menthone Thujone Linalool Geraniol Citronellol Lenzites betulina, Laetiporus sulphureus Fusarium sp., R. solani C. gloeosporioides, B. parva C. albicans F. verticillioides Phytophthora capsici C. camelliae C. camelliae Rhizopus stolonifer Terpine-4-ol Menthol A. ﬂavus, R. solani, P. commune, F. oxysporum F. verticillioides, R. stolonifer, Penicillium sp., Monilia sp. Thymol C. albicans, Fusarium sp., F. verticillioides Eugenol Zingiberene Curcumene Verbenone Verbenol Carvacrol L. betulina, L. sulphureus, T. mentagrophytes, C. albicans P. capsici C. gloeosporioides, B. parva R. solani, P. infestans, Cladosporium cucumerinum, Pythium ultimum R. solani R. solani Colletotrichum sp. Colletotrichum sp. Fusarium sp., Botrytis cinerea a-Cadinol T-muurolol R. solani, F. oxysporum R. solani, F. oxysporum Fenchone 1,8 Cineole Asarone Botanicals in Agricultural Pest Management 21 CH3 CH2 CH3 CH3 H3C CH2 H3C CH3 CH3 CH2 Myrcene CH2 H3C Caryophyllene H3C Limonene CH3 P-Cymene CH3 OH CH3 CH3 CH3 H CHO O CH2 H3C CH2OH H3C CH3 Linalool Camphor CH3 H H3C CH3 Citral Citronellol CH3 CH3 H O CH3 O H O H3C O CH3 H3C Pulegone Cinnamaldehyde H2C Menthone CH3 CH3 CH3 CH3 Carvone CH3 CH3 O O CH3 OH H3C α-Pinene CH3 CH3 Terpine-4-ol H3C Fenchone CH3 Thujone CH2 CH3 CH3 CH2OH H OH OCH3 OH Eugenol Fig. 2.1. 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Varma, J. and Dubey, N.K. (1999) Prospectives of botanical and microbial products as pesticides of tomorrow. Current Science 76, 172–179. Viuda-Martos, M., Ruiz-Navajas, Y., Lopez, J.F. and Pérez-Alvarez, J. (2008) Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. Food Control 19, 1130–1138. Wiktelius, S., Chiverton, P.A., Meguenm, H., Bennaceur, M., Ghezal, F., Umeh, E.D.N., Egwuatu, R.I., Minja, E., Makusi, R., Tukahirwa, E., Tinzaara, W. and Deedat, Y. (1999) Effects of insecticides on nontarget organisms in African agroecosystems: a case for establishing regional testing programmes. Agriculture, Ecosystems and Environment 75, 121–131. Williams, H.J., Phillips, T.D., Jolly, E.P., Stiles, K.J., Jolly, M.C. and Aggarwal, D. (2004) Human aﬂatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences and interventions. 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Zhang, Z.Z., Li, Y.B., Qi, L. and Wan, X.C. (2006) Antifungal activities of major tea leaf volatile constituents toward Colletorichum camelliae Massea. Journal of Agricultural Food Chemistry 54, 3936–3940. 3 Deleterious Effects of Fungi on Postharvest Crops and Their Management Strategies A.O. Ogaraku Plant Science and Biotechnology Unit, Department of Biological Sciences, Nasarawa State University, Kefﬁ, Nigeria Abstract Fungi inﬂuence our lives in many ways. The parasitic forms cause serious diseases in crop plants and pose hazards to the lives of animals and humans whenever they infect consumable crops. Most consumable crops are susceptible to fungal infection. The most prominent types of fungi attacking commodities are species of Aspergillus, Penicillium and Rhizopus, etc. Types of crop deterioration caused by fungi include discoloration, ﬂavours and odour, rotting and caking, destruction of viability and production of mycotoxins on food before infestation. Conditions that favour the development of fungi on harvested and stored crops include moisture, preharvest infection and lapses in the processing method. Method of control involves drying of produce to a safe moisture level, non-mixing of new produce with old ones, avoidance of pre-storage damage and use of chemicals, fungicides and medicinal plants in treating the produce. Introduction Fungi are one of the most important groups of organisms on the planet. They are microscopic, achlorophyllous and non-vascular plants. They cause deterioration of postharvest crops (Ogundana et al., 1970). Deterioration means that something is made to be of less value or worse in quality (Adebayo et al., 1994). It is a common phenomenon in agricultural crops, either on the farm, at harvest or during storage. Fungi are known to cause various types of deterioration and pose a hazard to humans and animals whenever they infect crops. Fungal deterioration can be deﬁned as any change resulting from the activities of fungi which renders a product unsuitable for its intended 28 use or reduces the economic value of the materials (Opadokun et al., 1979). It is also noteworthy to mention some other factors that have been identiﬁed as causing damage to crops, namely: ● ● ● ● ● insects and mites microorganisms, such as bacteria, actinomycetes, yeasts and virus rodents and birds physical factors, such as temperature and relative humidity of the storage environment harvesting, handling and transportation (Clarke, 1968). Before the 17th century, scientists concentrated on damage caused by insects on stored products. This was because damage by insects  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Effects of Fungi on Postharvest Crops was usually conspicuous, easy to quantify and these insects were visible to the naked eye. But, awareness of the losses caused by fungi, also referred to as ‘moulds’, came with the discovery of a toxic metabolite called aﬂatoxin in 1968 caused by a fungus called Aspergillus ﬂavus, which killed over 100,000 turkeys in Britain when fed with groundnut cakes that were infected by this organism. Studies in Nigeria have revealed the presence of aﬂatoxin in Nigerian groundnuts and livestock feed maize; hence, there is a need to take extremely good care of these products during storage (Akano and Atanda, 1989). Some crops in which fungal deterioration can take place are as follows: maize, sorghum, millet, cowpea, beans, groundnut, cocoa beans, palm kernels and tubers. Deleterious Effects of Fungi on Postharvest Crops Fungi occur everywhere and have a profound effect on their environment. Like other microorganisms, fungi may be good or harmful, depending on the species involved. The deterioration of postharvest crops by fungi can be either by destruction of the produce itself or by presenting a potential hazard to animals or humans. Some of the deleterious effects of fungi on postharvest crops are as follows: 29 in Alabama, while Macrophomina phaseoli causes ‘black mars’ in Gambian groundnuts. In fruits and seeds, the micropyle is the common place for infections to begin, but fungi, bacteria and actinomycetes can develop in any other region of the seed or fruit, causing abnormal colouring, either localized or generalized (Clarke, 1968). However, it is not all discoloration on produce that is caused by fungi; sometimes, it may be due to genetic mutations. Flavour and odour The ﬂavour and odour of produce caused by moulds usually affect the taste of the end products and are not acceptable to consumers. The change in the ﬂavour and odour is usually as a result of the biochemical change which takes place in the stored produce. For example, mouldy groundnuts have a very unpleasant and sour taste when consumed and these are usually spat out from the mouth as soon as they are chewed. Undesirable ﬂavour is easily noticed in mouldy cocoa beans, as it can be detected by tasting a sample of chocolate which has passed through all the normal manufacturing processes. Banana and plantain affected by mould also have a detectable ﬂavour and odour. Mouldy produce can also have an odour, ranging from the musty odour of mouldy grains to the foul smell of rotten grains (Ogundana et al., 1970). Discoloration Fungi come in various colours, i.e. green, brown, white, grey, black, etc. They impart these colours on postharvest crops, thereby changing the original appearance. Discoloured produce is often disliked by consumers and manufacturers in that the colours affect the end products from such produce. Cocoa beans, melon seeds, palm kernels, groundnuts, maize, yam and cassava are examples of produce in which deterioration is accomplished by marked discoloration. For instance, Lasiodiplodia theobromae is responsible for the disease which discolours cocoa, widely known as ‘concealed damage’ Biochemical effects The development of moulds leads to a great modiﬁcation in the chemical composition of the infected produce. One such effect is an increase in the free fatty acid (FAA) content of the produce. This acid is one of the intermediate products of spoilage in materials containing fats and oils and its formation results in rancidity. Many of the mould species infecting our crops are known to produce lipases, which can hydrolyse fats into fatty acids by a process called lipolysis, thereby increasing the free fatty acid content 30 A.O. Ogaraku of the produce and resulting in a decrease in oil content and a low protein content. Kuku (1972) isolated a number of moulds from palm oil and showed that many of these increased the FAA of palm oil in pure culture studies. Coursey et al. (1963) isolated a number of lipolytic fungi from Nigerian palm kernels. These included A. chevalieri, A. fumigatus, Paecilomyces variotii and P. steckii. The development of mould on produce causes other modiﬁcations; generally, an increase in reducing sugars and a loss in protein, which may lead to ﬂours unsuitable for bread making. Moreover, mouldy rice grain breaks easily during polishing. If we preserve damp grain in an anaerobic environment, fermentation results in the release of carbon dioxide, alcohol and other volatile substances. The compounds formed give a bad taste, which remains even after drying in the open air. Rotting and caking Extensive mould activities usually result in rotting and caking. Rotting and caking renders produce unsightly, decreases milling yield and quality. Studies carried out by several workers, including Adeniyi (1970) and Ogundana et al. (1970), revealed that a number of fungal species, for example A. niger, Fusarium moniliforme, P. exalicum, etc., caused rotting in Nigerian yams. Oyeniran (1970) and other workers carried out studies that showed that over 30 mould species could be associated with the deterioration of maize in Nigeria. Weight loss Fungi growing on plant parts or produce use them as food substrate. They produce a variety of enzymes, i.e. amylases, cellulases, pectinases and lipases, which hydrolyse the food substances into soluble forms. The food components easily absorbed and utilized are carbohydrates, proteins, fats and oil. This breakdown invariably leads to absolute weight loss. Scientists have reported up to 10% weight loss in rotting yam tubers during storage (Ogundana et al., 1970). Some of the fungi that can cause weight loss in maize are A. ﬂavus, A. niger, A. candidus, Mucor racemosus and P. pallitans. Destruction of viability Fungi reduce the viability of seeds by infecting and destroying their embryo. This in turn affects the germinability of the seeds during planting. Broadbent (1967) found samples of mouldy maize from government farms in southern Nigeria had only 7–14% germination, while the mould-free samples had 100% germination. Heating If crops with high water content are piled up together in one place, heat is generated and decay sets in. Heating or production of hot spots is one of the characteristics that results in rapid fungal development in moist stored produce, mostly grains and tubers. Heating in bulk storage is evidence of spoilage in progress or spoilage already completed. Growth abnormalities Groundnut and maize contaminated with A. ﬂavus produce deformed plants. The infected young groundnut or maize plant will have a greatly decreased growth. The follicles develop poorly and are elongated in form. During growth, a large number of sick plants die. Others are continually abnormal in appearance, while some evolve into normal plants. Preparation of the Material for Attack by Other Agents It is sometimes difﬁcult in many crops to separate deterioration or spoilage due to Effects of Fungi on Postharvest Crops insects from that caused by fungi, but that the two are interrelated is in no doubt. What is in doubt, however, is the exact sequence of events and the relative damage caused by the two agents. Invasion of stored produce by fungi prepare such commodities for attack by other agents of deterioration, especially bacteria, insects and mites. In fact, some insects are known to feed on fungi and in this way they help to spread the spores. These storage insects can live, develop and reproduce entirely on certain fungi and thus undoubtedly play an important part as carriers in the spread of the fungi. An example of such an insect is Adhasverus advena. Production of Toxic Metabolites (Mycotoxins) since 1960, when it was reported to have caused the death of about 100,000 turkeys in Britain when they were fed with groundnut cakes which was infected with A. ﬂavus. The toxic substance was therefore called ‘aﬂatoxin’. Different mycotoxins affect different sites of the body. Aﬂatoxins produced by A. ﬂavus are the commonest of all the toxins and affect the liver, causing aﬂatoxicosis or liver poisoning. High levels of aﬂatoxin have also been reported to cause infertility (abnormality in the spermatozoa) in samples of semen from men fed on diets contaminated with A. ﬂavus (Ibeh et al., 1994). The production of aﬂatoxins on maize grains and other consumable foods in Nigeria has been reported by many researchers, including Broadbent (1967), Oyeniran (1970), Opadokun et al. (1979) and Akano and Atanda (1989). Other common mycotoxins are: ● Toxic metabolite production is the most serious effect of microbiological deterioration of stored products because of its poisoning nature. There are two kinds of poisoning by fungi, mycetism and mycotoxicosis. In mycetism, the toxic substances are constituents of the fungi, large enough to be eaten alone. In mycotoxicosis, the fungus is a contaminant of and has produced toxic product in some food. The effects of mycetism include diarrhoea and jaundice, while mycotoxicoses were deﬁned by Clarke (1968) as diseases of animals and humans caused by ingesting poisonous metabolite fungi that have grown in the food previously before ingestion. Some notable examples of mycotoxicoses are: 1. Ergotism – diseases of cattle in central Europe caused by the fungi, Claviceps purpurea. 2. Yellow rice disease of humans in Japan caused by the fungi, P. citrinum. 3. Alimentary toxic aleukia (ATA) of humans and cattle caused by F. sporotrichioides. 4. Importantly, aﬂatoxicosis of poultry and livestock caused by A. ﬂavus. This last mentioned toxin disease, aﬂatoxicosis, has been receiving worldwide attention 31 ● Fumonism – this causes oesophageal cancer in horses and humans. It is produced by F. graminearum on maize. Ochratoxin – produced by A. ochareus, which causes serious nephropathy in pigs and humans. It is commonly found in milk and cereals (processed or raw). Some examples of mould species and the toxins they produce are shown in Table 3.1. Conditions that Favour Development of Fungi on Harvested and Stored Crops Fungi, like other living organisms, require certain conditions for growth and development. These conditions are as follows: Moisture It is not the moisture content as such that is the controlling factor in biological deterioration; it is the relative humidity of the air in and around the crop. Although relative humidity is the controlling factor, attention is usually focused on the moisture content because relative humidity of produce is difﬁcult to measure, while moisture content 32 A.O. Ogaraku Oxygen Table 3.1. Examples of mould species and the toxins they produce. Mould species Toxin produced Aspergillus ﬂavus A. ochareus A. chevalieri A. nidulans A. ruber A. niger Penicillium islandicum P. notatum P. rubrum P. citrinum P. patulinium Fusarium graminearum Aﬂatoxin Ochratoxin Xanthocillin Sterigmatocystin Rubratoxin Oxalic acid Islanditoxin Xanthocillin Rubratosin Citrinin Patulin Zearalenone is not. Moisture in stored produce is divisible into two main types: chemically bound water, which is the part of the intrinsic composition, and physically bound water, some of which is held loosely on the commodity. Moisture in terms of water is necessary for mould spores to germinate, and it also helps in the process of dissolution of food materials. The moisture level of a stored product therefore determines the development rate of the storage fungi. Mould species vary in their water requirement; for instance, there are those that thrive at low moisture levels and are said to be xerophytic. Examples are A. ﬂavus, A. chevalieri and A. repens. Others require high levels of moisture before they can survive and are said to be hydrophilic, e.g. Penicillium species. Most fungi are aerobic; they require oxygen to survive, like other living organisms. Any device which cuts off oxygen from the storage environment will reduce, if not totally eliminate, fungi. This is why storage at an inert temperature has been effective. Nutrients All biological systems, from microorganisms to humans, share a set of nutritional requirements with regards to the chemicals necessary for their growth and normal functioning. The great diversity of nutritional types required are energy, carbon, nitrogen, sulphur and phosphorus, metallic elements and vitamins. All these nutritional requirements are present in food substances, such as carbohydrates, proteins, fats and oil, which fungi need in soluble forms for metabolic processes. Fungi produce a variety of enzymes which break down complex food substances. Some of these enzymes are as follows: ● ● ● ● ● Cellulases – break down cellulose in plant materials. Amylases – hydrolyse carbohydrates. Lipases – hydrolyse fats to fatty acids and glycerol. Proteases – hydrolyse proteins. Pectinases – hydrolyse the pectic materials of plant tissues. Temperature Heating All living things have a minimum and maximum temperature for growth. Fungi are a co-exception. Most fungi will grow at temperatures between 5°C and 35°C. These are the mesophilic species. There are those that thrive at 35°C and above and are said to be thermophilic. Some thrive at very cold temperatures and are said to be psychrophilic. This means that fungi thrive well in a very wide temperature range, which gives room for existence in postharvest crops. If crops with a high water content are allowed to overlap or are piled together in one place, yam for example, heat is generated under moist conditions and decay will set in. Insufﬁcient drying Some crops grow mouldy if insufﬁciently dried. Fungi can creep in to destroy the crops. Effects of Fungi on Postharvest Crops 33 Preharvest infection Economic loss Produce destined for storage is sometimes infected by moulds before harvest. Most fungi species also invade, especially following natural or artiﬁcial wounds. Some examples are attack of cocoa beans by Lasiodiplodia theobromae and other moulds, attack of groundnut by Macrophomina phaseoli and attack of maize by F. moniliforme and P. citrinum. 1. There is a monetary loss because of inaccessibility to foreign trade due to the poor quality of the produce. There is also a monetary loss because of the poor health of animals fed with inferior feeds. 2. Some fungi, for example Fusarium species, can grow on stored animal feeds, generating products that are highly toxic to swine and other animals. 3. Infections leading to disease of crops are extremely important because of the famine, malnutrition and dietary deﬁciency they may cause. 4. Some plant pathogens cause food intoxication when eaten by humans or animals; for example, the fungus, C. purpurea, which grows on cereal grains and some grasses, replaces the feed kernels with compact masses of hardened fungus called sclerotia. These contain alkaloids that act on the nervous system of humans and other animals, causing gangrene, convulsions and death. Attack during preparation During the process of preparation, mould attacks some produce as a result of lapses in cultural practices; for example, during cocoa fermentation mould could infect and penetrate the beans if the fermenting mass of beans is not stirred or mixed thoroughly at intervals. In palm produce, mould can attack the fruits and sometimes the kernels when they are heaped on the ground just before de-husking. In groundnut, the crop has to be lifted at certain times to avoid mould contamination. Control of Fungal Deterioration in Postharvest Crops If, for instance, through economy a store is poorly constructed and the roof is holding water, it is possible to cause leakage and water will drip on to the commodity and thereby cause deterioration. There are other factors which contribute to the development of fungi in crops apart from those mentioned above and they are: If left uncontrolled, these fungi will cause deterioration of food products and many other articles of commerce and industry. For this purpose, a distinction can be made between postharvest produce that is stored dry, such as grains, cocoa, groundnuts, etc., and those which are stored with a high water content, such as yams and other tubers. However, some of the measures or suggestions listed below will certainly apply to both types: 1. The degree to which the grain has already been invaded by storage fungi before it arrives at a given site. 2. The amount of foreign material present in the grain. 3. The activity of mites and insects. Bored holes serve as an entry for mould spores. Some insects, such as A. advena, and mites feed on mould spores and therefore help to spread the fungi, as well as increase their activities in storage. 1. Proper drying of produce to a safe moisture level, either by retaining maize, millet or guinea corn on the cob and storing in a condition where gradual drying by heat or aeration takes place, or otherwise by providing artiﬁcial drying. 2. Prevention of damage or wounds on produce so as to forestall a source of entry for moulds. 3. Any produce to be stored must be wholesome and healthy. Bruised yam tubers, Types of stores 34 A.O. Ogaraku cassava, oranges and fruits should never be stored. 4. Avoid drying the produce on a bare ﬂoor because of infestation by soil fungi. 5. Hot produce should not be stored. After drying, allow produce to cool before storage. 6. New produce should not be mixed with an old consignment, to avoid crossinfestation. 7. Bagged produce should not be placed on the ground but on raised plank platforms. 8. Overfermentation should be avoided in produce like cocoa, cassava, etc. 9. The store or warehouse should be leakproof to prevent moisture reabsorption by the already dried produce. 10. Prevent pockets of heavy insect activity by proper application of insect control measure to avoid localized moisture increases and mould growth in the bulk of the grain. 11. In the case of fruits, harvesting should be done promptly as very old fruits are highly susceptible to fungi infection. 12. If possible, dried produce should be stored in airtight conditions to keep away from ﬂuctuating atmospheric relative humidity, which could lead to an increase in moisture content; for example, store in polythene bags or polythene-lined sacks. Other methods of controlling deterioration of dry produce are: 13. Use of fungicides – in the case of grains not desired for immediate consumption or use, some fungicides such as captan, benomyl, thiobendazole, borax, etc., have been used to control fungal attack, but their use has been limited because of their toxicity. 14. Use of plant materials – parts or roots with medicinal properties can also be used to suppress mould growth in stored crops. Williams and Akano (1985) reported on the efﬁcacy of dogonyaro (neem) as a ﬁltrate in suppressing rotting fungi growth in stored yam tubers. 15. Addition of chemical preservative agents – the addition of antiseptics to foodstuffs allows for better preservation under certain conditions. The use of these products is subject to regulations in most countries. Examples of such chemical preservatives are propionic acid, ascorbic acid, glycerol, sulphur dioxide and benzoic acid. Their use in many instances has been limited to livestock feeds. 16. Other technical methods of control – other methods by which fungal development in stored products can be controlled are refrigeration, irradiation (for yam) and storage in airtight containers and inert atmosphere for grains. Ogundana et al. (1970) found benomyl and thiabendozole effective in reducing the activities of fungi in causing yam rot during storage, but these chemicals are rather toxic. Research is currently in progress at the Nigerian Stored Products Research Institute on the use of safer fungistatic chemicals to preserve yams against microbiological rot during storage. Adesuyi (1973) stored yams successfully for up to 6 months by using a curing method, cutting off sprouts from healthy undamaged tubers and using low temperature and irradiation techniques. 17. Precautions in mycotoxicoses – it is very important to have a control measure in harvesting produce in order to eliminate the fungi causing mycotoxicoses diseases because of their devastating effect on humans and animals that consume such an infected crop. Standard safe limits should be determined and enforced levels of aﬂatoxin and other toxins in food and feed. Different countries have a wide variety of tolerance level of mycotoxin between 5 and 50 µg/kg (Hansen, 1993). In the USA, the Food and Drug Association has established an aﬂatoxin limit of 20 µg/kg for food and feed ingredients. A regular monitoring programme should be arranged for commodities that are susceptible to aﬂaxtoxin contamination. Processing, packaging, transportation and storage practices should be well managed to eliminate or reduce infestation by moulds, especially the toxigenic strains. Decontamination procedures are to be designed to remove or inactivate the toxins in feed and food. Mycotoxins can be removed from food by detoxiﬁcation using chemical agents. Effects of Fungi on Postharvest Crops Conclusions The role of fungi in the deterioration of postharvest crops is enumerated. The contribution of some workers in providing an insight into the deleterious effects of fungi on harvested and stored crops, economic loss, control of fungal deterioration in postharvest crops and precautions in mycotoxicoses diseases is also highlighted. Not forgotten 35 is the most important aspect, the precautions that need to be taken to control or eliminate the fungi causing mycotoxicoses in humans and animals. It is pertinent to say that knowledge is far from complete and experts should still endeavour to ﬁnd total solutions to the various aspects of these problems as the struggle of humans against the menace of fungi continues. References Adebayo, L.O., Idowu A. and Adesanya, O.O. (1994) Mycoﬂora and mycotoxins production in Nigeria corn based snacks. Mycopathologia 126, 183–192. Adeniyi, M.O. (1970) Fungi associated with storage decay of yam in Nigeria. Phytopathology 60, 590–592. Adesuyi, D.A.A. (1973) Curing techniques for reducing incidence of rot in yams. Nigerian Stored Products Research Institute Technical Report No. 12, 57–63. Akano, D.A. and Atanda, O.O. (1989) The present level of aﬂatoxin in Nigeria groundnut cake. Letters in Applied Microbiology 10, 187–189. Broadbent, J.A. (1967) The micoﬂora germination and seeding vigour of some maize seeds. Nigerian Stored Products Research Institute Technical Report No. 15, 113–114. Clarke, J.H. (1968) Fungi in stored produce. Tropical Stored Product Institute Technical Report 15, 2–14. Coursey, D.G., Summons, E.A. and Sheridan, A. (1963) Studies on the quality of Nigerian palm kernels. African Science Association 8,18–28. Hansen, T.J. (1993) Quantitative testing for mycotoxins in cereal foods. World 38, 346–348. Ibeh, I.N., Urath and Ogonar, J.I. (1994) Dietary exposure to aﬂatoxin in human male infertility in Benin City, Nigeria. International Journal of Fertility and Menopausal Studies 39, 208–214. Kuku, F.O. (1972) Some mould induced changes in palm kernels. Nigerian Stored Product Research Institute, Technical Report No. 9, 69–72. Ogundana, S.K., Haviq, S.H. and Ekundayo, J.A. (1970) Fungi associated with soft rot of yams (Dioscorea spp) in storage. Nigerian Stored Product Research Institute Technical Report No. 10, 41–45. Opadokun, J.S., Ikeorah, J.N. and Afolabi, E. (1979) The aﬂatoxin contents of locally consumed food stuffs. Nigerian Stored Product Research Institute Technical Report No. 12, 105–108. Oyeniran, J.O. (1970) Microbiological studies on maize used as poultry and livestock feeds at the research Farms in Kandan, Western State. Nigerian Stored Product Research Institute Technical Report No. 6, 47–49. Williams, J.O. and Akano, D.A. (1985) An assessment of wood ash for yam tuber (Dioscorea rotundata) in storage. Nigerian Stored Product Research Institute Report No. 2, 31–34. 4 Exploitation of Botanicals in the Management of Phytopathogenic and Storage Fungi Pramila Tripathi1 and A.K. Shukla2 1Department of Botany, D.A.V.-P.G. College, Kanpur, India; 2Department of Botany, Rajiv Gandhi University, Rono Hills, Itanagar, India Abstract Plants are known to contain a number of secondary substances like phenols, ﬂavonoids, quinines, essential oils, alkaloids, saponins, steroids, etc. Some of these plant-based metabolites have antimicrobial properties and are toxic to phytopathogens. They are also repellant to insects and have fumigant toxicity against pests. Currently, synthetic pesticides are the primary means of controlling pathogens. The adverse effects of synthetic pesticides on human health and from the food safety point of view has enunciated interest in ﬁnding an alternative means of controlling phytopathogens and pests. To reduce dependency on synthetic pesticides, the use of plant-based antimicrobial substances (essential oils, volatile aromatic compounds, glucosinolates, jasmonates and acetaldehydes) may help in the management of phytopathogens and pests as an alternative method for sustainable agriculture. Use of botanicals is still on a small scale compared to synthetic chemicals; therefore, it is timely to exploit and formulate low-cost, effective, free of human hazard and eco-friendly plant-based products for the management of pests and pathogens. Introduction To control fungal diseases, synthetic fungicides are usually applied as effective, dependable and economical control measures. However, the indiscriminate use of chemical fungicides has resulted in several problems, such as toxic residues in food, water and soil and disruption of the ecosystem, leading to the fear that their regular use may harm the environment further. Hardly 0.1% of the agrochemicals used in crop protection reach the target pest, leaving the remaining 99.9% to enter the environment to cause a hazard to non-target organisms, including humans (Pimentel and Levitan, 36 1986). According to WHO estimates, approximately 0.75 million people are becoming ill every year with pesticide poisoning. Further, the resistance of pathogens to fungicides has rendered certain fungicides ineffective, giving rise to a new physiological race of pathogens. Basic research for over more than 40 years in biology and biochemistry has made it possible to envisage not only how new pesticides may be synthesized but also has generated a completely new approach to the protection of plants using secondary plant products which may be toxic to a speciﬁc pest yet harmless to humans. Pesticidal plants have been in nature and its compounds for millions of years without having  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Exploitation of Botanicals any ill or adverse effects on the ecosystem and, because of their renewability, they have a distinct advantage in the management of disease-causing pests. Plants have a natural potential to withstand the aggressiveness of pathogenic species. Plants synthesize a dazzling array of structural variety, which inhibits an almost equally dazzling array of biological activities. A wide spectrum of secondary substances is contained in higher plants, namely phenols, ﬂavonoids, quinines, tannins, essential oils, alkaloids, saponins and steroids. The total number of plant chemicals may exceed 4000 and of these, 1000 are secondary metabolites. These secondary metabolites have a major defensive role for plants (Swain, 1977). The search for botanicals from plant species is one of the important areas where Indian scientists can take a lead and capture the global market. India enjoys the beneﬁts of a varied climate, from an alpine climate in Himalaya to a tropical one in the south and an arid one in Rajasthan to a highly humid climate in Assam and Bengal. This is consequently reﬂected in the rich and diversiﬁed ﬂora, which is often quite distinct, thanks to the natural barriers that India has all along its frontiers. It is estimated that India has about 17,000 species of angiosperms. There is a need for extensive screening programmes at different regional centres of the country so that knowledge on the various types of biological properties of angiospermic ﬂora may be gathered. This type of scientiﬁc testing would deﬁnitely be helpful in the conservation of plant resources and in proving our sovereign right over our plant biodiversity. Under these conditions, in any meaningful search for better and cheaper substitutes, plant resources for India are a natural choice. Hopefully, this will lead to new information on plant application and a new perspective on the potential use of these natural products. This chapter explores the potential to use a variety of botanicals in the form of plant extracts and essential oils to control various fungal phytopathogens and fungi related to the storage of grains and the postharvest pathogens of perishables. 37 Essential Oils Essential oils from different plant species are known to exhibit various kinds of biological activities. The volatility, ephemeral nature and biodegradability of such volatile components of angiosperms will be especially advantageous if they are developed as pesticides (French, 1985). Essential oils are a complex natural mixture of volatile secondary metabolites isolated from plants by hydro or steam distillation and by expression. The main constituents of essential oils are mono- and sesquiterpenes, along with carbohydrates, alcohols, ethers, aldehydes and ketones, polyphenolic compounds, oxides, nitrogen and sulphur compounds and organic acids, etc. The chemical composition of essential oils is extremely complex and varies with the geographical as well as the environmental conditions where the plants are grown (Bhaskara et al., 1998; Vanneste et al., 2002). The essential oils are extracted from various parts of plants such as ﬂowers, fruits, leaves and wood. They are normally formed in special cells or groups of cells or as glandular hairs. Oils occur as a globule or globules in the cell and may also be excreted from cells lining the schizogenous ducts or canals. They may be present in glandular regions such as leaves, bark or fruit and, when occurring in various organs in one plant, may possess different individual chemical compounds (Bonner, 1991; Hili et al., 1997). The general antifungal activity of essential oils is well documented (Tripathi et al., 2007, 2008). These essential oils are thought to play a role in plant defence mechanisms against phytopathogenic microorganisms (Mihaliak et al., 1991). The emerging picture is that certain speciﬁc oils and their chemical constituents have traditionally been used to protect stored grains and to repel ﬂying insects in the home and have demonstrable contact and fumigant toxicity to a number of economically important insects and mite pests, as well as to pathogenic fungi. The essential oils or their major constituents could be effective fumigants and also could be integrated with other pest management programmes. Natural pesticides based 38 P. Tripathi and A.K. Shukla on plant essential oils could represent alternative crop protectants. The essential oils produced by different plant species are, in many cases, biologically active and have antimicrobial, allelopathic, antioxidant and bioregulatory properties (Caccioni and Guizzardi, 1994; Vaughan and Spencer, 1994). Sometimes, the chemicals in the oil, as well as the oil itself, are registered as pesticide active ingredients. It is also fairly common for two or more oils to be used in the same commercial product. Since the essential oils as such are a mixture of different major and minor components which act synergistically in the biological efﬁcacy of the oil, there would be less chance of the development of physiological races of the target pathogens if the oils as such were formulated as botanical pesticides and fumigants. Essential oils as botanical pesticides may be produced easily, even by small-scale industries, as there is no sophisticated procedure for their distillation and most aromatic plants are available locally. They thus constitute a friendly, natural alternative in pest control. Essential Oils Against Phytopathogenic Fungi The antifungal activity of essential oils has been studied by a number of workers (Apablaza et al., 2004; Harish et al., 2004; Muller-Ribeau et al., 1995). Singh et al. (1980) found that essential oils from Cymbopogon spp. and Trachyspermum ammi L. exhibited strong antifungal activity against Bipolaris oryzae. Carvone, a monoterpene isolated from the essential oil of Carum carvi, was found to inhibit the sprouting of potatoes during storage. Carvone was also found to have fungicidal activity that helped to protect potato tubers from fungal rotting without exhibiting mammalian toxicity (Hartmans et al., 1995). It has been introduced in the Netherlands under the trade name TALENT. Besides, the essential oils of Salvia ofﬁcinalis have also shown practical potency in enhancing the storage life of some vegetables by protecting them from fungal rotting (Bang, 1997). Powdery mildew of Cucurbita maxima is caused by Sphaerotheca fuliginea. Reynoutria extracts and olive oil were found to be effective in controlling the disease (Cheah and Cox, 1995). Since olive oil is used in cooking, food additives and medicines, it does not cause any human health or environmental problems. Recent studies in Ghana conﬁrm that Ocimum gratissimum and Syzigium aromaticum are very effective in preventing fungal growth (FAO, 1999). Essential Oils Against Fungal Pathogens of Seeds The fungicidal effect of essential oils against pathogens of cereal grains has been tested successfully. It is especially signiﬁcant in the case of stored rice, where currently fungicides are not used to control fungal pests. Peppermint (Mentha piperata), thyme (Thymus copitatus) and caraway (C. carvi) oils have demonstrated effective control against fungal pathogens like Fusarium sp., Macrophomina phaseolina and Colletotrichum dematium (Abdelmonem et al., 2001). Essential oils from oregano (Origanum vulgare) and thyme were applied as fumigants against the mycelia and spores of Aspergillus ﬂavus, A. niger and A. ochraceus infesting wheat grains. Only oregano essential oil exhibited fungicidal activity (Paster et al., 1995). The antifungal activity of the essential and ﬁxed oils of thyme, clove, peppermint, soybean and groundnut were tested against A. ﬂavus, A. niger, F. oxysporum, F. equiseti and Penicillium chrysogenum in vitro on the cowpea (Vigna unguiculata) (Kritzinger et al., 2002). Thyme and clove oils inhibited growth of all the fungi signiﬁcantly at concentrations of 500 and 1000 ppm. Peppermint oil inhibited growth of the above-mentioned fungi successfully at 2000 ppm (Kritzinger et al., 2002). In blackgram (V. mungo), essential oil extracted from wood chips of cedar (Cedrus deodara) and that from seeds of T. ammi exhibited antifungal activity, inhibiting the mycelial growth of A. niger and Curvularia ovoidea, two storage fungi found on seeds (Singh and Tripathi, 1999). A. ﬂavus Exploitation of Botanicals was also found infesting seeds of guar (Cyamopsis tetragonoloba), a native plant of India which has main commercial value due to its seed gum (galactomannan gum). In this case, A. ﬂavus was controlled by cumin (Cuminum cyminum L.) oil extracted from its seeds (Dwivedi et al., 1991). Chemical studies indicated that the greater part of this antimicrobial activity might be attributed to the cuminaldehyde that is present in the dried fruit of this plant (De et al., 2003). The essential oils of Cassulia allaris and M. arvens have been reported as botanical fumigants for management of the biodeterioration of wheat from A. ﬂavus (Varma and Dubey, 2001). Essential Oils Against Aﬂatoxicogenic and Mycotoxicogenic Fungi The aﬂatoxins are well known for their carcinogenic, mutagenic and teratogenic effects on humans and domestic animals (Wyllie and Morehouse, 1978). A natural fungicide against aﬂatoxigenic fungi to protect stored rice using the essential oil of lemongrass (C. citrates) was developed by Paranagama et al. (2003). Lemongrass oil was tested against A. ﬂavus and the test oil was fungistatic and fungicidal against the test pathogen at 0.6 and 1.0 mg/ml, respectively. Aﬂatoxin production was inhibited completely at 0.1 mg/ml. Citral has been found as a fungicidal compound in lemongrass oil. During the fumigant toxicity assay of lemongrass oil, the sporulation and mycelial growth of the test pathogen were inhibited at a concentration of 2.80 and 3.46 mg/ml, respectively. Lemongrass oil could be used to manage aﬂatoxin production and to inhibit the fungal growth of A. ﬂavus in stored rice. Putative mycotoxicogenic fungi were partially or completely sensitive to different essential oils extracted from different medicinal plants (Soliman and Badeaa, 2002). Seed treated with cinnamon, palmarosa and lemongrass oils at 500 mg/kg showed antimycotoxigenic ability against fumonisin B1 accumulation produced by F. vesticillioides 39 and F. proliferatum (Marin et al., 2003). Velutti et al. (2004) reported antimycotoxicogenic activity of the essential oils against F. graminearum infested seeds. The essential oils of oregano, cinnamon, lemongrass, clove and palmarosa effect the growth rate of F. graminearum and mycotoxin Zearalenone (ZEA) and Deoxynivalenol (DON) production at two concentrations (500 and 1000 mg/kg). Plant Extracts Against Phytopathogenic Fungi The preservative nature of some plant extracts has been known for centuries and there has been renewed interest in the antimicrobial properties of extracts from aromatic plants. The application of the extracts of higher plants to control plant diseases was ﬁrst attempted by Democritus as early as 470 BC. Plant extracts have assumed special signiﬁcance nowadays as an eco-friendly method for plant disease management. Plants contain alkaloids, tannins, quinines, coumarins, phenolic compounds, phytoalexins and ipomeamarone in the extract, which are known for their antifungal property (Datar, 1999). Use of plant extracts for seed treatment is one of the alternative methods of preventing pathogen problems of agricultural crops. Plant materials as such can be used as soil amendments that can serve as both a nutrient as well as an antifungal agent. Plant extracts have also been reported to stimulate the growth of targeted plant species. This is probably due to some hormones and allied substances like IAA, IBA, etc. However, the active principles of some plants have been isolated phytochemically and have shown a strong inhibitory action against a number of fungi. Antifungal activity of plant extracts against a wide range of fungi has been reported by a number of workers (Grange and Ahmed, 1988; Davidson and Parish, 1989). Bhargava et al. (1981) screened extracts of some plant species and found O. canum to be most effective against A. ﬂavus and A. versiolor. Pandey et al. 40 P. Tripathi and A.K. Shukla (1982) evaluated the seed extract of 30 plants and found soybean, Leonotis nepetaefolis, Parpalum and Peltophorum to exhibit an inhibitory effect against the fungi, Alternaria alternata and A. niger. Ark and Thompson (1959) found the leaf extract of Allium sativum to be effective against various plant pathogens. Acacia nilotica (leaf and bark) and A. farnasiana (bark) of Mimosaceae showed high activity, while A. catechu of the same family did not show activity either from leaf or from bark (Tripathi, 2005). Four compounds, i.e. iritin A, iritin B, ﬂavononedehydroulogonin and sesquiterpene pygmol, were isolated with dichloro-methane extract of the aerial parts of Chenopodium procerum. These compounds have been found to inhibit the growth of the plant pathogenic fungi, Cladosporium cacumerinum (Bergeron et al., 1995). Kim et al. (2004) evaluated Achyranthus japonica and Rumex crispus for activity against various plant pathogenic fungi and control of powdery mildew. Methanol extract of the fresh material of 183 plants was screened in vivo for antifungal activity against Magnaporthe grisea, Corticium sasaki, Botrytis cinerea, Phytophthora infestans, Puccinia recondita and Erisiphe graminis. Among them, 33 plant extracts showed disease control efﬁcacy. The methanol extract of Achranthes japonica (whole plant) and R. crispus (roots) at a concentration greater than 11 g fresh weight of plant tissue per litre aqueous Tween 20 solution controlled the development of barley powder mildew caused by E. graminis effectively in an in vivo assay using plant seedlings. Some fungi like F. solani and Verticillium alboatrum have been shown to be susceptible to tannins extracted from the bark of various trees, including chestnut and wattle (Lewis and Papavizas, 1967). The effects of aqueous and methanol, petroleum ether, chloroform and ethyl acetate extracts of Cyprus rotundus were tested on spore germination of F. solani. Ethyl acetate extract exhibited an inhibitory effect on spore germination at 1000 µg/ml (Singh and Tripathi, 1999). In the ﬁeld, reduction of disease incidence has been recorded as a result of plant seed treatment with extract, and an increase in yield was also noted. Plant Extracts in the Management of Fungal Seed Diseases Cereal seeds carry a wide range of fungi that are known to play a signiﬁcant role in spoilage and probably rank second only to insects as a cause of deterioration and loss in all kinds of ﬁeld and storage crops throughout the world (Christensen and Kaufman, 1974). The information on fungal association with important cereal grains is relevant in assessing the potential risk of mycotoxin contamination. In recent years, the use of plant extracts for controlling fungal seed disease has also been of renewed interest. Carvone (monoterpene compound) completely inhibited F. oxysporum and A. pisi. African yam bean, Sphenostylis stenocarpa, is an important grain legume in most tropical African countries (Nwachukwu and Umechuruba, 2001). Major pathogenic fungi associated with this crop are A. niger, A. ﬂavus, Lasiodiplodia theobromae and F. moniliforme. Associated fungi could be controlled by using crude and aqueous extract of basil (O. basilicum), bitter leaf (Vernonia amydalina), neem and pawpaw (Carica papaya). Parimelazhagan and Francis (1999) reported reduction in the radial growth of Curvularia lunata associated with rice seeds when treated with leaf extract of Clerodendrum viscosum, which also increased seed germination, root and shoot length of the rice. The same results were observed by using plant extracts to control B. oryzae on rice seeds, which have a high natural infection of the fungus (Alice and Rao, 1986). In Bangladesh, use of the extract of Polygonum hydropiper, A. cepa, A. sativum and A. jidia demonstrated to be effective against B. oryzae at higher concentrations. Among them, neem and garlic were the most effective at 1:1 dilution and inhibited the occurrence of the pathogen by 91 and 83%, respectively (Ahmed et al., 2002). Alternaria padwickii, another important seedborne pathogen of rice, was also inhibited by aqueous extract of Strychnos nux-vomica, garlic bulbs, ginger rhizome, basil leaves and fruits of A. indica (Shetty et al., 1989). The ability of natural plant Exploitation of Botanicals extracts to prevent the growth of fungi naturally infesting grains was also studied. Before sowing, wheat seeds were soaked in an aqueous plant extract of O. gratissimum and disease transmission was evaluated. The rate of infection decreased with the extract at concentrations higher than 10% (Rodrigues et al., 2001). Leaf extracts of Delonix regia, Pongamia glabra and A. nilotica signiﬁcantly inhibit spore germination, mycelial growth and spore production of A. helianthi, M. phaseolina and F. solani from sunﬂower seeds (Tribuhavanaamala and Narsimhan, 1998). Melon seeds are very important as as condiment and constitute a very valuable source of oil and protein for many people of West Africa (Oyolu, 1977). After 6 months of incubation, all the melon seeds treated with leaf extract showed no infection except M. phaseolina. Ahmad and Prasad (1995) evaluated that post-infection treatment of sponge-gourd fruits with the extracts of Azadirachta indica, Lantana camara, Murraya exotica, O. sanctum, Datura ﬁstulosa and Catharanthus roseus almost fully inhibited the spread of disease caused by Helminthosporium spiciferum and F. scirpi. Application of Botanicals in Seed Storage Quality seed should have higher vigour and viability and these two characteristics cannot be maintained in storage because they deteriorate rapidly under storage conditions and suffer quantitative and qualitative losses due to pests and diseases. Therefore, treating seeds with synthetic chemicals is vital for successful storage. However, these chemicals are hazardous to humans. Therefore, use of natural plant products for long-term seed storage has multi-purpose beneﬁts as eco-friendly protection against the ageing process, prevention of insects and fungi and for their cost effectiveness (Vanangamudi et al., 2007). During storage, the enzymatic activity (amylase, catalase, peroxidase, superoxide dismutase and dehydrogenase) responsible for maintenance of seed quality 41 and the antioxidant (ascorbic acid) contents are maintained at optimum level in botanically treated seeds (Umarani, 1999). Common botanicals, arrapu (Abizia amaru), neem (A. indica), notchi (Vitex negundo), Prosopis sp., pungam (Pongamia glabra), moringa and tamarind, contain an auxin-like substance which regulates seedling growth and initial establishment. In botanicals, a gibberellin-like substance is also present in addition to saponin and other nutrients, which interact with amino acids, tryptophane to form the indole acetic acid (IAA), which leads to release of plant hormones that are responsible in cell elongation and vegetative growth. In botanical seed pelleting, the leaf powder acts as a water pad by absorbing/regulating soil moisture availability, which enhances a better seed–soil relationship (Narasimha, 1994). Seeds are stored by pelleting them with botanical products. The aim of botanical pelleting in seed storage is to extend storage potential, besides maintaining its ability to produce normal seedlings. Jegathambal (1996) found that sorghum seeds hardened and pelleted with arappu leaf powder could be stored for up to 2 weeks with higher germinability. Papaya seeds pelleted with botanicals or presoaked with botanicals gave improved germination, vigour index and ﬁeld emergence when compared to the control or water socking (Ananthakalaiselvi, 1995). Dry dressing of seeds with botanicals prolongs the storability of the seeds in many crops, especially in pulses, and acts as a dual-purpose technologically for seed storage by preventing biotic organisms attacking the seeds during storage. Sabir (1989) reported that soybean seeds treated with sambangi (Polianthes tuberose) seed powder at a ratio of 1:100 maintained a higher germination rate (70%), even up to 8 months after storage. Pea seeds dried and mixed with notchi (V. negundo) powder or sambangi seed powder at a ratio of 1:100 maintained a higher germination rate after up to 8 months in storage (Paramasivam, 1990). Umarani (1999) reported that dressing dried Casuarina seeds with neem leaf powder extended the storability of the seeds for up to 9 months. 42 P. Tripathi and A.K. Shukla Biocide Formulation of Essential Oils The formulation of plant metabolites must be introduced to overcome their degradation and to be used practically during handling and application as biocides. Such formulation could be used easily and diluted with water to form the appropriate concentrations in different applications. Study should be continued to evaluate the pesticidal activity of the produced formulated biocides against some plant pathogenic microorganisms. Narsimhan et al. (1988) demonstrated that neem oil (A. indica) and pungam oil (P. pinnata) emulsiﬁable concentrate formulation prevented sheath rot (Sarocladium oryzae) of rice. Gascon et al. (1999) showed that the essential oils of rosemary, jarilla, mendocina, tomillo mendocina, origanum, tarragon, lavandins and eucalyptus were emulsiﬁed with different formulations of water suspensions of wall support systems using both a handheld propeller blender and a high pressure, double effect homogenizer. Also, Bowers and Locke (2000) report that several commercial formulations of botanical extracts and essential oils have been investigated as possible alternatives for soil fumigation to control Fusarium wilt disease. Essential oils of fennel, peppermint and caraway have been formulated in the form of stable emulsiﬁable concentrates. occurrence as part of the diet, their ephemeral nature and their biodegradability suggest low toxic residue problems. Such compounds could be extracted and applied to other harvested perishables. Some of the volatile aromatic components, namely acetaldehyde, 6-carbon (C6) aldehydes, benzaldehyde, hexenel and hexanal, are of signiﬁcant importance. Aldehydes Vapours of acetaldehyde have been used to control B. cinerea (Prasad and Stadelbacher, 1973). Avissar and Pesis (1991) reported acetaldehyde to be active against B. cinerea and Rhizopus stolonifer causing rot to strawberry fruits. Benzaldehyde has been used in the laboratory to fumigate peaches and to protect them against Rhizopus rot. It inhibits spore germination of B. cinerea totally at 25 µl/l and germination of Monilinia fructicola at 125 µl/l (Wilson et al., 1987). The aldehydes, benzaldehyde, acetaldehyde and cinnamaldehyde, ethanol and benzyl alcohol were found to be the strongest growth inhibitors and the most lethal to fungal spores and mycelia of fruit and vegetable pathogens like P. digitatum, R. stolonifer and Colletotrichum during in vitro trials. Hexenal and hexanal Botanicals in the Management of Postharvest Diseases of Perishables Botanicals as antifungal agents in postharvest disease control of fruits Fruits and vegetables have a number of constituents and inducible volatile aromatic and ﬂavour compounds (Tripathi, 2007). These aromatic and ﬂavour components are generally produced by fruits during ripening and provide resistance to the fruits at the postharvest stage. The ﬂavour compounds are secondary metabolites having unique properties of volatility and low water solubility. As potential fungicides, their natural (E)-2-Hexenal and hexanal are two different volatile ﬂavour compounds. Hexenal vapours have a number of attributes that may be important in consumer demand for more natural measures to combat fruit diseases with fewer toxic residues. Hexenal vapour inhibited hyphal growth of apple slices (Song et al., 1996). Archbold et al. (1999) showed (E)-2-hexenal to be an efﬁcient fumigant in controlling mould on ‘Crimson Seedless’ table grapes. (E)-2-Hexenal has been found to be strongly antifungal in nature and its in vitro and in vivo activity against B. cinerea has been reported by a number of workers (Hamilton-Kemp et al., 1992; Fallik et al., 1998). The effect of trans-2-hexenal Exploitation of Botanicals on the control of blue mould disease (P. expansum) in reducing patulin content and on improving the fruit quality of ‘Conference’ pears has been evaluated and greater reduction of decay was obtained by treatment at 12.5 µl/l at 20°C for 24 or 48 h after inoculation (Neri et al., 2006). Acetic acid Acetic acid is a metabolic intermediate that occurs naturally in many fruits (Nursten, 1970). There are several advantages in using acetic acid fumigation. It is a natural compound found throughout the biosphere, posing little or no residual hazard. Low concentrations, i.e. 2.0 or 4.0 mg/l, of acetic acid in air have been found to be extremely effective for controlling B. cinerea conidia on apple (‘Red Delicious’) fruit (Sholberg and Gaunce, 1995). Acetic acid has been shown to be an effective fumigant for commercial use on apricot and plums (Liu et al., 2002), grapes (Sholberg et al., 1996) and sweet cherries (Sholberg, 1998; Chu et al., 1999, 2001). The use of acetic aid and vinegar is the better choice in most cases because it does not have an objectionable odour and has a long history of use on food (Sholberg et al., 2000). Jasmonates The term ‘jasmonates’ includes jasmonic acid (JA) and methyl jasmonate (MJ). These are naturally occurring plant growth regulators that are widely distributed in the plant kingdom and are known to regulate various aspects of plant development and responses to environmental stresses (Sembdner and Parthier, 1993; Creelman and Mullet, 1995, 1997). Droby et al. (1999) found that postharvest application of jasmonates reduced decay caused by grey mould, P. digitatum, either after natural or artiﬁcial inoculation of ‘Marsh Seedless’ grapefruit. When applied at low concentrations, jasmonates are potential postharvest treatments to enhance natural resistance and to reduce decay in fruit. Since they are naturally occurring compounds and are 43 given in low doses, jasmonates may provide a more environmentally friendly means of reducing the current chemical usage. Glucosinolates Among natural substances with potential antimicrobial activity are the glucosinolates, a large class of approximately 100 compounds produced by members of the family Crucifereae, with well-documented activity (Fenwick et al., 1983). Hydrolysis of glucosinolates produces D-glucose, sulphate ion and a series of compounds such as sothiocyanate (ITC), thiocyanate and nitril. The antifungal activity of six glucosinolates has been tested on several postharvest pathogens, namely B. cinerea, R. stolonifer, M. laxa, Mucor piriformis and P. expansum, both in vitro (Mari et al., 1993) and in vivo (Mari et al., 1996). Allyl-isothiocyanate (AITC), a naturally occurring ﬂavour compound in mustard and horseradish, has a well-documented antimicrobial activity. Exposure of pear fruit to an AITC-enriched atmosphere resulted in good control of blue mould, including a TBZ resistant strain on pears (Mari et al., 2002). The use of AITC, produced from puriﬁed sinigrin or from Brassica juncea, against P. expansum appears very promising as an economically viable alternative with moderately low impact on the environment. Essential oils The antimicrobial effects of essential oils (EOs) or their constituents on postharvest pathogens have been studied quite extensively (Bishop and Thornton, 1997; Tripathi et al., 2007). The advantage of EOs is their bioactivity in the vapour phase, a characteristic that makes them attractive as possible fumigants for stored product protection. Control of the storage pathogen, B. cinerea, on Dutch white cabbage (B. oleracea var. capitata) by the EOs of Melaleuca alternifolia in in vitro conditions has been investigated (Bishop and Reagon, 1998). Tripathi 44 P. Tripathi and A.K. Shukla et al. (2008) evaluated some EOs against moulds of grapes caused by B. cinerea. The effect of C. nardus EO on the growth and morphogenesis of A. niger has been tested (Bellerbeck et al., 2001). The potential of using EOs by spraying or dipping to control postharvest decay has been examined in fruits, namely cherries, citrus fruits, apple, peaches and cabbage (Tiwari et al., 1988; Smid et al., 1994; Dixit et al., 1995). Thymol is an EO component from thyme (T. capitatus). Fumigation of sweet cherries with thymol was effective in controlling postharvest grey mould rot caused by B. cinerea (Chu et al., 1999) and brown rot caused by M. fructicola (Chu et al., 2001). The shelf life and safety of some perishable foods treated with EOs have been improved remarkably (Ponce et al., 2004; Holley and Patel, 2005). The EO of S. ofﬁcinalis has also shown practical potency in enhancing the storage life of some vegetables by protecting them from fungal rot (Bang, 1995). Treatment of oranges by fumigation with the EOs of M. arvensis (100 µl/l), O. canum (200 µl/l) and Zingiber ofﬁcinale (200 µl/l) has been found to control blue mould, thereby enhancing shelf life (Tripathi et al., 2004). Plaza et al. (2004) evaluated the potential of thyme, oregano, clove and cinnamon EOs against P. digitatum and P. italicum on citrus fruits. The postharvest quality of strawberry and tomato fruit was evaluated after treatment with Eucalyptus and cinnamon volatile EO vapours (Tzortzakis, 2007). Plant extracts Some plants extracted in different organic solvents have shown inhibitory action against different storage fungi (Singh et al., 1993; Hiremath et al., 1996; Rana et al., 1999; Okigbo and Pandalai, 2005). The inhibitory effect of water-soluble extracts of garlic bulbs, green garlic, green onions, hot peppers, ginger, Chinese parsley and basil on the growth of A. niger and A. ﬂavus was examined. Garlic bulbs, green garlic and green onions showed an inhibitory effect against these two fungi (Yin and Cheng, 1998). Treatment of pineapple fruits infested with C. paradoxa by X. strumarium extract reduced the severity of the disease (Damayanti et al., 1996). The phytochemical investigation of a methanolic extract of A. nilotica resulted in isolation of kaempferol. It has shown antifungal activity against P. italicum at 500 µg/l (Tripathi et al., 2002). In vitro inhibition of B. theobromae causing Java black rot in sweet potato was induced by phenolic compounds, chlorogenic acid giving the highest in vitro inhibition, followed by pyrogallol, pyrocatechol, phenol and resorcinol. Low concentrations of phenols are required by the fungus during normal metabolism, but higher concentrations are inhibitory to growth (Mohapotra et al., 2000). The phytochemical investigations of most plants have resulted in the isolation of active principles. These compounds when tested against postharvest fungi have shown pronounced antifungal activity. A naturally occurring compound isolated from the ﬂavedo tissue of ‘Star Ruby’ grapefruit (Citrus paradise) identiﬁed as 7-geranoxy coumarins exhibited antifungal activity against P. italicum and P. digitatum during in vitro and in vivo tests (Agnioni et al., 1998). Arya (1988) controlled fruit rots by leaf extracts of medicinal plants. Mode of Action of Essential Oils The mechanism of action of EOs and other bioactive phytocompounds against microorganisms is a complex process and has not yet been fully explained. It is generally recognized that the antimicrobial action of essential oils depends on their hydrophobic or lipophilic character. Terpenoids may serve as an example of lipid-soluble agent that affects the activities of membrane catalysed enzymes; for example, their action on respiratory pathways. Compounds of EOs either affect the physiological function of microorganisms or cause structural changes of hyphae and spores (Thompson, 1986; Arras et al., 1993; Zambonelli et al., 2004). For instance, the effect of thyme oils and thymol on the hyphae cytomorphology of F. solani, R. solani and C. lindemuthianum Exploitation of Botanicals increased vacuolization of the cytoplasm and accumulation of lipid bodies, undulation of the plasmalemma and alteration of the mitochondrial and endoplasmic reticulum (Zambonelli et al., 2004). However, variations in the fungicidal action of the compounds seem to depend on solubility, as well as on the capacity to interact with cytoplasmic membrane. Conclusions Sustainable agriculture in the 21st centaury will rely increasingly on alternative interventions for pest management that are environmentally friendly and reduce the amount of human contact with chemical pesticides. The use of botanicals in crop protection has now gained popular ground in the world of agriculture as an alternative to the use of toxic, persistent and synthetic compounds. Several factors are now responsible for making the use of alternative methods more attractive. A number of studies have been conducted on the use of botanicals and several plants with promising biocidal properties have been identiﬁed. Most of these plants have also been used in vitro and in vivo in the control of various plant diseases. Certain plant EOs and plant extracts have a broad spectrum of activity against plant pathogenic and other fungi. They have considerable potential as crop protectants. Current information indicates that they are safe to the user and the environment, with few qualiﬁcations. With the modern techniques now available and the attention being given to this area, we look forward to intensifying development of the biological activity of botanicals so as to exploit them as fungicides. A consolidated and continuous search for natural products may yield safer alternative control measures like azadirachtin and pyrethroids, which are being used. However, in order to consider the use of any plant material seriously, further information is required. The use of locally available plants avoids the need to establish complex mechanisms for pesticide distribution; the community can collect or grow the plants 45 itself. Effective antifungal plant compounds that can ﬁll the void of phased-out chemicals will require some advances in the study of regional aromatic plants, their production, formulation and possible beneﬁcial mechanism to prevent or control fungal attack, better understanding of how they will ﬁt into integrated systems and their interaction with the environment and other IPM components and identiﬁcation of the optimum concentration of EOs that can control seedborne fungal pathogens without affecting seed germination and seedling growth. The information on the active principles present in various botanicals on germination and seedling vigour is to be elucidated. A proper study of the mode of action and structure activity relationship will bring about a new class of interesting compounds for future pest control. Despite the common belief that phytocompounds are safe, they all have inherent risk, just like synthetic compounds. Thus, it is within the scope of phytoscientists to elucidate the side effects and appropriate doses and identify bioactive phytocompounds and ways of extraction and conservation. As a cautionary note, the EOs that are the most efﬁcacious against pests are often the most phytotoxic. This phytotoxicity requires serious attention when formulating products for agricultural use. Also, selectivity among invertebrates is not well documented. Honeybees appear somewhat susceptible (Lindberg et al., 2000). The susceptibility of various natural enemies has yet to be reported, although the lack of persistence of EOs under ﬁeld conditions could provide some information on temporal selectivity favouring non-target species. Finally, we should maintain our efforts in considering and valorizing our natural patrimony, as well as conducting more scientiﬁc research on aromatic plants for chemical analysis and biological, toxicological and pharmacological investigation of therapeutic aspects. It is important to remember that just because a pesticide is derived from a plant does not mean that it is safe for humans and other mammals, or that it cannot kill a wide variety of other life. Some botanical pesticides can be quite toxic to humans and should not be used on plants 46 P. Tripathi and A.K. Shukla for human consumption. For example, methyl salicylate (oil of wintergreen) is commonly used as food ﬂavouring, but it can be quite toxic in large doses (Jonathan and Davis, 2007). Few systematic studies have been conducted to determine how farmers use plant protectants, their effectiveness and method of application. 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Perithecial development by Gibberella zeae: a light microscopy study. Mycologia 92,130-138. © Mycological Society of America) Plate 2. Diaporthe phaseolorum (anamorph Phomopsis sojae) causing seed rot on soybean seeds. (Courtesy M. C. Rollán) Plate 3. Fusarium sp. Infecting soybean seeds. (Courtesy M. C. Rollán) Plate 4. Germinating onion seed affected by Botrytis allii. (Courtesy L. du Toit, Diseases in vegetable seed crops: Identification, biology, and management [Online]. Available at: http://www.seedalliance.org/uploads/pdf/VegSeedDiseases.pdf) Plate 5. Seedborne wilt of spinach by Verticillium dahliae. (Courtesy L. du Toit) 6 7 8 9 10 Plate 6. Spinach seed showing stromatisation due to pseudothecia of Pleospora herbarum (anamorph Stemphylium botryosum). (Courtesy L. du Toit) Plate 7. Rice seed discoloration caused by a fungi complex. Plate 8. Wheat seed discoloration caused by a fungi complex. Plate 9. Open pod of soybean showing purple discoloration caused by Cercospora kikuchii. (Courtesy M. C. Rollán). Plate 10. Conidiophores and conidia of Cladosporium variabile on spinach seed. (Courtesy L. du Toit) 5 Use of Plant Extracts as Natural Fungicides in the Management of Seedborne Diseases Gustavo Dal Bello and Marina Sisterna Comisión de Investigaciones Cientíﬁcas de la Provincia de Buenos Aires, Centro de Investigaciones de Fitopatología (CIDEFI) – Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, La Plata, Argentina Abstract Seedborne fungi can cause substantial losses to grains, rendering them unﬁt for human consumption and sowing. Several methods have been used for the control of seedborne diseases and among them chemical control has been the most widely adopted over many decades. The use of most of these fungicides has been restricted because of high and acute toxicity, long degradation periods and bad effects on human health, plants and animals, which is harmful to our environment. Moreover, recent increases in the production and sale of organic seed has heightened the scrutiny of organic seed quality and in particular brought attention to concerns of seedborne disease contamination. In order to meet the demands of consumers and growers alike, exploration of alternative methods for managing fungal diseases is under way. One such eco-friendly approach of controlling seed fungal diseases is the use of natural products, speciﬁcally plant-derived compounds. They have played a signiﬁcant role in reducing the incidence of seedborne pathogens and in the improvement of seed quality and the emergence of plant seeds in the ﬁeld. It has long been recognized that several plant compounds, such as essential oils, have antifungal activity against both pathogens and spoilage fungi. As a rich source of bioactive chemicals, plants may provide potential alternatives to synthetic fungicides for seed treatment to protect them against seedborne pathogens. Therefore, this chapter discusses the current status of the use of plant extracts to control seedborne fungi. Introduction Almost 90% of all the world’s food crops are grown from seeds (Schwinn, 1994), which are widely distributed in national and international trade. Many plant pathogens can be seed transmitted and seed distribution is a very efﬁcient means of introducing plant pathogens into new areas, as well as a means of survival of the pathogen between growing seasons. Disease-causing organisms may be carried with, on or in seeds and, in suitable environmental conditions, may be transmitted to cause diseases in developing seedlings or plants. With some diseases, the pathogen attacks the germinating seedling, which affects seedling establishment and hence plant populations; with others, disease symptoms are not seen until a later stage of growth (Rennie and Cockerell, 2006). Furthermore, seedborne pathogens such as bacteria, fungi, viruses and nematodes have the  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 51 52 G. Dal Bello and M. Sisterna potential to spread disease to the subsequent crop. Seedborne infection of fungal pathogens is important not only for its association with the seeds but also contamination of the soil by permanently establishing its inocula. Additionally, fungi are signiﬁcant destroyers of foodstuffs and grains during storage, rendering them unﬁt for human consumption by retarding their nutritive value and often by producing mycotoxins (Satish et al., 2007). It is, therefore, necessary to search for control measures that are economical, ecologically sound and environmentally safe to eliminate or reduce the incidence of these important pathogens so as to increase seed germination and obtain healthy and vigorous plants with better yield (Hasan et al., 2005). Seed treatment is the oldest practice in plant protection. Its origin can be traced to the 18th century with the use of brine to control cereal smuts (Neergaard, 1979). The modern era of seed treatments began with the introduction of organomercury fungicides in 1912, which were widely used for several decades. The post-World War II period saw the development of new fungicide chemistry and the ﬁrst use of seed treatment for insect control. Today, the most widely used application of seed treatment is the traditional one of protecting the germinating seedling against seed- and soilborne fungi in the period immediately after planting (McGee, 1995). Chemical fungicides can control plant diseases but they have bad effects on human health, plants and animals, which is harmful to our environment. Besides, using conventional seed treatment with synthetic fungicides to kill pathogens is a practice not allowed in organic production. Additionally, resistance by pathogens to fungicides has rendered certain fungicides ineffective. Worldwide ecological awareness requires more natural foods and products, which has inﬂuenced the improvement and utilization of integrated pest management. In this kind of control, alternative methods are used to protect seeds to decrease the use of chemical products. Moreover, recent increases in the production and sale of organic seed has heightened the scrutiny of organic seed quality, and in particular brought attention to concerns of seedborne disease contamination. The number of alternative crop production systems has increased in the past decade in response to growing concerns about agricultural concentration and interest in a more ecological, farmbased agriculture. In these low-input systems, some non-chemical substances, such as plant extracts, may be used successfully as a contact fungicide seed treatment for organic crops. As a rich source of bioactive chemicals, plants may provide potential alternatives to be used as pathogen-control agents. Hamburger and Hostettmann (1991) report that the total number of plant chemicals may exceed 400,000 and of this, more than 10,000 are secondary metabolites whose major role in plants is defensive in nature. Thus, plant-based secondary metabolites that have a defensive role may be exploited for the management of diseases and pests. However, most species of higher plants have never been surveyed. Their chemical or biologically active constituents that have the potential to be used as new sources of commercially valuable pesticides remain to be discovered. This is due mainly to the lack of information on the screening/evaluation of diverse plants for their antifungal potential (Satish et al., 2007). Nevertheless, several higher plants and their constituents have shown success in plant disease control and have proved to be harmless and non-phytotoxic, unlike chemical fungicides. Essential Oils to Reduce Seedborne Fungi Plant extracts have played a signiﬁcant role in reducing the incidence of seedborne pathogens and in the improvement of seed quality and the emergence of plant seeds in the ﬁeld (Hasan et al., 2005). In recent years, much attention has been paid to essential oils, a group of plant-derived compounds, for seed treatment to protect them Use of Plant Extracts as Natural Fungicides against seedborne fungi (Sisterna and Dal Bello, 2007). The essential oils arise from a secondary metabolism of the plant, normally formed in special cells or groups of cells as glandular hairs, found on many leaves and stems. Oils occur as a globule or globules in the cell and may also be secreted from cells lining the schizogenous ducts or canals. Plant volatile oils are generally isolated from nonwoody plant material by several methods, usually distillation, and are a variable mixture of principally terpenoids, speciﬁcally monoterpenes [C10] and sesquiterpenes [C15], although diterpenes [C20] may also be present. A variety of other molecules can also occur, such as aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones and, exceptionally, nitrogen- and sulphur-containing compounds, coumarins and homologues of phenylpropanoids (Dorman and Deans, 2000). Faleiro et al. (2003) have shown that the antimicrobial action is determined by more than one component. In such cases, the major component is responsible not only for the antimicrobial activity, but also the synergistic effect that may take place. The mixtures are extremely complex and vary with environmental and genetic factors (Asplund, 1968; Cabo et al., 1986; Arras, 1988; Bhaskara et al., 1998; Vanneste et al., 2002). Moreover, the composition of essential oils from a particular species of plant can differ between harvesting seasons and between geographical sources (Di Pasqua et al., 2005; Di Pasqua, 2006). Major active compounds from essential oils are known for their broad-spectrum antifungal activity against both human and plant pathogens. These constituents can either affect the physiological functions of microorganisms or cause structural changes of hyphae and spores (Arras et al., 1993; Zambonelli et al., 2004; Kishore et al., 2007), and different fungi appear to react differently to these components (Szczerbanik et al., 2007). The antifungal essential oils reduce hyphal growth and also induce lysis and cytoplasmic evacuation in fungi. Growth inhibition by essential oils often involves induction of changes in cell wall composition (Ghﬁr et al., 1997), plasma membrane disruption, 53 mitochondrial structure disorganization (de Billerbeck et al., 2001) and interference with enzymatic reactions of the mitochondrial membrane, such as respiratory electron transport, proton transport and coupled phosphorylation steps (Knobloch et al., 1989). The active components vary between oils. For example, the main component is l-carvone in spearmint (Mentha spicata L.), terpinen-4-ol in tea tree (Melaleuca alternifolia (Maiden. & Betche.) Cheel.) oil and α-terpineol in pine (Pinus spp.) (Knobloch et al., 1989). The essential oils of Cinnamomum zeylanicum Blume (cinnamon) and Syzygium aromaticum (L.) Merr. & Perry (syn. Eugenia cariophyllata Thunb.), consisting of cinnamaldehyde and eugenol, respectively, as major components (Paranagama, 1991), are known to be potent antifungal materials (Beg and Ahmad, 2002; Ranasinghe et al., 2002). Citral and geraniol are the major components in essential oils of Cymbopogon citratus (DC.) Stapf (lemongrass) and C. martinii (Roxb.) Stapf var. motia (palmarosa), respectively, which are antifungal compounds (Paranagama et al., 2003; Velluti et al., 2004). Thymol was identiﬁed as the active ingredient of Ocimum gratissimum L. (wild basil) and has been found to suppress fungal growth (Adekunle and Uma, 2005). Linalool is a major component in the essential oil of Thymus mastichina L. subsp. mastichina, with antimicrobial activity (Faleiro et al., 2003), and both limonene and linalool are the minor components in the essential oils derived from different plants. The majority of these essential oils and their components have proved valuable in protection against postharvest fungal diseases which cause build-up of toxic fungal metabolites in stored foods (Kishore et al., 2007). Therefore, essential oils might substitute agrochemicals or contribute to the development of new agents to inhibit both fungal growth and the production of mycotoxins affecting grain and seed crops. This chapter discusses the current status of plant extracts and the potential use of essential oils as natural antifungal agents to control the main seedborne pathogens and spoilage fungi. 54 G. Dal Bello and M. Sisterna Symptoms on Seeds Caused by Fungi Seedborne mycoﬂora comprise a large number of saprobes and pathogenic fungal species. Pathogenic fungi grown on seeds can cause heavy damage and reduce yields of seed, both quantitatively and qualitatively (Neergaard, 1979). Other fungi, including saprophytes and very weak parasites (Sisterna and Lori, 2005), may lower the quality of seeds by causing discoloration, which may seriously depreciate the commercial value of seeds, particularly of grain when graded for consumption. Disease and disorder The following types of disease and disorder are encountered, often in combination (Neergaard, 1979): Seed abortion The most prominent examples of fungi producing abortion are the smut fungi, which infect cereals and grasses systemically, and the ergot fungi. The ﬂoral parts of the hosts are replaced by the fructiﬁcations of the parasites. Other examples are different species of Fusarium (in wheat, maize and rice); Ascochyta rabiei in chickpea may kill the young seeds; Drechslera verticillata causes death of seed primordia in brome grass and in wheat. Shrunken seeds, reduced in size Examples of more or less heavy reduction of seed size are: Alternaria brassicicola and Phoma lingam in crucifers, Septoria linicola in ﬂax, D. teres in barley, F. graminearum and S. nodorum in wheat. B. maydis and B. oryzae in cereals; Colletotrichum graminicola, Diaporthe phaseolorum (Plate 2) and Fusarium spp. in soybean (Plate 3); Botrytis allii on onion (Plate 4); Verticillium dahliae on spinach (Plate 5) and B. cinerea in the seeds of many hosts, including forest trees. Sclerotization and stromatization Transformation of ﬂoral organs or seed into sclerotia or stromata is an important disease condition in certain categories of fungi and host. Ergots produced by Claviceps purpurea and other species of Claviceps in cereals and grasses exemplify sclerotia of this type. Another example is Phomopsis viterbensis in chestnut, Pleospora herbarum in spinach (Plate 6) and Ciboria spp. in the seeds of forest trees and grasses. Seed necroses Many seed-rotting fungi produce superﬁcial necroses in the seed; other fungi never penetrate deeply into the tissues, most seedborne fungi usually not beyond the protective layers, the seed coat or pericarp. Anthracnose fungi, Colletotrichum spp. as well as Ascochyta spp., often penetrate into the ﬂeshy cotyledons, producing conspicuous necrotic lesions in the seeds of bean, soybean, pea, cowpea and other hosts. Seed discoloration Discoloration of seeds is a very important degrading factor, both for consumption (grain) or for industrial purposes (oil seed). It may be a general indication of poor quality (Plates 7 and 8). Well-known examples are the effects of A. pisi in pea; C. lindemuthianum in bean; B. sorokiniana in wheat, B. oryzae in rice, Cercospora kikuchii (Plate 9) in soybean, etc. Seed rot Reduction or elimination of germination capacity, lowered viability Many seedborne fungi produce seed rot either in the crop or during germination. Examples are F. avenaceum, F. graminearum (Plate 1), F. moniliforme, Bipolaris sorokiniana, Obviously, necroses or more deeply penetrating rots in seeds reduce the viability of the seeds, their longevity in storage and their emergence in the ﬁeld. Use of Plant Extracts as Natural Fungicides Physiological alterations or effects in seed Metabolic products of seedborne microorganisms may affect the seed itself or may have other, sometimes serious consequences such as toxicity to animals and humans (Aspergillus spp., Penicillium spp., Fusarium spp.). Moreover, seed fungi are classiﬁed as ﬁeld and storage fungi (Christensen and Kaufmann, 1965). Genera such as Alternaria, Cladosporium (Plate 10), Fusarium and Bipolaris invade seeds as they are developing on the plants in the ﬁeld or after they have matured, but before they are harvested, and for this reason, they have been designated ‘ﬁeld fungi’. These fungi require moisture content in equilibrium with a relative humidity of more than 90% to grow and usually do not continue to grow in grains after harvest, since grains and seeds are stored with moisture contents below those required by the ﬁeld fungi. The storage fungi consist mainly of several species of Aspergillus. Species of Penicillium are encountered at times, usually in lots of grain stored at low temperatures and with moisture contents above 16%. The storage fungi do not invade grains to any appreciable degree or extent before harvest. Fungicidal Effects of Plant Extracts Against Seed Fungi Numerous studies have described the use of botanicals with a view to exploiting their potential as natural fungicides against seedborne fungi. The following section discusses this alternative method, with particular emphasis on the main seedborne fungal pathogens. Alternaria A. padwickii, an important seedborne pathogen of rice (Oryza sativa L.), was inhibited by aqueous extracts of Strychnos nux-vomica L. (strychnine tree), garlic (Allium sativum L.) 55 bulbs, ginger (Zingiber ofﬁcinale Roscoe) rhizomes, basil (O. basilicum L.) leaves, and fruits of Azadirachta indica A. Juss. (neem) (Shetty et al., 1989). Positive effects have been recorded on the same fungus with essential oils of C. citratus, O. gratissimum L. and Thymus vulgaris L. (thyme) (Nguefack et al., 2004). The researchers investigated the ability to control seedborne infection and seed–seedling transmission in naturally infected seeds. The essential oils increased the germination capacity of the treated seeds. Bipolaris Hasan et al. (2005) demonstrated that plant extracts, namely Z. ofﬁcinale, A. sativum, A. cepa L. (onion), Adhatoda vasica Nees (vasaka), Achyranthes aspera L. (devil’s horsewhip), A. indica, Lawsonia alba Lamarck (henna), Cuscuta reﬂexa Roxb. (giant dodder), Vinca rosea L. and Nigella sativa L. (black cumin), signiﬁcantly reduced seed infection of wheat by B. sorokiniana (Triticum aestivum L.). Alcoholic extracts of neem and garlic inhibited the presence of B. sorokiniana completely, whereas the highest percentage of the fungus was recorded from untreated seeds (control). Water extract of all tested plants had the ability to control seedborne fungi of wheat var. Kanchan, which showed 100% inhibition of B. sorokiniana with the application of extracts from Z. ofﬁcinale, A. sativum, A. cepa, A. indica, C. reﬂexa and N. sativa, whereas the highest fungal incidence (11.67%) was observed on untreated seed. After treatment with the water extract of L. alba and A. aspera, only 4.84% and 7.16% incidence of the pathogen, respectively, was recorded. Seeds of wheat treated with A. vasica and V. rosea gave statistically identical results (5.83% and 5.90% incidence of B. sorokiniana). Alice and Rao (1986) reported good results using plant extracts to control B. oryzae on rice seeds which have high natural infection of the fungus. After soaking in the ﬁltrates of different extracts, A. sativum and M. piperita (peppermint) reduced seed 56 G. Dal Bello and M. Sisterna infection by 68%. In Bangladesh, use of extracts of Polygonum hydropiper L. (waterpepper), A. cepa, A. sativum and A. indica was demonstrated to be effective against B. oryzae at higher concentrations. Among them, neem and garlic were the most effective at 1:1 dilution and inhibited the occurrence of the pathogen by 91 and 83%, respectively (Ahmed et al., 2002). Neem and pungam (Pongamia pinnata (L.) Pierre) oil-based emulsiﬁable concentrate (EC) formulations were evaluated for their efﬁcacy to inhibit the mycelial growth of the fungus Helminthosporium oryzae (syn. B. oryzae) causing grain discoloration of rice under in vitro conditions. All three formulations, namely neem oil 60 EC (acetic acid), neem oil 60 EC (citric acid) and neem oil + pungam oil 60 EC (citric acid), inhibited mycelial growth of the pathogen; they were effective even after 9 months of storage. These formulations controlled the grain discoloration on rice effectively (Rajappan et al., 2001). The efﬁcacy of essential oils such as clove, ginger, lemongrass, basil, peppermint, anise (Pimpinella anisum L.) and cinnamon at different concentrations on growth inhibition of B. oryzae was examined by Palaoud (2006). Treatments with clove, anise, ginger and cinnamon oils at 500 ppm provided the best results in controlling the fungus and, after storage for 4 months, seed viability was as high as 97–98%. Also, the extracts of C. citratus, O. gratissimum and T. vulgaris applied to rice seeds infected with B. oryzae controlled fungal growth and seedling transmission of the pathogen (Nguefack et al., 2004). Colletotrichum Abdelmonem et al. (2001) screened oils of M. piperita, T. capitatus (L.) Hoffmans. and Link and Carum carvi L. (caraway) against various seedborne fungi of soybean (Glycine max (L.) Merr.) and lentil (Lens culinaris Medik.) and found all plant extracts to be highly effective in controlling C. dematium. Among the ﬁbre-producing species, a study on garlic bulb extract reported a fungicidal effect of the oil against numerous seedborne fungal pathogens of white jute (Corchorus capsularis L.), one of the most important crops from Bangladesh, India and China. The essential oil produced inhibition in both mycelial growth and spore germination of fungi, including C. corchori (Ahmed and Shultana, 1984), which was also strongly inhibited in in vitro tests by using crude leaf extracts from Eupatorium triplinerve Vehl. (yapana) (Rahman and Junaid, 2008). Several studies carried out in Burkina Faso underlined the antifungal properties of extracts from some Cymbopogon spp. against C. graminicola, the causal agent of anthracnose on sorghum (Sorghum bicolor (L.) Moench and pearl millet (Pennisetum glaucum (L.) R. Br.). Somda et al. (2007) demonstrated that the essential oil of C. citratus at a concentration of 6% was effective in controlling seedborne infection and seed– seedling transmission of C. graminicola without affecting seedling development. Similarly, the essential oils extracted from C. giganteus (Hochst.) Chiov., C. nardus (L.) Rendle and C. schoenanthus Spreng. reduced sorghum seed infection by the pathogen signiﬁcantly. The lowest rates of infected seeds were recorded on seeds treated with 10 µl and 15 µl of C. nardus oil/g seeds. These doses were more efﬁcient than chemical control (Elisabeth et al., 2008). Curvularia In blackgram (Vigna mungo L.), essential oils extracted from wood chips of cedar (Cedrus deodara (Roxb. ex Lamb) G. Don) and that from seeds of Trachyspermum ammi (L.) Sprague ex Turrill (ajowan) exhibited absolute toxicity, inhibiting the mycelial growth of C. ovoidea, storage fungi found on seeds (Singh and Tripathi, 1999). Parimelazhagan and Francis (1999) reported reduction in the radial growth of C. lunata associated with rice seeds when treated with leaf extracts of Clerodendrum viscosum Vent. (glory tree), which also increased seed germination and root and Use of Plant Extracts as Natural Fungicides shoot lengths of rice. Considerable research activity has occurred in the Asian-Paciﬁc region on the potential for plant extracts to control seedborne fungi including maize. The oils of cassia (C. cassia Blume) and clove inhibited the growth of established seedborne infections of C. pallescens (Chatterjee, 1990). Fusarium The essential oils and their constituents have been found effective as antifungal agents against the main species of Fusarium. Among several plant extracts, Sitara et al. (2008) found that essential oils from seed of neem, black cumin and asafoetida (Ferula asafoetida L.) showed fungicidal activity of varying degree against F. oxysporum, F. moniliforme (syn. F. verticillioides), F. nivale and F. semitectum. Of those oils, asafoetida oil at 0.1% and 0.15% inhibited the growth of all test fungi signiﬁcantly. A variety of wild plants from Mexico were evaluated against several cereal seedborne fungi in in vitro tests (Tequida-Meneses et al., 2002). Extracts from leaves and stems of Larrea tridentata (Sessé & Moc. ex DC.), Coville (creosote bush) and Datura discolor Bernh. (desert thorn apple) in methanol or ethanol inhibited the radial growth of F. poae completely. Next to these extracts, Proboscidea parviﬂora (Woot.) Woot. & Standl. (double claw) also showed good fungal inhibition (86.6%), followed by Baccharis glutinosa Pers. (saltmarsh baccharis) (79.6%), compared to the alcoholic controls (0% inhibition). In legumes, soybean and lentil, carvone (monoterpene compound), among other tested compounds, manifested the highest antimicrobial inﬂuence with complete inhibition to F. oxysporum. It showed broadspectra activity against all the tested isolates of fungal strains at low concentrations. Peppermint, T. capitatus and caraway oils also demonstrated a high control effect against Fusarium sp. (Abdelmonem et al., 2001). Also, the antifungal activity of the essential oils of thyme, clove, peppermint, soybean and peanut (Arachis hypogaea L.) were 57 tested against F. oxysporum and F. equiseti in vitro on cowpea (V. unguiculata (L.) Walp.) (Kritzinger et al., 2002). Likewise, plant leaf extracts (crude and aqueous) of basil, bitter leaf (Vernonia amygdalina Del.), neem and pawpaw (Carica papaya L.) reduced the incidence of F. moniliforme signiﬁcantly and increased seed germination and seedling emergence of African yam bean (Sphenostylis stenocarpa (Hochst ex. A. Rich) Harms) when compared with the untreated controls (Nwachukwu and Umechuruba, 2001). Regarding cereals, several natural plant compounds have been identiﬁed as having antifungal activity against seedborne fungi. The essential oils of C. citratus, O. gratissimum and T. vulgaris have proved valuable in protection against the seedborne fungus, F. moniliforme in rice. This study evaluated the ability to control seedborne infection and seed–seedling transmission in naturally infected seeds (Nguefack et al., 2004). The extracts applied controlled seed infection and seedling transmission of the pathogen and increased the germination capacity of the treated seeds. In the ﬁeld, as a result of extracts seed treatment as compared to the non-treated control, reduction of disease incidence and important increases in yield were recorded. After rice seeds inoculated with F. moniliforme were soaked in seven plant essential oils at ten different concentrations, anise, ginger, clove and cinnamon oils at 500 ppm provided the best results in controlling the fungus. The percentage of seed germination and the number of normal seedlings was signiﬁcantly high when compared with the control. Anise and clove also showed the highest seedling dry weight (Palaoud, 2006). In another study on wheat, ten plant extracts were tested for their efﬁcacy in vitro against seedborne fungi; alcoholic extract of neem and garlic controlled the infection of Fusarium sp. completely. Good results of these treatments contributed to increased seed germination (Hasan et al., 2005). Furthermore, botanicals from male fern (Dryopteris ﬁlix mas (L.) Scott.) suppressed completely the population of F. oxysporum in the seed mycoﬂora of wheat (Rake et al., 1989). Putative mycotoxicogenic fungi, such 58 G. Dal Bello and M. Sisterna as F. moniliforme, were partially or totally sensitive to different essential oils extracted from 12 medicinal plants (Soliman and Badeaa, 2002). Results indicated that oils of thyme, cinnamon and anise (< or = 500 ppm), marigold (Calendula ofﬁcinalis L.) and caraway (< or = 2000 ppm), spearmint and basil (3000 ppm) inhibited this fungus completely. Bioassays using a poisoning technique were carried out with C. citratus, O. gratissimum and T. vulgaris for the control of seedborne fungi infecting maize (Zea mays L.) seeds. The results disclosed the fungicidal properties of theses oils against F. verticillioides. These natural products control the seedborne inoculum of the pathogen by 90% to 100%. Field trials conducted in the humid forest and the warm savannah zones of Cameroon have shown that these products are potential seed treatments that could be used as substitutes for synthetic fungicides, which are usually unaffordable to resource-limited farmers (Tagne et al., 2008). Seed treatment with cinnamon, palmarosa and lemongrass oils at 500 mg/kg showed antimycotoxigenic ability against fumonisin B1 accumulation by isolates of F. verticillioides and F. proliferatum (Marín et al., 2003). Furthermore, different effects of oregano (Origanum vulgare L.) and herb Louisa (Aloysia triphylla (L’Herit) Britton) essential oils were observed on F. verticillioides M 7075 fumonisin B1 production in corn grain in Argentina (López et al., 2004). As alternative preharvest natural fungicides, Velutti et al. (2004) showed the antimycotoxigenic activity of the essential oils against F. graminearum on corn infested seed. The effect of oregano, cinnamon, lemongrass, clove and palmarosa on growth rate, zearalenone (ZEA) and deoxynivalenol (DON) production was assessed at two concentrations (500 and 1000 mg/kg), at different water activity and temperature levels. DON production in general was inhibited by all essential oils at 30°C and, although palmarosa and clove were the only essential oils with statistically signiﬁcant inhibitory effect on ZEA production, an inhibitory trend was observed when cinnamon and oregano oils were added to maize grain. Studies on the efﬁcacy of indigenous plant extracts against seedborne infection of F. moniliforme on maize demonstrated that aqueous extracts of leaves of O. gratissimum, Acalypha ciliata Forssk., V. amygdalina, M. indica L. (mango tree) and A. indica had signiﬁcant inhibitory growth effects on the fungal pathogen. A. ciliata extract was more effective than other plant extracts and compared favourably with benomyl in the control of the pathogen (Owolade et al., 2000). Furthermore, to determine whether essential oils can be used as a contact fungicide seed treatment for organic corn, the essential oils of 18 plants were screened for their fungicidal properties. Five oils, cinnamon, clove, O. minutiﬂorum O. Schwarz and P.H. Davis, savoury (Satureja montana L.), and thyme, controlled Fusarium completely in vitro. The minimum inhibitory concentration (MIC) was 800 µl/l and seedlings presented no phytotoxicity symptoms in the germination test at rates up to 64 µl/kg active ingredient (MIC × 20). Field emergence of inbred and hybrid seeds treated with the essential oils were signiﬁcantly lower than seeds treated with the commercial fungicides, Maxim XL {ﬂudioxonil [4-(2,2-diﬂuoro-1,3benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile] 21.4%, mefenoxam [(R)-2-[(2,6-dimethylphenyl) methoxyacetylamino] propionic acid methyl ester] 8.4%}, which is a conventional fungicide, and Natural 2 (proprietary ingredients), which is an organic fungicide, but were not different from the organic fungicide, Yield Shield (Bacillus pumilus GB34 0.28%) or an untreated control (Christian and Goggi, 2008). Bioactivity of different plant extracts on F. thapsinum pathogen of sorghum was evaluated on seeds contaminated with the fungi. Cinnamon, clove, epazote (Teloxys ambrosioides (L.) Weber), oregano and thyme, singly and in combination, as well as the essential oils of Mentha sp. and rue (Ruta chalepensis L.) and the combination of clove with cinnamon, had a fungicidal effect. Nevertheless, only thyme did not affect either seed germination or sorghum seedling height. The rest of the oils were phytotoxic (MontesBelmont and Flores Monctezuma, 2001). Additionally, plant extracts were also tested Use of Plant Extracts as Natural Fungicides on naturally infected sorghum seeds for controlling F. moniliforme. More than 50% of the growth of this fungus was reduced by C. citratus essential oil on seeds, whereas Eucalyptus camaldulensis Dehnh. (Eucalyptus) essential oil was less efﬁcient, even at high concentrations (Somda et al., 2007). Elisabeth et al. (2008) investigated the efﬁcacy of essential oils extracted from C. schoenanthus, C. nardus and C. giganteus in controlling Fusarium sp. on seeds of sorghum and pearl millet. The results indicated that all the essential oils reduced seed contamination of both cereals signiﬁcantly. The lowest rates of infected seeds were recorded on seeds treated with 10 µl and/or 15 µl of essential oil/g seeds. Most of the time, these doses were as efﬁcient as the chemical control and oil of C. giganteus used at 15 µl/g seeds eliminated pearl millet seed infection by Fusarium completely. In another experiment, de Souza et al. (2003) analysed the mycoﬂora and physiological quality of cotton (Gossypium hirsutum L.) seeds treated with chemical fungicides and aroeira (Astronium urundeuva L.) extract. Pure extract did not control the fungal population but, when mixed with the fungicides, captan, benomyl and tolylﬂuanid, they showed reduction in the incidence of Fusarium sp. From Leguminosae members, leaf extracts of Delonix regia (Bojer) Raf., ﬂamboyant tree, Pongamia glabra Vent. (Karanja) and Acacia nilotica (L.) Willd. ex Delile (gum arabic tree) signiﬁcantly inhibited spore germination, mycelial growth and spore production of F. solani from sunﬂower (Helianthus annuus L.) seeds (Thiribhuvanamala and Narasimhan, 1998). The same pathogen could be controlled using crude leaf extracts of A. indica and O. gratissimum to protect egusi melon (Cucumeropsis mannii Naudin) seed. After 6 months incubation, all the seeds treated with leaf extracts showed no Fusarium infection (Adekunle and Uma, 2005). Efﬁcacy of some plant extracts in controlling seedborne Fusarium infections of mustard (B. nigra (L.) W.D.J. Koch) was evaluated. It was found that garlic and neem extracts were the most effective in controlling the pathogen among the plant extracts 59 studied. The effectiveness of garlic extract was comparable to the fungicide, Rovral (Latif et al., 2006). Macrophomina Several natural plant compounds have been identiﬁed as having antifungal activity against M. phaseolina. The work of Ahmed and Shultana (1984) reported that garlic oil produced inhibition in both mycelial growth and spore germination of M. phaseolina, an important seedborne fungal pathogen of jute. In sunﬂower seeds, the leaf extracts of the ﬂamboyant tree, karanja and gum arabic tree signiﬁcantly inhibited the germination of fungal spores, mycelial growth and spore production as well (Thiribhuvanamala and Narasimhan, 1998). In vitro experiments conducted by Dwivedi and Singh (1999) conﬁrmed the fungitoxicity of some higher plant extracts against the mycelial growth of M. phaseolina. Among the plant products, the essential oils of T. ammi exhibited absolute fungicidal effect at an MIC of 200 ppm. Studies of Abdelmonem et al. (2001) also showed the inhibitory effect of the essential oils of M. piperita, T. capitatus and C. carvi against M. phaseolina associated with the seeds of soybean and lentil. Furthermore, when tested in infected cowpea seeds, A. indica extract was found to inhibit the incidence of the pathogen. After naturally infected seeds were immersed in a suspension containing neem tree oil at a concentration of 0.5% for 16 h, the infection incidence decreased to 50% in relation to controls using only water (Mello et al., 2005). Aspergillus and Penicillium Putative mycotoxicogenic fungi of wheat grains were partially or completely sensitive to different essential oils extracted from 12 medicinal plants (Soliman and Badeaa, 2002). They were tested for inhibitory activity against A. ﬂavus, A. parasiticus and 60 G. Dal Bello and M. Sisterna A. ochraceus. Results indicated that oils of thyme, cinnamon (< or = 500 ppm), marigold (< or = 2000 ppm), spearmint and basil (3000 ppm) inhibited all the tested fungi completely. Caraway was inhibitory at 2000 ppm against A. ﬂavus and A. parasiticus and at 3000 ppm against A. ochraceaus. Also, the three species were suppressed by anise at < or = 500 ppm. An in vitro initial screening of a range of several spice hydrosols on inhibition of mycelial growth of A. parasiticus revealed that hydrosols of anise, cumin (Cuminum cyminum L.), fennel (Foeniculum vulgare Mill.), Mentha sp., oregano, savoury and thyme caused a stronger inhibitory effect on mycelial growth (Özcan, 2005). When essential oil from oregano was applied as a fumigant against the mycelia and spores of A. ﬂavus, A. niger and A. ochraceus on wheat, the oil vapour exhibited a fungicidal effect and a signiﬁcant reduction in the per cent of infested grain was observed (Paster et al., 1995). Plant extracts of Z. ofﬁcinale, bulbs of A. sativum and A. cepa, leaves of A. vasica, L. alba, A. indica, A. aspera, stem of C. reﬂexa, root of V. rosea and seeds of N. sativa were tested for their efﬁcacy in vitro against Aspergillus sp. and Penicillium sp. in wheat. All the plant extracts reduced the incidence of seedborne fungi signiﬁcantly and increased seed germination, the number of healthy seedlings and the vigour index. Neem and garlic extracts controlled the intensity of the fungi completely (Hasan et al., 2005). A natural fungicide against aﬂatoxigenic fungi to protect stored rice using the essential oil of C. citratus was developed by Paranagama et al. (2003). Lemongrass oil was tested against A. ﬂavus and the test oil was fungistatic and fungicidal against the test pathogen at 0.6 and 1.0 mg/ml, respectively. Aﬂatoxin production was completely inhibited at 0.1 mg/ml. The results obtained from the thin layer chromatographic bioassay and gas chromatography indicated citral a and b as the fungicidal constituents in lemongrass oil. During the fumigant toxicity assay of lemongrass oil, the sporulation and the mycelial growth of the test pathogen were inhibited at concentrations of 2.80 and 3.46 mg/ml, respectively. Therefore, lemongrass oil could be used to manage aﬂatoxin formation and fungal growth of A. ﬂavus in stored rice. Besides, the essential oil of lemongrass inhibited growth of moulds like A. ﬂavus, A. fumigatus and P. chrysogenum of maize and cowpea grains. Within a storage period of 10 days, seeds of maize and cowpea treated with lemongrass powder and essential oil showed no physical deterioration. Off-colour, off-odour and mouldiness, however, characterized untreated control seeds (Adegoke and Odelusola, 1996). Another assay on A. ﬂavus determined optimal levels of dosages of 11 plant essential oils for maize kernel protection, effects of combinations and residual effects (MontesBelmont and Carvajal, 1998). Bankole (1997) showed that essential oils from A. indica and Morinda lucida Benth. (brimstone tree) inhibited the growth of a toxigenic A. ﬂavus and reduced aﬂatoxin B1 synthesis signiﬁcantly in inoculated maize grains. Studies in experimental grain bins have demonstrated that soybean oil alone also reduces infection by storage fungi (White and Toman, 1994). After 12 months, kernel infection by Penicillium spp. and Aspergillus spp. was 83% and 63.7%, respectively, in untreated corn, compared to 60% and 46.2%, in soybean oil-treated corn at 200 ppm (McGee, 1989). Essential oils from aromatic plants such as cinnamon, clove, oregano, savoury and thyme inhibited the growth of the corn pathogen Penicillium sp. completely in vitro. The MIC of the essential oils in the laboratory was 800 ppm. The growing seedlings were not affected and no phytotoxicity symptoms were seen at rates up to 16,000 ppm concentration of the oils (Goggi et al., 2008). A previous work was undertaken by Chatterjee (1990) to screen some essential oils for their inhibitory activity against fungal infection and mycelial growth in postharvest maize grains during storage. It was observed that the oils of Cassia sp. clove (30 ml/g grain and above), star anise (Illicium verum Hooker ﬁl.) (40 ml/g grain and above), Geranium sp. (30 ml/g grain and above) and basil (50 ml/g grain) inhibited the in vivo mycelial growth of established seedborne infections of A. ﬂavus, as well as Use of Plant Extracts as Natural Fungicides preventing infection following inoculation with A. ﬂavus, A. glaucus, A. niger and A. sydowi. These oils also preserved the grain from natural A. ﬂavus infection during the experimental period. Christian and Goggi (2008) studied whether essential oils could be used as a contact fungicide seed treatment for organic corn. In vitro, the essential oils of cinnamon, clove, oregano, savoury and thyme controlled Penicillium completely. Soybean oil, applied at a rate used to suppress grain dust, reduced storage fungi growth in maize and soybeans in ﬁeld storage bins. After 12 months, soybean seed infection by Penicillium spp. and Aspergillus spp. was 45.7% and 39.2%, respectively, in untreated seeds, 17.7% and 8.2% in soybean oil-treated seeds and 1.7% and 2% in soybean oil + thiabendazole-treated seeds (McGee, 1989; White and Toman, 1994). Also, soybean oil demonstrated its effectiveness in decreasing by 50% the levels of seed infection and physiological ageing by the storage fungus, A. ruber, on garden pea seeds (Pisum sativum L.) (Hall and Harman, 1991). Peppermint, thyme and clove oils were tested in vivo against A. ﬂavus, A. niger and P. chrysogenum on different seed cultivars of cowpea. Antifungal activity was observed for the three oils, depending on cultivar and concentrations (Kritzinger et al., 2002). In blackgram, essential oils extracted from wood chips of cedar and that from the seeds of ajowan exhibited absolute toxicity, inhibiting the mycelial growth of A. niger on storage seeds (Singh and Tripathi, 1999). Major seedborne fungi associated with African yam bean like A. niger and A. ﬂavus could be controlled by using leaf extracts (crude and aqueous) of basil, bitter leaf, neem and pawpaw. All the plants’ leaf extracts reduced signiﬁcantly the incidence of fungi tested and increased seed germination and seedling emergence when compared with the untreated controls. The crude extracts were most effective, mainly neem, which gave complete control of A. niger and A. ﬂavus. In addition, seed germination was enhanced by this extract and reached nearly 90% (Nwachukwu and Umechuruba, 2001). A. ﬂavus is also found infesting seeds of guar, Cyamopsis tetragonoloba (L.) Taub., a 61 native plant of India, whose main commercial value is due to its seed gum (galactomannan gum). In this case, A. ﬂavus was reduced by cumin oil extracted from seeds (Dwivedi et al., 1991). Studies carried out have shown that cumin has powerful antimicrobial properties against diverse species of bacteria and fungi. The chemical studies indicated that the greater part of this antimicrobial activity might be attributed to the cuminaldehyde [p-isopropil benzaldehyde] that is present in the dried fruit of this plant (De et al., 2003). Another study (de Souza et al., 2003) investigated the mycoﬂora and physiological quality of cotton seeds treated with chemical fungicides and aroeira extract. Pure extract did not control the fungal population but, mixed with the fungicides, captan, benomyl and tolylﬂuanid, it showed reduction in the incidence of Aspergillus sp. Garlic extract was also found to be effective in removal of the seedborne pathogens of mustard, including species of Aspergillus and Penicillium (Latif et al., 2006). Fungi of the genera Aspergillus and Penicillium are widely distributed storage fungi of egusi melon seeds, causing seed discoloration, decreased nutritive value, increase in free fatty acid and peroxide values, decreased seed germination and producing a number of toxic metabolites, including aﬂatoxin. Four mould species, A. ﬂavus, A. niger, A. tamarii and P. citrinum, were inoculated on to shelled melon seeds. The essential oil of C. citratus at 0.1 and 0.25 ml/100 g seeds reduced deterioration and aﬂatoxin production signiﬁcantly in shelled seeds inoculated with A. ﬂavus. At higher dosages (0.5 and 1.0 ml/100 g seeds), the essential oil prevented aﬂatoxin production completely. After 6 months in farmers’ stores, unshelled melon seeds treated with 0.5 ml/100 g seeds of essential oil had a signiﬁcantly lower proportion of visibly diseased seeds and Aspergillus spp. infestation levels and signiﬁcantly higher seed germination compared to the untreated seeds. The efﬁcacy of the essential oil in preserving the quality of melon seeds in stores was statistically on a par with that of fungicide (iprodione) treatment (Bankole et al., 2005). 62 G. Dal Bello and M. Sisterna Conclusions While modern agricultural practices have resulted in higher and more stable yields, they have also weakened the natural balance between pests and their antagonists and have reduced soil fertility and health. Harmful chemicals threaten both the environment and human health alike. The beneﬁts of pesticides, in terms of reduced crop losses, are often overestimated because the viability of alternative pest management approaches is not fully understood. Conversely, the costs of relying predominantly on synthetic pesticides in pest control, in terms of health, environment, agroecology and trade, are also not known completely and consequently are often underestimated (SP-IPM, 2008). Integrated pest management (IPM) has emerged as a way towards maintaining or increasing agricultural productivity without over-reliance on synthetic chemical pesticides, emphasizes the growth of a healthy crop with the least possible disruption of agroecosystems and encourages natural pest control mechanisms (FAO, 2002). In this context, development of simple and eco-friendly seedborne disease management methods is necessary to improve the quality of seed in general and farmers’ saved-seed in particular (Elisabeth et al., 2008). Plant-derived compounds as crop protectants represent a vast and rapidly progressing resource. Botanical fungicides are best suited for use in industrialized countries when strict enforcement of pesticide regulations is impractical, or in the case of organic production. However, they can play a much greater role in protecting crops in developing countries, where human pesticide poisonings are most prevalent. Among the plant products, essential oils especially are a very attractive method of controlling plant diseases. Essential oils and their components are gaining increasing interest because of their relatively safe status, their wide acceptance by consumers and their exploitation for potential multi-purpose use. Besides, the problem of developing resistant strains of fungi may be solved by the use of essential oils of higher plants as fumigants in the management of fungal pathogens because of synergism between different components of the oils (Varma and Dubey, 1999; Dubey et al., 2008). In recent years, tremendous strides have been made in advancing the study of the natural control of plant pathogens, particularly seedborne fungi. As is shown in this chapter, plant metabolites and plant-based fungicides appear to be one of the better alternatives, as they are known to have minimal environmental impact and danger to consumers in contrast to synthetic pesticides. Despite the potential of these naturally occurring biochemicals as biorational fungicides, their practical development and implementation will require more detailed studies. Efforts should be made to search for indigenous plants as a source of antifungal compounds and to bioprospect the antifungal properties of these plant products, especially essential oils, towards seed fungi. Field trials are required to assess the practical applicability of botanical pesticides, together with bulk production, extensive usage of active compounds and interaction with other IPM components. Biosafety studies should be conducted to ascertain their toxicity to humans, animals and crop plants. Additional screenings might be focused on the quality assurance of botanicals and its regulation. While it is unlikely that biopesticides will replace chemical pesticides completely in the foreseeable future, we can expect that there will be some decline in the use of chemicals, particularly in developed countries. Exploitation of naturally available chemicals from plants, which retard the reproduction of undesirable microorganisms, would be a more realistic and ecologically sound method for plant protection and will have a prominent role in the development of future commercial pesticides for crop protection strategies, with special reference to the management of plant diseases (Varma and Dubey, 1999; Gottlieb et al., 2002). The prospect of botanical products as fungicides includes plant compounds with broad-spectrum activity to provide protection against a range of pathogenic fungi that attack the plant at the same or subsequent growth stages following their application. Furthermore, essential oils are made up of many components that may have synergistic effects; it may therefore be Use of Plant Extracts as Natural Fungicides expected that blends of essential oils or oil components will be produced to control a wide range of fungal species (Szczerbanik et al., 2007). 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Introduction Bread wheat (Triticum aestivum L.) is the most widely grown and consumed food crop in the world. It is the staple food of nearly 35% of the world population and the demand for wheat will grow faster than for any other major crop (Rajaram, 1999). The forecast global demand for wheat in the year 2020 varies between 840 (Rosegrant et al., 1995) to 1050 Mt (Kronstad, 1998). To meet this demand, global production will need to increase by 1.6–2.6% annually from the present production level of 620 Mt. Wheat breeding is focused on developing widely adapted, disease-resistant genotypes with high yields that are stable across a wide range of environments. Incorporating durable resistance is a priority since breeding for stable yields without adequate resistance against the major diseases would be impossible (Rajaram, 1999). Diseases of wheat, mostly caused by fungal pathogens and a few by viruses and bacteria, are important production constraints in almost all wheat-growing environments (Rajaram and van Ginkel, 1996; McIntosh, 1998). Globally important fungal diseases of wheat caused by obligate parasites include the three rusts (leaf rust, caused by Puccinia triticina Eriks., yellow rust caused by P. striiformis West f. sp. tritici Eriks. and stem rust caused by P. graminis Pers. f. sp. tritici Eriks & Henn); powdery mildew caused by Blumeria graminis tritici (DC) Speer; asexual form Oidium monilioides (Nees) Link; stinking smut (Tilletia caries (DC) Tul. and C. Tul. and T. foetida (Wallr. Liro.); loose smut (Ustilago tritici (Pers.) Rostr.); U. nuda (J.L. Jensen) Kellerm. and Swingle. Those caused by facultative parasites include leaf blotch, M. graminicola (Fuckel) J. Schröt., in Cohn, asexual form S. tritici Rob ex Desm.; glume blotch (Phaeosphaeria nodorum, asexual form Stagonospora nodorum blotch); spot blotch (Cochliobolus sativus (Ito and Kuribashani) Drechs. ex Dastus, asexual form Bipolaris sorokiniana (Sacc.) Shoem.); tan spot,  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 69 70 M.R. Simón Pyrenophora tritici-repentis (Died.) Drechs., asexual form Drechslera tritici-repentis (Died.) Shoemaker; Alternaria spp. (belonging to the A. infectoria species groups); scab, Fusarium graminearum Schwabe, take all (Gaeumannomyces graminis (Sacc.) von Arx and Olivier var. tritici Walker). Leaf blotch causes important yield losses in many countries. Yield reductions range from 31 to 54% (Eyal et al., 1987), from 10 to 45% (Caldwell and Narvaez, 1960) and even yield losses higher than 60% have been reported (Shipton et al., 1971). Sanderson (1972) proved the connection between the two stages and the sexual (teleomorph) form has been reported in several countries (Hunter et al., 1999). The sexual stage in Argentina was reported by Cordo et al. (1990). Mycosphaerella graminicola is a hemibiotrophic pathogen; early infection is biotrophic, followed by a switch to necrophic growth just prior to symptom expression. The sexual stage is also known to play a role in the disease cycle. It causes most of the initial infection of winter wheat crops during autumn in the UK (Shaw and Royle, 1989) and the USA (Schuh, 1990). In Argentina, an increase in ascospores at harvest time has been reported, suggesting that the sexual stage may be important to initiate the infection in the next growing season. Following stem elongation, infection of the upper leaves of a crop has been thought to be entirely due to the asexual stage of the fungus, in which pycnidia give rise to splash-dispersed pycnidiospores, which are splash-dispersed from infected basal tissue to the upper leaves by raindrops. However, more recent work has shown that upward movement of inoculum can occur in the absence of splashy rainfall, being inﬂuenced by the position of developing leaves in relation to infected leaf layers (Lovell et al., 1997). Another possible means of spread within a crop during summer is by airborne ascospores, which may play a role more important than previously recognized (Hunter et al., 1999). Types of Resistance Although several control methods, including cultural practices and the use of fungicides, may reduce the effect of S. tritici blotch, genetic resistance is the most cost-effective and environmentally safe technique to manage the disease. Monogenic or oligogenic and polygenic resistance coexist in the pathosystem T. aestivum/M. graminicola. Monogenic or oligogenic resistance is generally near complete, isolate speciﬁc, follows the ‘gene-for-gene’ mode of inheritance and has been found in several genotypes (Rillo and Caldwell, 1966; Rosielle and Brown, 1979; Lee and Gough, 1984; Somasco et al., 1996; Arraiano et al., 2001; Brading et al., 2002; McCartney et al., 2002). Polygenic resistance is generally partial and isolate non-speciﬁc and is also present in several genotypes (Jlibene et al., 1994; Simón and Cordo, 1997, 1998; Brown et al., 2001; Zhang et al., 2001; Chartrain et al., 2004b). Partial resistance is expressed as a reduced epidemic development and is supposed to be durable. Several components contribute to the epidemic-retarding effect. Parlevliet (1979) mentioned four partial resistance components: infection frequency, latent period, spore production and infection period. The earliest studies on this type of resistance, previous to the mapping of genes and QTLs, investigated the gene effects conditioning these components. Several of the components of partial resistance to M. graminicola may be controlled by just a few genes (Jlibene et al., 1994). Danon and Eyal (1990) determined that additive effects for pycnidial coverage were the major variance component, although dominance effects were also signiﬁcant. Jlibene et al. (1994) found that general combining ability (GCA) effects accounted for most of the variation of percentage pycnidial coverage, although speciﬁc combining ability (SCA) effects were detected in some crosses. Simón and Cordo (1997, 1998) determined that GCA was preponderant for incubation period, latent period, pycnidial coverage and spore production, although SCA was also signiﬁcant. Incubation period was inherited independently of maturation period and pycnidial coverage. Those components that are genetically different and independent could be combined into the same genetic background by crossing (van Ginkel and Rajaram, Resistance to Septoria Leaf Blotch 1999), increasing the level of durable resistance. Signiﬁcant correlations were found between pycnidia/cm2 and spore/ml, indicating the feasibility of selecting for a lower pycnidial density in order to obtain a reduction in spore production (Simón and Cordo, 1998). Heritability tends to be only moderate (Simón et al., 1998), but progress in breeding for resistance may still be possible. Major genes are interesting because of the high level of resistance and thus an almost complete absence of symptoms in the host; partial resistance, however, is very important due to its putative durability and its expression under a broad spectrum of isolates of the pathogen. A few genes may be enough to confer resistance that will hold up in farmers’ ﬁelds (Dubin and Rajaram, 1996). Resistance conditioned by a single dominant gene was assigned to some cultivars as Bulgaria 88 (Rillo and Caldwell, 1966), Oasis (Shaner and Buechley, 1989), Veranopolis (Wilson, 1979) and others. Later, genes were located and it was found for example that Stb1 conditioned resistance in Bulgaria 88 and Oasis, Stb2 in Veranopolis, etc. Some other cultivars showed resistance conditioned by several major genes as Kavkaz 4500 L.6.A.4. (Jlibene et al., 1992) and the genes were identiﬁed (Stb6, Stb7, Stb10 and Stb12; Chartrain et al., 2005a). Also, three major genes were identiﬁed in the Portuguese line TE 9111 (Stb6, Stb7 and Stb11; Chartrain et al., 2005b). Furthermore, commercially grown cultivars range from moderately resistant to susceptible, indicating the presence of partial resistance. Chartrain et al. (2004b) found high partial resistance levels in several wheat cultivars from Europe and Mexico (Arina, Milan, Senat). Simón et al. (2005a) also found high levels of partial resistance in some Argentinian cultivars effective to several isolates (Klein Volcán, Klein Dragón) in adult stage. Some germplasm as the Portuguese line TE 9111 (Chartrain et al., 2005b) also has been proved to carry several major genes together with partial resistance. 71 investigated the chromosomal location of resistance using substitution lines. Resistance was found to be located on chromosome 7D from a synthetic hexaploid wheat (T. dicoccoides × T. tauschii) in seedling and adult stage to some speciﬁc isolates (Simón et al., 2001, 2005b). Also, resistance was found in chromosomes 1B at the seedling stage and on 5D at the seedling and adult stage of the T. aestivum cv. Cheyenne (Simón et al., 2001, 2005b); on the 2B, 3A and 3B of the T. aestivum cv. CappelleDesprez at the seedling stage and on 6D and 7D of T. spelta with some speciﬁc isolates (Simón et al., 2001, 2005b). During the past decade, several genes (Table 6.1) and QTLs (Table 6.2) have been located. Some of them have proved to be effective to isolates from several regions in the world. Simón et al. (2007) tagged, using isolates from Argentina, a gene in the 7D chromosome of Aegilops tauschii, which is likely Stb5. This would indicate that the presence of Stb5 ensures resistance against some isolates from both Europe (Portugal, The Netherlands) (Arraiano et al., 2001) and South America (Argentina). Breeding for Resistance The incorporation of resistance to the pathogen has been slow for several reasons, among them: 1. The high variability of the pathogen population. 2. The lack of knowledge of the virulence spectrum. 3. The lack of relationship in the expression of resistance in seedling and adult stage. 4. The inﬂuence of heading date and plant height on resistance and the difﬁculty in assessing real values in breeding programmes. Variability of the pathogen population Location of the Resistance Studies on the location of resistance began during the past decade. Some of them The population of the pathogen has been studied and a high variability has been found. Variation in virulence patterns within 72 M.R. Simón Table 6.1. Major genes conditioning resistance to Mycosphaerella graminicola identiﬁed in hexaploid wheat. Locus Chromosomal location Stb1 Stb2 5BL 3BS Stb3 Stb4 Stb5 Stb6 Stb7 Stb8 Stb9 Stb10 Stb11 Stb12 Stb13 Stb14 Stb15 6DS 7DS 7DS 3AS 4AL 7BL 2B 1D 1BS 4AL 7BL 3BS 6AS Linked markers Reference Xbarc74, Xgwm335 Xgwn389, Xgwm533.1, Xbarc133, Xbarc75, Xgwm493 Xgdm132 Xgwm44, RC3, Xgwm11, Xgwm437, Xgwm121 Xgwm44, RC3, Xgwm111, Xgwm437, Xgwm121 Xgwm369, Xwmc11 Xgwm160, Xwmc219, Xwmc313 Xgwm146, Xgwm577, Xgwm611 Adhikari et al., 2004c Adhikari et al., 2004b Xgwm848 Xbarc008, Xbarc137 Xwmc219, Xwmc313 Xwmc396-7B Xwm632-3B Xpsr563a, Xpsr904 Adhikari et al., 2004b Adhikari et al., 2004a Arraiano et al., 2001 Brading et al., 2002 McCartney et al., 2002 Adhikari et al., 2003 Chartrain, 2004 Chartrain et al., 2005a Chartrain et al., 2005b Chartrain et al., 2005a Cowling et al., 2007 Brule Babel, 2007 Arraiano et al., 2007 Table 6.2. Quantitative trait loci (QTLs) conditioning resistance to Mycosphaerella graminicola in hexaploid wheat. Locus Chromosomal location Linked marker Reference QStb.risø-2B QStb.risø-3A.1 QStb.risø-3A.2 QStb.risø-3B QStb.risø-6B.2 QtStb.risø-7B QStb.ipk-1D QStb.ipk-2D QStb.ipk-6B QStb.ipk-3D QStb.ipk-7B 2BL 3AS 3BL 3B 6B 7B 1D (seedlings) 2D (seedlings) 6B (seedlings) 3D (adult) 7B (adult) Xwmc1575a-Xwmc175a Xgwm369 Xwmc489-Xwmc505 M62/P38-373 Xwmc397-Xwmc341 M49/P38-229-M49/P11-229 Xmwg938a Xcdo405a Xksuh4b Xbcd515 Xksud2a Eriksen et al., 2003 Eriksen et al., 2003 Eriksen et al., 2003 Eriksen et al., 2003 Eriksen et al., 2003 Eriksen et al., 2003 Simón et al., 2004a Simón et al., 2004a Simón et al., 2004a Simón et al., 2004a Simón et al., 2004a and between populations was shown by assessing host response on a selected set of cultivars, with little similarity between the results obtained with various sets of differentials (Eyal et al., 1995). Evidence for speciﬁcity was also conﬁrmed by several researchers (Danon and Eyal, 1990; Kema and van Silfhout, 1997; Simón et al., 2005a). Non-speciﬁc resistance to a wide set of isolates was also found (Simón et al., 2005a). During the past decades, the population has been studied using molecular markers and a high variability within populations has been conﬁrmed (Chen and McDonald, 1996; Zhan et al., 2001, 2003; Cordo et al., 2007). The sexual state might have an impact on the virulence spectrum in regions where pseudothecia were found and ascospore dispersal coincided with the wheat growing cycle (Shaw and Royle, 1989; Lovell et al., 1997). No attempts to determine races have been carried out. Recently, the genome of the pathogen was sequenced completely (Goodwin et al., Resistance to Septoria Leaf Blotch 2007). The essentially ﬁnished sequence contains 18 chromosomes from telomere to telomere, plus ﬁve fragments, which presumably make up two additional chromosomes. A comparative bioinformatics analysis of M. graminicola with seven other sequenced fungal genomes revealed that it possessed fewer enzymes than expected for degrading plant cell walls. The frequency of transposable elements in the genome of the pathogen was intermediate between those of other sequenced fungi. Availability of the ﬁnished genome for M. graminicola should aid research on this organism greatly and will help in the understanding of its interaction with wheat. Expression of resistance in seedlings and adults Resistance is sometimes expressed in seedlings, sometimes at adult stage and sometimes at both stages (Kema and van Silfhout, 1997). Some germplasm with resistance at both stages have been found (Arama, 1996; Somasco et al., 1996; Simón et al., 2005a). Inﬂuence of heading date and plant height on resistance One complicating factor for the assessment of resistance level has been the inﬂuence of heading date and plant height on the expression of resistance. Several scientists have reported an increased disease level in earlier heading or shorter cultivars (Eyal et al., 1987; van Beuningen and Kohli, 1990; Camacho Casas et al., 1995; Chartrain et al., 2004a). Baltazar et al. (1990) suggested a genetic association between shortness and susceptibility, while Eyal (1981) and Rosielle and Boyd (1985) assumed a genetic association between earliness and susceptibility. Arama et al. (1999), Simón et al. (2005a) and Arraiano et al. (2006) reported no genetic association among those traits. Simón et al. (2004b, 2005a) determined that there was no inﬂuence of heading date when cultivars were evaluated at the same development stage under similar weather conditions and found 73 that the relationship between those traits was caused mainly by environmental and epidemiological factors. Associations between pycnidial coverage percentage and days to heading were positive or negative, depending on whether weather conditions before the evaluations were more conducive to the development of the disease in late or early heading cultivars, respectively. Negative associations with plant height were only present in the years where weather conditions were less conducive to the development of the disease. Inconducive conditions and longer distances between leaves in tall cultivars could have reduced the rainsplash dispersal of pycnidiospores, thus causing this negative association, mainly when the sexual form is not present. In most cases, previous reported associations between heading date and resistance could be attributed to the fact that the disease was scored at the same time but not at the same growth stage, causing early maturing lines to be exposed to inoculum for a longer period than later maturing leaves. Simón et al. (2009 unpublished) mapped a population derived from T. spelta 7D/Chinese Spring where QTLs conditioning resistance were found, but no genes for heading date were present. Also, some QTLs for resistance were mapped in a Synthetic 6 × (T. tauschii × Altar 84) × Opata 85 (Simón et al., 2004a), which did not coincide with the regions where QTLs for ﬂowering time were previously mapped. Eriksen et al. (2003) located in a double haploid population originated from the cross of Savanah and Senat, a QTL for increasing plant height linked to a QTL for resistance. Although associations could exist in some germplasm, pleiotropic effects have not been detected and breeders can select for S. tritici blotch resistance within a range of heading dates and plant heights. Resistance and Integrated Management It is necessary to consider that integrated management can contribute to the durability of resistance. Epidemiological advantages can be derived by combining management 74 M.R. Simón practices and through disease management on a regional scale. Diversifying sources of partial resistance, on a ﬁeld or regional basis, might slow pathogen adaptation. Populations of M. graminicola sampled from mixtures of a susceptible and a partially resistant wheat cultivar were all less ﬁt than populations derived from the same cultivars grown in pure stand (Mundt et al., 2002). Cultural practices such as adequate tillage method, planting density and Nfertilization conditions, together with fungicide applications, are important to the appropriated expression of resistance. The planting of no-till wheat may increase the level of Septoria leaf blotch because increasing levels of crop residue on the soil surface potentially increase primary inoculum of plant pathogens, mainly under continuous wheat production or wheat/soybean sequences in the same year. Since the pathogen can survive in infested wheat residues for about 2 years, a rotation where wheat is planted in only 1 of 3 years is recommended. Although there are contrasting results, several reports indicate that, under conducive conditions for the development of the disease, an increase in N-fertilization causes a slight increase in severity (Hayden et al., 1994; Howard et al., 1994; Leitch and Jenkins, 1995; Simón et al., 2002, 2003). Conclusions Research on Septoria leaf blotch has expanded greatly in the past decades. New molecular tools enable the exploration of biological issues associated with the pathogen, the host and the host–pathogen interaction. Several genes and QTLs have been identiﬁed and mapped. The major challenge to wheat breeders and plant pathologists is the selection and development of cultivars with durable resistance. To achieve this goal, the incorporation of marker-assisted selection into breeding programmes will speed pyramiding several genes or QTLs effective at different stages of plant development into single wheat cultivars to develop broad-spectrum and durable resistance. Management of cultivars should be optimized to minimize the associations between heading date, height and resistance, but selection of early and short lines with high levels of quantitative resistance is possible. Progress in the analysis of variability and virulence patterns of the pathogen population is also necessary to test the available germplasm with representative isolates. Durability of the resistance can be enhanced by appropriate cultural practices and diversifying sources of resistance. References Adhikari, T.B., Anderson, J.M. and Goodwin, S.B. (2003) Identiﬁcation and molecular mapping of a gene in wheat conferring resistance to Mycosphaerella graminicola. Phytopathology 93, 1158–1164. 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Rogers Laboratorio de Biología Funcional y Biotecnología (BIOLAB)-CEBB, Facultad de Agronomía, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNICEN), Buenos Aires, Argentina and Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET), Argentina Abstract The genetic control of resistance to Fusarium head blight (FHB) in barley and wheat is reviewed. This disease, which can reach epidemic proportions under certain climatic conditions, is caused by various Fusarium species and affects grain yield and quality detrimentally, resulting in important economic losses in both crops. Furthermore, FHB infection poses a serious threat to human and animal health, due to the presence of toxic trichothecenes, of which deoxynivalenol and its derivatives appear to be the most important. Marker-based mapping studies have identiﬁed numerous quantitative trait loci (QTLs) for FHB resistance, located on all the chromosomes of both species. Only a relatively small number of these can be detected consistently over a wide range of different environments and genetic backgrounds. None the less, where genetic effects have been characterized, they have been shown to be mainly additive in nature, meaning that the accumulation of several QTL factors in a single line ought to be effective in achieving raised levels of resistance. Indeed, marker-assisted selection has been directly shown to be feasible for some QTL. A number of QTLs for FHB resistance are associated with other agronomic characters, such as heading date (HD), ﬂowering time and plant height. In some cases, QTL alleles favourable for resistance are associated detrimentally with alleles for these characters, although there appear to be sufﬁciently large numbers of QTLs for resistance acting independently of these characters to imply that reasonable genetic gains for resistance ought to be achievable in the future. While most studies in barley have addressed Type I resistance (initial infection) and in wheat Type II (spread between spikelets), or a combination of both Type I and Type II, more recent studies have addressed other types of resistance, such as Type III (effects on kernel size and characteristics), Type IV (yield tolerance) and Type V (decomposition or non-accumulation of mycotoxins such as deoxynivalenol). Besides identifying additional QTLs, these latter studies offer insights into the mechanisms of the different types of resistance observed, in some cases blurring the distinctions between them. Other prospects for improvement in FHB resistance, additional to those offered by marker-assisted selection, are also discussed. Introduction Fusarium head blight (FHB) or scab is a destructive disease of wheat and barley in 78 environments with prolonged wet climatic conditions from ﬂowering through the softdough stage of kernel development (Parry et al., 1995; McMullen et al., 1997). The  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Barley and Wheat Resistance Genes disease is of worldwide importance. FHB epidemics have been documented in 26 US states and ﬁve Canadian provinces. Economic losses in wheat since 1990 were estimated at US$2.5bn (Windels, 2000). Wheat yields in 1993 were reduced by about 50% in north-eastern North Dakota and 40% in north-western Minnesota, compared with 1992 (National Agricultural Statistics Service, 1993–1999). Barley losses have been equally devastating, with estimated losses from 1993 to 1999 totalling in excess of US$400m (Windels, 2000). In China, FHB has affected more than 7m ha wheat and has caused yield losses of more than 1 Mt in severe epidemics (Leonard and Bushnell, 2003). In Argentina, during the past 60 years, several FHB epidemics of varying severity have occurred in the central-north area, where yield losses were estimated to average between 20 and 50%. FHB is a preharvest disease, but Fusarium species can grow in postharvest phase if wet grain is not dried efﬁciently and quickly. More than 17 Fusarium species Fig. 7.1. 79 have been isolated from naturally infected wheat or barley spikes and have been associated with FHB (Parry et al., 1995; Leonard and Bushnell, 2003). Fusarium graminearum (teleomorph Gibberella zeae) is the most frequently encountered pathogen and the most virulent species, although F. avenaceum (teleomorph G. avenacea), F. culmorum and F. poae are reported to be prevalent in some European and North and South American countries (Leonard and Bushnell, 2003; Barreto et al., 2004; Bourdages et al., 2006). The distribution and predominance of a Fusarium species in a region is thought to be determined by climatic factors, competition among various Fusarium spp. sharing the same ecological niches, fertilizer use, cropping sequence and practices and vegetation type (Snyder and Nash, 1968; Nelson et al., 1981; Doohan et al., 2003). FHB reduces kernel set and kernel weight. Invasion of the kernel by Fusarium destroys the starch granules and cell walls and affects endosperm storage proteins, resulting in a poor quality product (Fig. 7.1). Shrivelled lightweight seeds of wheat affected by FHB (left) and healthy wheat seeds (right). 80 S.A. Stenglein and W.J. Rogers Germination rate and seedling vigour are reduced when the seeds are infected. In addition to causing signiﬁcant yield losses, FHB is of greater signiﬁcance under certain conditions because of the associated mycotoxin accumulation which can occur in infected grain. Fusarium graminearum, F. avenaceum, F. culmorum and F. poae can produce a range of mycotoxins and contaminated grain is unsuitable for animal and human consumption because of the adverse effects of such toxins on health (Placinta et al., 1999; Gutleb et al., 2002). Within Fusarium mycotoxins, some of the most important from the point of view of animal health and productivity, are the trichothecenes, zearalenone and the fumonisins (D’Mello et al., 1999). Type A and B trichothecenes represent the most important members of these mycotoxins. Type A trichothecenes include T-2 toxin, HT-2 toxin, neosolaniol (NEO) and diacetoxyscirpenol (DAS), while type B trichothecenes include deoxynivalenol (DON, also known as vomitoxin) and its 3-acetyl and 15-acetyl derivates (3-DON and 15-DON, respectively), nivalenol (NIV) and fusarenon-X (FUS-X). A common feature of many Fusarium species is their ability to synthesize zearalenone (ZEN or F-2 toxin) and its co-occurrence with certain trichothecenes raises important issues regarding additivity and/or synergism in the aetiology of mycotoxicosis in animals (Placinta et al., 1999). Fumonisins are an increasingly important group of toxins as they have been postulated as the causative agent for several endemic diseases, both in humans and animals (Sydenham et al., 1990; Chu and Li, 1994). Host resistance has long been considered the most practical and effective means of disease handling, but breeding for FHB resistance has been hindered by a lack of effective resistance genes and by the complexity of the resistance in identiﬁed sources (Mesterhazy, 1997). No source of complete resistance is known and current sources provide only partial resistance. Resistance types are generally classiﬁed as either morphological or physiological. Head anatomy or positioning that contributes to higher humidity around the spikelets is often associated with more diseases. Generally, awned genotypes with short peduncule and a compact spike have faster disease spread than genotypes that are awnless, have a long peduncule and a lax spike (Rudd et al., 2001). In addition, short saturated genotypes with a long grain-ﬁlling duration generally get more disease than tall genotypes that have rapid grain ﬁll (Mesterhazy, 1995). These morphological characteristics contribute to resistance, but are often considered nuisance factors in screening nurseries, and it is generally agreed that they are of minor signiﬁcance compared with physiological resistance (Rudd et al., 2001). However, morphological traits have also been associated with FHB resistance in barley. Two-rowed barley is more resistant to FHB than six-rowed barley and, in crosses between six-rowed and two-rowed genotypes, tworowed progenies are most resistant, followed by genotypes heterozygous for spike type. Six-rowed types are most susceptible (Takeda and Heta, 1989; Xihang et al., 1991). Mesterhazy (1995) described ﬁve types of physiological resistance, expanding the two types described by Schroeder and Christensen (1963). These include Type I resistance to initial infection. It may be passive, involving morphological characteristics of wheat head. Alternatively, Type I resistance may be active and include defence reactions such as the activation of enzymes degrading the fungal cell wall or pathogenesis-related (PR) proteins (Nicholson et al., 2005). This type of resistance is estimated by spraying a spore suspension over ﬂowering spikes and counting diseased spikelets. Type II refers to the resistance of movement of the pathogen from one infected spikelet to another via the rachis. The mechanisms involved in Type II resistance are thought to be active, but again may be due to morphological characteristics. This type of resistance is estimated by delivering conidia into a single ﬂoret of a spike and counting the blighted spikelets after a period of time. The other types of resistance include: kernel size and number retention (Type III), yield tolerance (Type IV) and decomposition or non-accumulation of mycotoxins (Type V). Type III resistance is measured by threshing infected spikes and Barley and Wheat Resistance Genes observing the damage to the kernels. Kernel number reduction, kernel weight, test weight, or visual estimates of Fusarium-damaged kernels (tombstones) are common measurements used to assess this resistance. Type IV resistance, or yield tolerance, can be assessed by measuring grain yield of naturally or artiﬁcially inoculated spikes or plots and comparing the data with spikes or plots that do not show disease symptoms (Rudd et al., 2001). Finally, Type V resistance is identiﬁed by measuring DON concentration at a given level of FHB (Rudd et al., 2001). This resistance is important from a grain utilization perspective, for example for malting barley, because even trace levels of DON may reduce beer quality signiﬁcantly. Considerable progress in the search for host resistance has been made. Improvement of cultivar resistance has become a major breeding objective worldwide. Recent developments in genomic research and biotechnology hold promise for understanding the genetic mechanisms of FHB resistance and allow more effective utilization of FHB resistance genes to develop new resistant wheat and barley cultivars. Genetics of FHB Resistance in Barley Few sources of FHB resistance have been found in barley and the level of their resistance is modest. Although FHB in barley usually does not spread from spikelet to spikelet within a spike (up and down the spike), barley seems to be very susceptible to initial infection. Severe disease usually results from multiple initial infections in the spike. Of primary importance to barley breeders are data on FHB severity and DON concentration, since these are traits that affect the marketing of grain in malting most severely. The ﬁrst sources of resistance used were the breeding lines Gobernadora from ICARDA/CIMMYT in Mexico and Zhedar 1 and Zhedar 2 from China. All three lines had the two-rowed spike morphology. Other tworowed barley with low DON content were CI 4196, Svanhals and Imperial. CI 4196 81 was identiﬁed as one of the most resistant two-rowed barley accessions and also accumulated low concentrations of DON (Urrea et al., 2005). Six-rowed types are preferred for malting, but they are generally more susceptible to FHB than two-rowed barley. Chevron, an old cultivar from Switzerland, is a six-rowed malting barley and a popular parent in barley breeding programmes. It has high resistance to kernel discoloration, which is a disease complex caused by several different fungal pathogens, including Fusarium. In China and Japan, over 10,000 barley accessions from different countries have been screened for FHB resistance, but only several dozen accessions have a low level of FHB (Xihang et al., 1991; Zhou et al., 1991). To date, no wild species of Hordeum have shown greater resistance than that of tworowed barley. DON content in even the best sources of resistance are still well above the speciﬁcation for the brewing industry (< 0.5 mg/kg), but much lower than that of current commercial malting barley cultivars (Leonard and Bushnell, 2003). Investigation of the genetics of resistance to FHB in barley has not been very extensive and published reports on the identiﬁcation of loci controlling FHB resistance and DON accumulation are limited (Rudd et al., 2001). Barley producers currently attempt to manage the disease through crop rotation and fungicide application. However, these measures alone are not sufﬁcient to reduce the risk of the disease. Resistant barley cultivars are the most cost-effective measures for controlling the disease, but breeding for FHB resistance has been difﬁcult for several reasons. One, genetic resistance is complex. There seem to be many QTLs that have relatively small effects and are subject to genotype × environment interactions. Two, FHB screening experiments are labour-intensive and expensive. Three, assessing FHB severity in both the ﬁeld and the greenhouse is difﬁcult. Disease severity is correlated strongly with HD and other agronomic and spike morphology traits. Since infection can occur only after the spike emerges from the boot, differences in HD make it difﬁcult to distinguish ‘true’ disease 82 S.A. Stenglein and W.J. Rogers resistance from ‘apparent’ resistance that is due to host escape from the pathogen. Both of these problems necessitate the identiﬁcation of molecular markers linked to QTLs for FHB resistance that can be used in markerassisted breeding. In addition, since disease expression is inﬂuenced strongly by the environment, comparisons among barley genotypes that differ in HD are themselves confounded by the effect of the environment on disease development. However, because of the complex nature of genetic resistance to FHB, QTL identiﬁcation is not always very robust. Therefore, validation of these QTLs is important before implementing markerassisted selection in a breeding programme. To gain a genetic understanding of FHB resistance in barley, multiple sources of resistance including Chevron (de la Pena et al., 1999; Ma et al., 2000), Gobernadora (Zhu et al., 1999), Fredrickson (Mesﬁn et al., 2003; Smith et al., 2004), Zhedar 2 (Dahleen et al., 2003) and CI 4196 (Horsley et al., 2006) have been used in QTL mapping studies. QTLs providing resistance to FHB and DON accumulation in barley have been identiﬁed on all seven chromosomes. QTLs for FHB resistance were identiﬁed on chromosomes 1(7H), 2(2H), 3(3H), 4(4H), 5(1H) and 7(5H) in the Chevron (resistant)/M69 (susceptible) population (de la Pena et al., 1999). A major QTL on chromosome 2(2H) explains 13.5% of the phenotypic variation for FHB resistance. However, this QTL is also associated with HD and the resistant allele is linked to late heading. Ma et al. (2000) used a population derived from the cross Chevron/ Stander and reported nine QTLs for FHB resistance located on chromosomes 1(7H), 2(2H), 3(3H), 6(6H) and 7(5H). A QTL on chromosome 2(2H) was detected consistently in ﬁve environments and explained 11.8– 20.7% of the phenotypic variation for FHB resistance. This QTL, in addition to the QTL on chromosome 2(2H) discovered by de la Pena et al. (1999), is also associated with days to heading. Using a population derived from the two-rowed parents, Gobernadora and CMB 643, Zhu et al. (1999) found QTLs for FHB resistance on all barley chromosomes except chromosome 7(5H). The largest QTL explained 33% of the phenotypic variation and was found on chromosome 2(2H). The QTL on chromosome 4(4H) explains 4–12% of the phenotypic variation for FHB resistance. This QTL was also associated signiﬁcantly with morphological traits including plant height, seeds per inﬂorescence, inﬂorescence density and lateral ﬂoret size. In each of the previous mapping studies, QTLs for accumulation of DON in harvested grain were also detected. These QTLs were also distributed throughout the genome and were, in some cases, coincident with FHB QTL. Taken together, these studies indicate resistance is conditioned by many loci and that there is a strong association between certain morphological traits and FHB resistance. Two major traits associated with FHB severity are spike type and HD. The Vrs1 and Int-c loci control lateral ﬂoret fertility and hence determine whether a spike is tworowed (Vrs1; int-c/int-c) (Lundqvist and Franckowiak, 1997) or six-rowed (vrs1/vrs1; Int-c/Int-c) (Hockett and Nilan, 1985). In several studies, the two-rowed spike type has been associated with FHB resistance (Chen et al., 1991; Xihang et al., 1991; Steffenson et al., 1996; de la Pena et al., 1999). In a genetic study, Takeda (1990) demonstrated an association between the Vrs1 locus and FHB resistance. In two-rowed barley (Vrs1) with the Int-c/Int-c genotype, the laterals can be inﬂated and lateral ﬂoret size has been associated with FHB severity (Zhu et al., 1999). The FHB mapping studies published to date have used populations derived from either six-rowed × six-rowed or two-rowed × tworowed crosses (de la Pena et al., 1999; Zhu et al., 1999; Ma et al., 2000). Therefore, the Vrs1 locus was not segregating in these populations. HD may also strongly inﬂuence the severity of FHB on barley and QTLs for HD and FHB resistance are coincident (de la Pena et al., 1999; Ma et al., 2000). Generally, late heading plants tend to have lower severity, while early heading plants have higher severity, indicating that the late heading plants are exposed to the inoculum for a shorter period of time (Leonard and Bushnell, 2003). In all of these studies except the one using Gobernadora, the bin 8 region of the long arm of chromosome 2H designated by Barley and Wheat Resistance Genes Horsley et al. (2006) as Qrgz-2H-8 was associated consistently with FHB severity, HD and DON concentration. The approximate size for the overlapping QTL region ranged from 22cM in the Fredrickson/Stander population (Mesﬁn et al., 2003) to 45cM in Chevron/M69 (de la Pena et al., 1999) and CI 4196/Foster (Horsley et al., 2006) populations. Depending on the population and the environment, Qrgz-2H-8 explained 7–60% of the variation in FHB resistance, 12–30% of the variation in HD and 10–30% of the variation in DON concentration. In all of the studies, FHB severity and DON concentration were correlated negatively with HD. In a validation study of this QTL, the Chevron introgression at the Qrgz-2H-8 region reduced FHB by 42% and increased HD by 3.8 days (Canci et al., 2004). The association between lower FHB severity and late heading may be due to shorter inoculum exposure (pleiotropy) or tight linkage of separate genes for ﬂowering time and disease resistance (Leonard and Bushnell, 2003). To determine if the association between late HD and FHB resistance is due to linkage or pleiotropy, Nduulu et al. (2007) constructed a ﬁne map for the chromosome 2(2H) QTL region using recombinant near isogenic lines (rNILs) derived from a cross between a BC5 line carrying the Chevron alleles for markers at the Qrgz-2H-8 region and the recurrent parent M69, and concluded that the relationship between FHB and HD at the Qrgz-2H-8 region was likely due to tight linkage rather then pleiotropy. Genetics of FHB Resistance in Wheat Besides similar considerations as for barley regarding the detrimental effects of FHB on grain yield and quality in general, and the effects of mycotoxins on human and livestock health, the fact that the disease results in the degradation of the endosperm storage proteins means speciﬁcally that the quality of bread, biscuit, pasta and other industrial products can be seriously prejudiced. Worldwide, the species F. graminearum predominates, but F. avenaceum, F. culmorum and 83 F. poae are also the cause of the disease in some environments. Epidemics may cause major losses when climatic conditions are favourable after ﬂowering (Paillard et al., 2004). As in barley, agricultural management and fungicide treatments, while reducing the damage (Gervais et al., 2003), are not wholly effective (Stack, 1989; Bai and Shaner, 1994; Parry et al., 1995). Unfortunately, complete FHB resistance is unknown, although longterm control of the disease is probably most likely to be achieved through genetic resistance research, involving QTL mapping and other procedures (see below), and its consequent application in the breeding of resistant cultivars. This appears to be the case, in spite of the complexity of the genetic control involved, the presence of confounding environmental effects, the inﬂuence of genotype × environment interaction and the fact that laborious inoculation and evaluation procedures in mature host plants are required in order to identify useful marker associations (Snidjers, 1990; van Ginkel et al., 1996; del Blanco et al., 2003). A further complication is that associations between FHB resistance with HD, ﬂowering time (FT) and plant height (PH) have also been observed (Mesterhazy, 1997; Hilton et al., 1999; Buerstmayr et al., 2000). For breeding purposes, three broad origins of resistant germplasm have been recognized (Gilbert and Tekauz, 2000; Paillard et al., 2004): (i) spring wheat from Asia (e.g. cv. Ning 7840 [China], cv. Sumai 3 [China], cv. Nobeokabozu [Japan]); (ii) spring wheat from South America (e.g. cv. Frontana [Brazil]); and (iii) winter wheat from Europe (e.g. Arina, Praag-8, Novokrumka). Further examples of individual resistant cultivars are given in the studies described below, which are all concerned with bread wheat, unless speciﬁed otherwise. In contrast to barley, FHB generally spreads between spikelets (although it is currently unclear whether this is so for F. poae) and most genetic research has therefore concentrated on Type II resistance (most frequently evaluated after single-spikelet inoculation with F. graminearum), although combined evaluation of Type I and Type II resistance through spray inoculation has 84 S.A. Stenglein and W.J. Rogers also been widely carried out. However, there are an increasing number of studies that address other types of resistance, such as the ability to detoxify DON (Type V) and the ability to maintain grain yield in spite of disease symptoms (Type IV). The ﬁrst QTL mapping studies were carried out in the mid-1990s (Bai, 1995; Moreno-Sevilla et al., 1997), involving the use of RFLP and RAPD markers to map Type II resistance. However, the marker associations identiﬁed individually accounted for only a small proportion of the variation, perhaps due to the relatively low level of polymorphism observed for the markers employed (Bai et al., 1999). Subsequently (Bai et al., 1999), AFLP markers were applied to a mapping population involving the relatively resistant cv. Ning 7840 (Type II resistant cultivar), where the main speciﬁc character measured was the area under disease progress curve (AUDPC) after F. graminearum single-spikelet inoculation. One major QTL was identiﬁed accounting for up to 60% of the observed variation, which, although originally thought to be located on 7B, was identiﬁed subsequently as being equivalent to the QTL identiﬁed on chromosome arm 3BS (designated Qfhs.ndsu-3BS) (Waldron et al., 1999) and present in one of the ancestral cultivars of cv. Ning 7840, namely cv. Sumai 3. Two years later (Anderson et al., 2001), the same group veriﬁed the presence of this QTL (up to 41.6% of the variation accounted for) in Sumai 3 and located two further QTLs from Sumai 3 on 6AS (up to 11.6%) and 6BS (up to 9.2%). The susceptible parent, cv. Stoa, was also shown to carry two QTLs for resistance, on 2AL (up to 14.3%) and 4BS (up to 7.2%). A further QTL from a third line, ND2603 (partially resistant), was located on 3AL (up to 9.1%), in this case in a cross with the susceptible cv. Butte 86. These studies referred to Type II resistance (0–100% FHB severity scale after F. graminearum single-spikelet inoculation). During this period, in crosses between six resistant Chinese bread wheat cultivars with two susceptible cultivars (Bai et al., 2001), where AUDPC was evaluated after F. graminearum single-spikelet inoculation, it was shown, from joint scaling tests aimed at explaining the differences in means between parental, F1, F2 and backcross generations (Mather and Jinks, 1982), that most of the observed genetic variation could be explained by additive effects, where dominant and epistatic effects accounted for only a small proportion of the genetic effects present in the crosses analysed. The authors pointed out that this implied that it should be possible to accumulate different genes to improve resistance to FHB. The mainly additive nature of genetic effects was also observed in the soft red winter wheat, Ernie (Liu et al., 2005). In a subsequent study involving Type II resistance after inoculation with F. graminearum and F. culmorum (applied separately) of a mapping population derived from the bread wheat cross cv. CM-82036 (resistant, a line derived from Sumai 3) × cv. Remus (susceptible) and using RFLP, AFLP, SSR and endosperm storage protein markers (Buertsmayr et al., 2002), the large effect of Qfhs.ndsu-3BS (up to 60% of variation accounted for) was again conﬁrmed and two further QTLs were located to 5A and 1B. The 3BS and 5A QTLs were ﬂanked with SSR markers and the 1B QTL associated with the Glu-B1 locus encoding high molecular weight glutenin subunits. In a second part of this study (Buertsmayr et al., 2003), the authors extended the analysis to include combined Type I and Type II resistance; they found that, under spray inoculation, Qfhs. ndsu-3BS had a much larger effect than the 5A QTL, which they named Qfhs.ifa-5A, whereas after single-spikelet inoculation, the two loci showed effects of similar magnitude. Qfhs.ndsu-3BS appeared to be associated mainly with resistance to fungal spread (Type II), whereas Qfhs.ifa-5A appeared to be associated principally with fungal penetration, and might contribute primarily towards Type I resistance and, to a lesser extent, towards Type II. In both these studies, no isolate × wheat genotype interaction was observed, consistent with the previously observed non-speciﬁc or horizontal nature of resistance (Mesterhazy, 1995; van Eeuwijk et al., 1995), which was particularly interesting in this case since the two isolates used belonged to different species Barley and Wheat Resistance Genes (F. graminearum and F. culmorum). The authors concluded that FHB resistance depended on a few (2–3) major QTLs, operating together with unknown numbers of minor genes. They pointed out that marker-assisted selection (MAS) for the major QTLs ought to be a feasible method of accelerating the development (through breeding that included use of backcrosses) of resistant cultivars that combined Type I and Type II resistance. They felt that marker-mediated transfer of the QTL to durum wheat also ought to be feasible, given that no D genome chromosomes were involved in the QTL identiﬁed. The effect of Qfhs.ndsu-3BS was also observed in several other studies (Kolb et al., 2001; Zhou et al., 2002; Bourdoncle and Ohm, 2003; del Blanco et al., 2003; Shen et al., 2003a; Xie et al., 2007). Effects on 2A and 2B have also been observed in analyses involving Sumai 3 (Zhou et al., 2002). In one study (Yu et al., 2006), it was suggested that the 3BS, 5AS and 6BS resistance QTLs of Sumai 3 were derived from the Chinese landrace, Taiwan Xiaomai. QTLs on chromosomes 2A, 3A, 3B and 5A, which had been observed previously in Asian wheats, were also observed in RILs derived from a cross between the European winter wheat cultivars, Renan (resistant) and Récital (susceptible), using spray inoculation of F. culmorum (Gervais et al., 2003). In the same study, new QTLs were identiﬁed on 2BS and 5AL. Although co-localization of QTLs for resistance with awnedness (5A), PH (5A) and FT (2B) was observed, the authors considered that it should be possible to produce resistant lines independent of these characters. In RILs obtained from the Swiss winter wheat cross cv. Arina (resistant) × cv. Forno (susceptible) characterized with microsatellite and RFLP markers and subjected to spray inoculation with F. graminearum (combined Type I and II resistance), eight QTLs were identiﬁed that together explained 47% of the variation (Paillard et al., 2004). Three of these were considered of major effect: 6DL (22%), 5BL (14%, contributed by the susceptible parent) and 4AL (10%). The authors considered that these were different from QTLs previously reported. The other QTLs 85 detected were located on 2AL, 3AL, 3BL, 3DS and 5AL. The authors concluded that FHB resistance was polygenic, rather than the bimodal distribution observed in some previous studies (Bai et al., 1999; Waldron et al., 1999; Buertsmayr et al., 2002). The 2AL QTL was located at the same map position as one originating from cv. Stoa (Waldron et al., 1999; Anderson et al., 2001) and the 5AL QTL in the same position as one identiﬁed previously (Gervais et al., 2003). In contrast, the 3DS QTL was located differently compared to one identiﬁed previously on this arm (Shen et al., 2003a). The major 6D and 5B QTL overlapped completely with a QTL for HD and the 6D QTL overlapped partially with a QTL for PH. However, QTLs for PH were identiﬁed that were not associated with FHB resistance. The data could not distinguish pleiotropic effects from linkage. A further study involving cv. Arina (crossed to cv. NK93604) failed to detect the same QTL (Semagn et al., 2007); instead, QTLs on 1BL and 6BS from Arina and on 1AL and 7AL from NK93604 were detected. A study of Arina crossed to the susceptible UK cultivar, Riband, identiﬁed at least 10 QTLs, very few of which were coincident with the other Arina studies; the most consistent was a major QTL on 4DS (Draeger et al., 2007), detected in four of the ﬁve environments evaluated. In the winter wheat cross cv. Patterson × cv. Fundulea F201R (resistant cultivar from Rumania), QTLs for Type II resistance were found on 1B, 3A, 3D and 5A, with the 1B and 3A consistent over experiments (Shen et al., 2003b). It appears that, whereas Sumai 3 and its derivatives have major QTLs on 3B and 5A, the three winter wheat populations so far characterized seem to depend more on the accumulation of moderate and minor QTLs. The 3BL QTL located in the Renan/Récital population may be the same as that observed in the Arina/Forno population. In a cross of the resistant Brazilian cv. Frontana with the susceptible cv. Remus (Steiner et al., 2004) inoculated with F. graminearum and F. culmorum, a major QTL accounting for 16% of the variation in FHB severity and incidence was located on 86 S.A. Stenglein and W.J. Rogers 3A and a QTL accounting for 9% of the variation in FHB severity was located on 5A. Smaller effects for severity were located on 1B, 2A, 2B, 4B, 5A and 6B. The resistance of Frontana was found to be due principally to the inhibition of fungal penetration (Type I), but with a minor effect on fungal spread (Type II). PH, FT and spike morphology inﬂuenced FHB reaction, but co-localization of QTLs was observed only for minor QTL, and sufﬁcient QTLs for FHB resistance acting independently of these characters were observed in order to allow selection of resistant lines with any height, ﬂowering date and spike morphology. Seven QTLs for Type I and II resistance were found on 1BS, 1DS, 3B, 3DL, 5BL, 7BS and 7AL in a cross between cv. Cansas (moderately resistant) and cv. Ritmo (susceptible). The 1DS QTL seemed primarily to involve resistance to fungal penetration, while the other QTLs were concerned mainly with resistance to fungal spread (Klahr et al., 2007). Signiﬁcant correlations with PH and HD were observed. The Qfhs.ndsu-3BS region of Sumai 3 has been ﬁne mapped and named Fhb1 (Cuthbert et al., 2006), as well as being validated by near-isogenic line studies (Cuthbert et al., 2007). A second region on 6BS has also been ﬁne mapped and named Fhb2 (Pumphrey et al., 2007). Over recent years, attention has turned towards other types of resistance. For example, in the partially resistant cultivars, Wuhan-1 and Maringa, QTLs for the accumulation of DON (Type V) were located on 2DS and 5AS (as well as QTLs for FHB resistance on 2DL, 3BS and 4B) (Somers et al., 2003). QTLs were located on 5A (12.4%), 2A (8.5%) and 3B (6.2%) for low DON content in the Chinese landrace, Wangshuibai (as well as QTLs for Type II resistance on 3B and 2A (Ma et al., 2006)). In the previously cited study on Arina × NK93604 (Semagn et al., 2007), the QTLs located on 1AL and 2AS were associated with DON content, although only 1AL was associated with FHB resistance. In the additional Arina study cited, involving Arina × Riband (Draeger et al., 2007), the major 4DS QTL identiﬁed was found to affect AUDPC, DON content, fungal DNA content (FDNA), relative spikelet weight (RSW) and per cent of Fusariumdamaged kernels (FDK); although this may be due to linked genes, the authors considered it more likely to represent one resistance gene (which appeared to be linked to the Rht-D1 locus, an association that may prejudice attempts to improve resistance in germplasm containing the Rht-D1b (Rht) semi-dwarﬁng allele). In this study, further QTLs were observed as follows, whose detected presence varied over environments: AUDPC: 1BL, 2B, 6BL, 7AL, 7BL, 7DL; DON content: 6BL, 7DL; FDNA: 3DL, 6BL, 7BL; RSW: 1BL, 2AS, 6BL, 7DL; FDK (Type III): 5AS, 7AL; yield loss (Type IV): 7AL. In a study involving lines derived from the cross CM-82036 × Remus (Lemmens et al., 2005), the QTL on 3BS derived from Sumai 3, closely associated with resistance to spread of the disease (Type II), appears to convert DON to DON-3-O-glucoside. The authors hypothesized that the 3BS QTL encoded a DON-glucosyl-transferase or regulated the expression of this. In a cross involving cv. CJ 9306 (Jiang et al., 2007), two QTLs were found for resistance to DON accumulation, QFhs.nau-2DL, explaining up to 20% of the observed variation, and QFhs.nau-1AS, explaining 4–6%. The QTLs, QFhs.ndsu-3BS (up to 23% of the variation) and QFhs.nau-5AS (4–6%) were also validated. QTL × environment interaction was found for QFhs.nau-2DL only. The authors suggested that marker-assisted selection would be effective and made suggestions for the particular markers to be used, either singly or in combination. They also validated QFhs.ndsu-3BS for resistance to grain yield loss (Type IV). No QTL independent of Type II resistance was found. In many of the above studies, markers closely linked to the FHB resistance QTL were identiﬁed, enabling MAS to be contemplated. For example, SSR markers for the 3A and 5A QTL in Frontana have been identiﬁed, allowing these to be combined through MAS with the QTL in Sumai 3 and its derivatives. The feasibility of MAS has been directly demonstrated (Wilde et al., 2007), involving the 3B and 5A resistance of Sumai 3 and the 3A resistance of Frontana; Barley and Wheat Resistance Genes MAS for the two Sumai 3 QTLs gave signiﬁcant reductions in FHB severity and DON content, although MAS for the Frontana QTL had no effect. Additional phenotypic selection acting on other unmarked QTLs should give additional gain. Some markers have been used extensively in breeding programmes (Guo et al., 2006). Some of the above reports are particularly illuminating, since they appear to be showing that the various types of resistance are not necessarily truly distinct categories. For example, the Sumai 3 3BS resistance generally has been regarded as being of Type II. However, this locus may in fact be involved in detoxifying DON (Type V resistance). That is, it may be that at least a part of the mechanistic basis of the Type II resistance associated with this locus is its Type V nature. The map-based cloning of QTL ought to contribute to understanding resistance mechanisms further (Liu and Anderson, 2003; Shen et al., 2006). An expressed sequences tag (EST) rich in leucine and with low similarity to a protein kinase domain of the Rpg1 gene in barley was identiﬁed on 3BS and might represent a portion of a gene for FHB resistance (Shen et al., 2006). This EST could be used in MAS and for map-based cloning. Resistance gene analogues (RGA) associated with 1AL have been identiﬁed (Guo et al., 2006); all RGA markers studied contained a heat shock factor that initiated the production of heat shock proteins. Other promising areas for improvements in FHB are: (i) the introduction of genes from related species (QTLs for FHB resistance have been identiﬁed on 3A in Triticum dicoccoides (Otto et al., 2002) and on 4A in T. macha (Steed et al., 2005)); and (ii) the genetic engineering of FHB resistance by, for example, the expression in wheat of Arabidopsis NPR1 (Makandar et al., 2006). The above studies (and others not included here due to space conﬁnes, some of which are cited in the ‘Catalogue of gene symbols for wheat’ [Mclntosh et al., 2003] and subsequent annual supplements published in the Annual Wheat Newsletter) demonstrate that QTLs for FHB resistance have been identiﬁed on all the chromosomes 87 of wheat, whose detected presence and magnitude of effects depend greatly on environmental factors and the particular genetic background in which they are evaluated. In this sense, the genetic control of resistance appears to be complex, even though genetic effects appear to be mainly additive in nature. The situation may be set to become more complicated still: although, as mentioned previously, FHB resistance is thought to be non-speciﬁc or horizontal, recent studies indicate that interactions may be more complex (Xihang et al., 1991). Conclusions Although handling of FHB requires the application of several different disease management strategies, substantial progress has been made in understanding the genetic basis of resistance to FHB in wheat and barley. Quantitative resistance usually is caused by the simultaneous segregation of several to many genes and diverse non-genetic factors. Of the several types of resistance that have been hypothesized or reported, Type II resistance is the most stable and well studied. The Chinese wheat cultivar, Sumai 3, and its derivates are one of the best sources of resistance to FHB and may provide the maximum degree of Type II resistance. The major QTL on chromosome 3BS is found in most of the resistant cultivars from China. However, QTLs located on all the other chromosomes have also been reported but, for many of them, their expression is not stable over different environments or in all genetic backgrounds. Only a few barley cultivars have a relatively higher level of FHB resistance. Most of these resistant cultivars are two-rowed barley. Within six-rowed barley, which is preferred for malting, the cultivar, Chevron, has the best degree of resistance, but its DON level is still too high and far from meeting the safety requirements of the brewing industry. In contrast to wheat, Type I resistance is the major resistance type in barley. Molecular mapping indicates that many QTLs, spread over many chromosomes 88 S.A. Stenglein and W.J. Rogers and with minor effects, control this resistance. Correlation between FHB severity and other spike-related traits has presented a major barrier to breeding for FHB resistance in barley. Using MAS for the Chevron allele at the Qrgz-2H-8 locus should help breeders surpass this barrier (Nduulu et al., 2007). 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Basu4 1Bioproducts and Bioprocesses, Lethbridge Research Center, Agriculture and Agri-Food Canada, Lethbridge, Canada; 2Bidhan Chandra Krishi Vishavidalay, Nadia, India; 3Department of Weed Science, NWFP Agricultural University, Peshawar, Pakistan; 4Department of Biological Sciences, University of Lethbridge, Lethbridge, Canada Abstract Rice blast fungus (Magnaporthe grisea (Hebert) Barr) as a species has a very broad host range, infecting more than 40 Graminaceous hosts and some other non-grass hosts. The seedling stage, the rapid tillering stage after transplanting and the ﬂower emergence stage have been identiﬁed as the most susceptible to rice blast. In developing countries, poor farmers cannot afford to control blast disease by the application of expensive fungicides. Therefore, sustainable rice blast disease management is more important for environmental concern, as well as for better ﬁnancial returns to farmers in Third World countries. During the past few decades, a substantial amount of research has been conducted all over the globe to cope with blast fungus. In this chapter, we emphasize speciﬁcally the molecular biological aspect of the study on rice blast fungus over the past 50 years. Abbreviations used: BRV: blast-resistant varieties; HR: hypersensitive response; RBD: rice blast disease; RBF: rice blast fungus; RGAs: resistance gene analogues; ROI: reactive oxygen intermediates; PCR: polymerase chain reaction; RAPD: random ampliﬁcation of polymorphic DNA; RFLP: restriction fragment length polymorphism. Introduction Many rice researchers consider blast to be the most important disease of rice worldwide (Valent and Chumley, 1994). This is because the disease is widely distributed (85 countries) and can be very destructive when environmental conditions are favourable. Rice blast causes between 10–30% yield losses worldwide in rice, posing a constant 92 threat to the supply of this staple food for nearly one-half of the world’s population (Zhu et al., 2000; Talbot, 2003). The rice blast fungus (RBF), scientiﬁcally known as M. grisea (Hebert) Barr (anamorph: Pyricularia grisea Sacc.), is a ﬁlamentous Ascomycetous fungus that parasitizes over 40 grasses, including economically important crops like wheat, rice, barley and millet (Ou, 1985), but the pathogen is best known  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Sustainable Management of Rice Blast as the casual agent of the rice blast disease (RBD). RBD is one of the most serious diseases in all rice-growing regions of the world. Under heavy dew, all aerial parts of the plant can be affected; leaf surfaces become speckled with oval to globular lesions and severely infected plants are liable to lodging if stems are infected. The infected panicle results in severe yield loss (Ou, 1985). The fungus has the capacity to overcome resistance in a short period of time, soon after the release of a resistant cultivar, and thus has made breeding for resistance a constant and difﬁcult challenge to address for rice breeders and pathologists (Shao et al., 2008). Analysis of the existing genetic variation in plant pathogen populations is an important prerequisite for understanding the mechanism of co-evolution in the plant pathological system (McDonald et al., 1989). Several populations of rice blast pathogen all over the globe have been studied for their characteristic phenotypic and genotypic variations (Levy et al., 1991, 1993; Shull and Hamer, 1994; Chen et al., 1995; Kumar et al., 1999). Blast disease was ﬁrst reported in China (1637) and then in Japan (1704), Italy (1828) and in the USA (1996) (Asuyama, 1965; Ou, 1985; CRRI Annual Report, 2001–2002). In this chapter, we discuss the 50 years of research on M. grisea and the available sustainable disease resistance management in rice. Epidemiology of Blast Disease Seedling stage, rapid tillering stage after transplanting and ﬂower emergence stage were identiﬁed as the most susceptible to rice blast. The fact that the age of the leaves inﬂuences the susceptibility to blast was also brought out. The older the leaves on the plant, the more they are resistant to blast (Ou, 1985; CRRI Annual Report, 2001–2002). Excessive exposure to nitrogen and cold night temperatures predisposed susceptible varieties, but did not show any effect on highly resistant varieties. The critical range of temperature for penetration and establishment of infection was around 25–26°C, whereas germination of spores and appressoria formation 93 occurred within 6–10 h at 20–30°C in the presence of water on the surface of the leaf (Asuyama, 1965; Ou, 1985). The formation of dew or a little rainfall or the occurrence of fog provided the necessary water required for the germination of spores. Analysis of the intensity of infection recorded in different long-term experiments of several years revealed that blast infection had occurred under natural conditions when the minimum temperature during the night was 26°C and below, with the concomitant occurrence of relative humidity of 90% and higher (CRRI Annual Report, 2001–2002). Grouping of Blast Fungal Isolates M. grisea as a species has a very broad host range, infecting more than 40 Graminaceous hosts and some other non-grass hosts (Asuyama, 1965; Ou, 1985). Ou (1980) studied variability in the pathogen and the host resistance of M. grisea. Monoconidial cultures showed continued segregation for virulence pattern and generated diverse lesion types on individual leaves. Conidial and mycelial cells of M. grisea were reported to contain nuclei with a different number of chromosomes. These observations offered the best genetic explanation for the variation. Latterell and Rosi (1986) studied the longevity and pathogenic stability of M. grisea for 30 years. They suggested that the species comprised a wide range of pathotypes (races), each characterized by its capacity to attack certain cultivars of rice, and that these races were basically stable and mutations (or parasexual recombination) were the exception rather than the rule, resulting in broader host range or increased sporulating capacity. The detection of parasexual DNA exchanges in wildtype strains and the existence of merodiploids in nature suggest that parasexual recombination occurs in ﬁeld populations of M. grisea (Zeigler et al., 1997). Three DNA probes were developed by Hamer et al. (1989), which reliably and speciﬁcally identiﬁed the genetic backgrounds of the full spectrum of the rice blast fungal pathotypes. One of these probes consists of 94 S. Nandy et al. cloned fragments of repeated DNA obtained from the RBF genome and which are called MGR586 (M. grisea repeat elements, previously referred to as PCB586). The probe hybridizes with approximately 50 EcoRI fragments, ranging in size from 1.5–20.0 kb in the genome of all M. grisea isolates pathogenic to rice. Worldwide conservation of MGR586 sequences in RBF suggests that they descend from a common ancestral source, genetically isolated from other host-limited forms of M. grisea. The use of MGR shows that sequences are dispersed randomly on all chromosomes of the pathogens and segregate as genetic loci (Zeigler et al., 1997; Suzuki et al., 2007). Borromeo (1990) studied the Philippine isolates of RBF with MGR586 and MGR613. Valent and Chumley (1994) discussed the recent application of tools for molecular genetic analysis of M. grisea and past and current research in the problem areas. Iwano (1990) and Chen (1993) reported that the racial composition in a ﬁeld in Yongnan province, China, and the Philippines showed wide yearly ﬂuctuations. Iwano (1990) claimed that isolates from the same lesion changed their reaction on a set of several cultivars annually. Silue et al. (1992) studied the patterns of inheritance of avirulence in M. grisea in seven different rice cultivars. Avirulence to four cultivars has been reported as being controlled by one gene, whereas for the other three cultivars, it was controlled by two genes. In another study using DNA polymorphism, common ancestral patterns were found among Magnaporthe infecting rice isolates and their associated weed hosts (Borromeo et al., 1993) However, the pathogenic populations infecting the weed hosts do not supply pathogenic inoculums for the rice. Weeds can act as alternative hosts for the disease in greenhouse tests; but their role in the ﬁeld is not yet quite clear (Kato, 2001). Rice, as a widely and intensively cultivated crop, could be a potential target for parasitic ‘host shifts’ and a potential agent for ‘shifts’ to accompanying weeds (Couch et al., 2005). The authors also reported the single origin of rice-infecting M. oryzae after a ‘host shift’ from a Setaria-millet and that it was probably closely followed by additional ‘shifts’ to weeds of rice, cutgrass and torpedo grass. Levy et al. (1993) studied the genetic diversity of RBF in a disease nursery in Colombia. DNA ﬁngerprints using MGR586, 115 haplotypes from 151 fungal isolates were identiﬁed and partitioned into six discretely distinct genetic lineages. Xia et al. (1993) conducted a DNA ﬁngerprinting study to examine microgeographic variations in the M. grisea population in two different rice ﬁelds in Arakans in South-east Asia. The DNA ﬁngerprints of 113 isolates were grouped based on restriction fragment length polymorphism (RFLP) similarity. Seven distinct ﬁngerprint groups were identiﬁed and four ﬁngerprint groups were common in both ﬁelds. A study examining the relationship between phylogeny and pathotypes for isolates of the RBF in the Philippines revealed that the distribution of virulence was nonrandom with respect to lineage for the cultivars under study (Zeigler et al., 1995). Sivaraj (1995) reported six different lineages (L, A, B, E, F and H) from Karnataka in southern India, using the MGR DNA ﬁngerprinting approach. The repetitive DNA element, MGR586, has been widely used for ﬁngerprinting and phylogenetic analyses of M. grisea. George et al. (1998) developed a polymerase chain reaction (PCR)-based marker to DNA ﬁngerprint the Magnaporthe species coming from different biogeographic zones. Roumen et al. (1997) studied the genetic variability among 41 isolates of the blast pathogen from ﬁve rice-growing countries from the European Union, including Spain, France, Hungary, Italy and Portugal. DNA ﬁngerprinting grouped the isolates into ﬁve discrete lineages, which typically showed less than 65% band similarity. Srinivasachary et al. (1998) classiﬁed 27 single spore isolates of M. grisea from Karnataka in southern India over three different locations using random ampliﬁed polymorphic DNA (RAPD) primers. They found three clear groups at 70% similarity level. But Srinivasachary et al. (2002a,b) used 27 isolates from Ponnampet, Mandya and Bangalore for genetic analysis using 30 RAPD primers. Three distinct lineages were reported by the authors. Chadha and Gopalakrishna (2005) also used 20 isolates from seven different locations in India using Sustainable Management of Rice Blast 123 RAPD primers for cluster analysis. Scientists have sequenced the M. grisea genome and it is now available online at http:// www-genome.wi.mit.edu/annotation/fungi/ magnaporthe/. It is, however, important to note that for the ﬁrst time in the USA, the genomic structure of a signiﬁcant plant pathogen has been made publicly available. Physiology of Disease Resistance Plants develop defence mechanisms to recognize pathogens and protect them from attack. These defence reactions are triggered by the recognition of pathogens by plant disease resistance (R) genes. After the recognition of pathogens, a signalling pathway is activated, resulting in resistance to pathogens (Hammond-Kosack and Jones, 1997). During the early steps in R gene-mediated disease resistance, reactive oxygen intermediates (ROI) such as O2– and H2O2 are generated rapidly after infection; and, subsequently, hypersensitive response (HR) leading to cell death has been observed. An understanding of how pathogens induce disease, how the plants become diseased and how they defend themselves against the pathogens would help us to understand the functions of the genes governing resistance, which remains unknown, and eventually to develop novel methods for controlling RBD. The nature of resistance to blast disease operating at both the pre- and post-penetrative stages of the disease was investigated using several models involving cultivars differing in their reaction to the disease, nitrogen fertilization and temperature-induced tissue susceptibility and resistance induced by certain chemicals (CRRI Annual Report, 2001–2002). Four different mechanisms govern blast resistance in rice: (i) the epicuticular wax present on the surface of the leaves inﬂuences the infection by suppressing the appressorium formation by the pathogen, thus offering a partial resistance resulting in a reduced number of lesions being formed; (ii) free phenolic compounds and their oxidases toxify the tissue in the infected region: the speed and magnitude at which the toxiﬁcation 95 takes place in response to infection determines the tissue resistance to the pathogen; (iii) the presence of two toxic cinnamate derivatives (ferulate and coumarate) in the cell walls forming toxic oxidized products/ polymers like lignin and melanin-like compounds on oxidation forming a mechanical barrier for the fungus and thereby arresting the spread of the pathogen to adjacent cells, thus restricting disease lesions; and (iv) the synthesis and accumulation of antimicrobial compound(s) (diterpenoid in nature) known as ‘phytoalexins’ in response to infection toxic to the growth of the pathogen. However, none of these mechanisms seemed to be universal in nature and the defence mechanism was dependent on the varieties tested (CRRI Annual Report, 2001–2002). Finding the Right Gene The generation of cultivars that possess non-speciﬁc resistance to M. grisea would provide an economically effective and environmentally sound approach to rice blast control. One promising approach to the achievement of non-speciﬁc resistance to M. grisea is to incorporate genes that elicit general defence responses in rice (Dang and Jones, 2001; Stuiver and Custers, 2001). Much effort has been devoted to understanding the genetic and molecular basis of resistance in RBF and several genes have been cloned (Parson et al., 1987; Leung et al., 1990; Khang et al., 2008; Shao et al., 2008). Although earlier studies focused on pathotypic variability (Ou, 1985), later studies focused extensively on molecular markers to characterize population diversity (Nandy et al., 2004). Extensive use of the MGR586 heterodispersed element (Roumen et al., 1997; Kumar et al., 1999; Correll et al., 2000; Viji et al., 2000; Srinivasachary et al., 2002a,b; Chadha and Gopalakrishna, 2005) to delineate DNA ﬁngerprint lineages has helped to identify and classify the genetic structure of this important pathogen. PCR-based molecular markers are useful tools for detecting genetic variation within populations of important plant pathogens (Vakalounakis and 96 S. Nandy et al. Fragkiadakis, 1999; Kolmer and Liu, 2000; Srinivasachary et al., 2002a,b; Chadha and Gopalakrishna, 2005). RAPD (Welsh and McClelland, 1990; Williams et al., 1990) and markers have been widely used for estimating genetic diversity in wild populations (Annamalai et al., 1995), mainly because the technique does not need previous molecular genetic information and increases marker density for evaluating genetic kinship. The RAPD technique has also been used to study genetic diversity among RBF from different geographical locations in the world (Lima, 1999; Suzuki et al., 2007). The dynamic virulence of the rice blast pathogen could be the main cause for the breakdown of resistance in several rice varieties. The diversity and variability of the pathogen population may originate from the clonal mode of reproduction, coupled with mutation, migration, selection or random drift, heteroploidy and parasexuality of the fungus (Gesnovesi and Magill, 1976; Dayakar et al., 2000; Noguchi et al., 2007). A repeat sequence termed MGR586 was identiﬁed in the genome of rice-infecting strains of M. grisea (Shull and Hamer, 1994). This sequence has been widely used for DNA ﬁngerprinting of M. grisea to investigate the epidemiology of the RBD (Roumen et al., 1997; Kumar et al., 1999; Correll et al., 2000; Viji et al., 2000; Chadha and Gopalakrishna, 2005). Molecular analysis of isolates of M. grisea from different regions within a state (West Bengal, India) revealed the occurrence of a high level of polymorphism, indicating a wide and diverse genetic base (Mandal et al., 2004). Overall, a high genetic diversity was also obtained in Indian RBF (Roumen et al., 1997; Kumar et al., 1999; Correll et al., 2000; Mandal et al., 2004, Chadha and Gopalakrishna, 2005). Genetic mechanisms, namely simple mutations, meiotic recombination and parasexual recombination, could explain such genetic diversity (Yamasaki and Niizeki, 1965; Zeigler, 1998; Zeigler et al., 2000, Khang, 2001). Some indirect evidence suggests that M. grisea has the potential for sexual reproduction in speciﬁc geographic zones and localities (Viji et al., 2000, Adreit et al., 2007). There have been few investigations on the perfect state of M. grisea in India (Dayakar et al., 2000; Mandal et al., 2004). The sexual cycle does not seem to be a source of variation for the rice blast pathogen in India (Kumar et al., 1999). Similar results have also been reported from other corners of the globe (Valent et al., 1986). The wide range of diversity among collected isolates of M. grisea from different locations in West Bengal can be explained mainly by evolution resulting from natural and stressinduced transposition (Ikeda et al., 2001). Other mechanisms like horizontal gene transfer between RBF and its host (Kim et al., 2001) may also be of importance because varieties deployed within a region are based on crop seasons, along with several other biotic and geographic factors (Babujee and Gnanamanickam, 2000). Using Genetic Diversity of Disease Resistance Genetic studies of qualitative resistance were started when Goto (1970) established the differential system for races of P. grisea or M. grisea in Japan. Thirteen major genes for qualitative resistance have been reported by several researchers (Kiyosawa et al., 1981). Several rice cultivars with durable blast resistance have been identiﬁed and ‘Moroberekan’ have been cultivated in the world for many years without high losses from blast (Notteghem, 1985). These plants have been used as resistance donors in breeding programmes. Major resistance genes have been used successfully for developing blast resistance cultivars (Khush, 2004) and several dominant resistance genes have been identiﬁed which confer complete blast resistance (Kiyosawa et al., 1981). Atkins and Johnson (1965) identiﬁed two independent genes designated Pi-1 and Pi-6. Hsieh et al. (1967) in China found four dominant genes for pathogen resistance in japonica cultivars, named as Pi-4, Pi-13, Pi-22 and Pi-25 using RFLP techniques. Yu et al. (1991) mapped three major resistance genes, namely Pi-1, Pi-2 and Pi-4 in the Philippines. Several genes from tropical cultivars like ‘Tetep’, ‘Pai-kan Sustainable Management of Rice Blast tao’, ‘5173’, ‘LAC23’, Moroberekan and ‘Apura’ were identiﬁed and mapped using RFLPs (Yu et al., 1991; Miyamoto et al., 1996; Rybka et al., 1997) (Table 8.1). Recent reports identiﬁed at least four clusters, with ﬁve to eight loci each, located on chromosomes 4, 6, 11 and 12 (Roumen et al., 1997; Rybka et al., 1997, Tabien et al., 2000; Gao et al., 2002). Many pathogenic races have been identiﬁed in M. grisea and pathogenic variability has been cited as the principal cause for the breakdown of resistance in rice varieties (Baker et al., 1997). Therefore, an artiﬁcial inoculation study can be practised in place 97 of natural screening, which is quite cumbersome, time-consuming and season speciﬁc. There has been considerable achievement in the development of blast-resistant varieties (BRV), particularly using vertical-resistant genes (Nandy et al., 2004). Nevertheless, durable resistance alone can protect irrigated rice crops in the tropics adequately. Exploitation of durable resistance has been proposed for less blast-conducive environments (Buddenhagen, 1983; Notteghem, 1985; Parlevliet, 1988; Bonman et al., 1992). Artiﬁcial inoculation in Karnataka, southern India, was also carried out by Srinivasachary et al. (2002a) to study involving the Table 8.1. List of blast disease-resistance genes with chromosome numbers, donor varieties and linked markers of rice. Gene symbol Chromosome number Donor variety Pi-1(t) 11 Pi-2(t) 6 Pi-4(t) 12 Pi-5(t) 4 Linked marker Reference(s) LAC23, C101LAC Npb181, RZ536 BL245, C101A51, 5173 RG64 Tetep, Pai-kan-tao, BL245, C101PKT RG869, RZ397 RIL 45, RIL 249, Moroberekan – RG498 RG103 RG16 Atkins and Johnson (1965); Yu et al. (1991); Leung et al. (1998) Yu et al. (1991); Sridhar et al. (1999) Yu et al. (1991); Hittalmani et al. (1995); Tabien et al. (2000) Wang et al. (1994); Sridhar et al. (1999) Causse et al. (1994); Atkins and Johnson (1965) Wang et al. (1994) Leung et al. (1998); Khush et al. (1999) Naqvi et al. (1995), Tabien et al. (2000) Zhu et al. (1992); Roca et al. (1996); Khush et al. (1999) Khush et al. (1999) Miyamoto et al. (1996); Khush et al. (1999) Fukuoka and Okuno (1997); Leung et al. (1998); Sridhar et al. (1999) Chao et al. (1999); Bryan et al. (2000) Shigemura and Kitamura (1954); Rybka et al. (1997); Leung et al. (1998); Bryan et al. (2000) Pi-6 12 RG869 Pi-7(t) Pi-9 11 6 Pi-10 5 Moroberekan, RIL 29 O. minuta derivative WHD-IS-75-1-127 Moroberekan Pi-11 8 Oryzica Llanos 5 RRF6, RRH18, OPF6(2700) BP127, RZ617 Pi-12 Pi-b 12 2 Moroberekan, RIL 10 F-145-2 RG869 RZ123 Pi-z5 6 C101A51 RG64, RG612 Pi-k 11 F-129-1 – Pi-ta and Pi-ta2 12 Taducan, C101PKT, IR64, F-124-1, F128-1 RZ397, RG241 98 S. Nandy et al. reaction of representative single-spore culture PPT-4 to rice varieties Moroberekan, isolines of Co39, namely Pi-1, Pi-2, Pi-4, Pi-2 + Pi-1, Pi-1 + Pi-4, along with IRAT177, Apura and Doddi showed resistant reaction. Of these, Pi-1, Pi-2, Pi-4, Pi-2 + Pi-1 and Pi-1 + Pi-4 are known to contain major genes conferring resistance to blast disease. Yamada et al. (1976) and Kiyosawa et al. (1981) selected 12 differential varieties for resistance genes Pi-ks, Pi-a, Pi-k, Pi-km, Pi-z, Pi-ta (Pi-4), Pi-ta2, Pi-zt, Pi-kp, Pi-b and Pi-t. These differential varieties were used in Japan especially, but were not readily available in other countries. Monogenic lines including only a single gene in each genetic background and targeting for 24 different resistance genes – Pi-a, Pi-b, Pi-i, Pi-ks, Pi-k, Pi-k-h, Pi-km, Pi-kp, Pi-sh, Pi-t, Pi-ta (Pi-4), Pi-ta2, Pi-z, Pi-z5 (Pi-2), Pi-zt, Pi-1, Pi-3, Pi-5(t), Pi-7(t), Pi-9, Pi-11(t), Pi-12(t), Pi-19 and Pi-20 – were developed by Tsunematsu et al. (2000) as the ﬁrst international standard differential variety set. The polymorphic RG-64 marker was used by Hittalmani et al. (2001) to identify rice plants carrying Pi-2(t) from an F2 population derived from the cross between Co39 and C101A51. More than 30 blast-resistant genes (Babujee and Gnanamanickam, 2000) and QTLs have been identiﬁed in rice by conventional genetic studies based on linkage analyses and recombination frequencies (Kinoshita, 1991; Mackill et al., 1993). Some major genes for blast resistance have been identiﬁed in recombinant inbred lines (RILs) (Wang et al., 1994). Zeigler et al. (1995) proposed that organization of the blast fungus population into well-deﬁned lineages and their distribution in speciﬁc geographic locations have led to the employment of resistance genes targeted against pathogen populations prevalent in that region. This has been known as the ‘lineage exclusion’ hypothesis. Sivaraj et al. (1996) proposed a model to support gene pyramiding based on lineage exclusion. They consider traditional plant breeding as a strategy of pathotype exclusion, which leads to frequent resistance breakdown when appropriate pathotypes appear within 1 or 2 years after such resistance is deployed in large areas. In lineage exclusion, the conventional strategy is modiﬁed as a phylogenetic pathotype exclusion. Lineage exclusion presumes that lineage-speciﬁc avirulences represent an evolutionary genetic barrier to pathotype diversiﬁcation within the lineage. IRAT212/ N22, RR18-3/Bala, Bala/Tetep, Azucena/Gaurav and several lines from the natural cross of CR314-5-10 were resistant to leaf blast disease (CRRI, Annual Report 2000–2001). A combination of genes is also considered useful to confer resistance to the pathogen lineages prevalent in China, the USA and Latin America (Babujee and Gnanamanickam, 2000). Molecular Genetic Analysis of the Pathogen Plant disease resistance (R) genes confer resistance to a wide range of pathogens (fungi, viruses, bacteria and nematodes); they share various conserved motifs, suggesting the existence of a common defence signal transduction pathway in different plant–microbe interaction systems (Dang and Jones, 2001; Martin et al., 2003). In general, the R genes fall into six distinct classes, the most prevalent of which is the nucleotide-binding site plus leucine-rich repeat (NBS–LRR) genes (Martin et al., 2003; Qu et al., 2006). The LRR domains are generally thought to be involved in the interaction with avirulence (AVR) proteins and to be the major determinant of resistance speciﬁcity (Hulbert et al., 2001). The AVR-Pita avirulence gene family has been cloned recently at Kansas State University, USA, by Khang et al. (2008). They have studied isolates of the M. grisea species complex from diverse hosts and have found that AVR-Pita is a member of a gene family, which led them to rename it AVRPita1. Using the dominant DNA markers derived from portions of the Pi-ta gene, 141 rice germplasm accessions were rapidly determined and the results were conﬁrmed by inoculating rice germplasm with an M. grisea strain containing AVR-Pita (Wang et al., 2007). The Pi-ta gene was found in accessions from major rice-producing countries, including China, Japan, Vietnam, the Philippines, Iran and the USA. Sustainable Management of Rice Blast In another recent study, Shao et al. (2008) have reported that the expression of a hairpin-encoding gene (hrf1), derived from Xanthomonas oryzae pv. oryzae, confers non-speciﬁc resistance in rice to the blast fungus, M. grisea. Transgenic plants and their T1–T7 progenies were highly resistant to all major M. grisea races in rice-growing areas along the Yangtze River, China. The expression of defence-related genes was activated in resistant transgenic plants and the formation of melanized appressoria, which is essential for foliar infection, was inhibited on plant leaves. These results suggest that hairpins may offer new opportunities for generating broad-spectrum disease resistance in other crops. However, occurrence of clustered multigene families is a major obstacle in the cloning of R genes (Dixon et al., 1996; Ori et al., 1997), which makes it even more difﬁcult to determine the functional copy of these genes. Therefore, ﬁne mapping of R-gene analogues on different chromosomes would be helpful in the identiﬁcation of multigene families in rice, which in turn will lead to the establishment of correlation between the chromosomal position of known R genes and their analogues. Recently, Kumar et al. (2007) cloned and also carried out in silico mapping of resistance gene analogues (RGAs) isolated from rice lines containing known genes for blast resistance. They have ampliﬁed RGAs from the genomic DNA of 10 rice lines having varying degrees of resistance to M. grisea by using degenerate primers. Twenty RGAs were mapped near to the chromosomal regions containing known genes for rice blast, bacterial leaf blight and sheath blight resistance. Thirtynine RGA sequences also contained an open reading frame representing the signature of potential disease-resistance genes. Control Measures Kato (2001) suggests burning and composting of infected plant parts; use of non-infected or certiﬁed healthy seeds and disease-resistant cultivars; appropriate regulation of fertilizer application; proper cultural control and 99 avoiding damp or most soil with high moisture content for seed sowing, etc. However, chemical control is the most commonly used approach in most parts of the globe for effective disease control. Several fungicides are used against blast disease, including benomyl, fthalide, edifenphos, iprobenfos, tricyclazole, isoprothiolane, probenazole, pyroquilon, felimzone (= meferimzone), diclocymet, carpropamid, fenoxanil and metominostrobin, and antibiotics such as blasticidin and kasugamycin (Kato, 2001). The composition, quantity, time and application method of fungicides applied in ﬁeld trials are dependent on the disease forecast for a particular region or zone, or on the local disease prevalence rate (Kato, 2001). Carbendazim, chlorobenthiozone, coratop, fungorene, hinosan and kitazin fungicides and antibiotic kasumin were effective against foliar and neck blast in India (CRRI Annual Report, 2001–2002). Rice seed treatment with Carbendazim + TMTD 25 was effective in controlling seedborne blast (CRRI Annual Report, 2001–2002). The control of rice blast relies on the use of resistant cultivars and the application of fungicides, but neither approach is particularly effective in different geographic locations (Shao et al., 2008) because management of rice blast via breeding BRV has had only short-term success due to the frequent breakdown of resistance under ﬁeld conditions (Valent and Chumley, 1994). The frequent appearance of new races (or pathotypes) of the fungus that are capable of infecting previously resistant varieties has been proposed as the principal cause for the loss of resistance (Ou, 1980). Host resistance in rice to M. grisea functions via a classical gene-for-gene interaction in which a single dominant resistance gene corresponds with a dominant avirulence gene in the pathogen (Hammond-Kosack and Jones, 1997; Talbot, 2003). Because of the apparent instability in the genome of M. grisea, new pathogenic races evolve rapidly and thus host resistance typically lasts for a few years only (Zhu et al., 2000; Talbot, 2003). Few fungicides are available for the effective control of rice blast, but rapid mutation in the pathogen leads to the emergence of fungicide-resistant variants (Takagaki 100 S. Nandy et al. et al., 2004); thus, higher-dose applications of fungicides pose risks both to humans and the environment. have been used for rice blast management for the past 50 years. Conclusions Sustainable Rice Blast Disease Management In developing countries, poor farmers cannot afford to control blast disease by the application of fungicides. Chemical control of plant pathogens is most effective and yet the use of chemicals is not generally desired due to the serious environmental threat it poses. Environmental effects and resistance are not considered a major concern in developing countries. Farmers are more interested in short-term strategy for disease control. However, the continuous use of fungicides leads to the resurgence of resistant races of the pathogen under selection pressure. Therefore, sustainable rice blast disease management is more important for environmental concern. Figure 8.1 shows the basic components that We have reviewed here the past 50 years of research progress in the genetics and molecular biology of rice blast disease, but different approaches can be taken for sustainable disease control with recent advances in genomics, proteomics and diverse genetic resistance mechanisms. Liu et al. (2002) recently reported the application of candidate defence genes to develop blast-resistant breeding lines with resistance to diverse pathogen populations. Several biocontrol agents for blast have been deployed successfully to combat the disease in the laboratory, greenhouse and ﬁeld tests (Chatterjee et al., 1996; Krishnamurthy et al., 1998; Gnanamanickam et al., 1999). The feasibility of such strategies on a commercial scale still remains to be tested. Hence, use of Using the genetic diversity of disease resistance Finding the right gene Sustainable rice blast disease management Understanding the physiology of disease resistance Molecular genetic analysis of the pathogen Fig. 8.1. The four basic components of sustainable rice blast management. Sustainable Management of Rice Blast resistant cultivars is the best available alternative to overcome severe yield losses. The objective of the green revolution has not changed; there is the added impetus that crop protection should be conducted in the context of improving the livelihood of rural people and preserving limited natural resources (Leung et al., 2003). However, the gene revolution has opened up newer and better possible ways of preventing yield loss from pathogen attack, conservation and utilization of wild species for resistance genes. The variability of the pathogen and the history of resistance breakdown have led to the development of a number of different plant breeding and molecular approaches to achieve durable blast resistance. Combinations of resistance genes are thought to provide broader spectra of resistance through both ordinary gene action and quantitative 101 complementation that result in durable resistance. Gene pyramiding is one of the strategies recommended to increase the durability of blast disease resistance (Robinson, 1973; Nelson, 1978; Buddenhagen, 1983; Pedersen and Leath, 1988). Pyramided resistance will be durable in places where compatibility to the component resistance genes is distributed among the prevalent lineages. Agricultural practices such as soil preparation, low nitrogen fertilization, low sowing density, optimized use of water and seed selection contribute to reduce the virulence of M. grisea populations. Optimized integration of genetic resistance in agricultural management is the preferred strategy to protect cultivated rice from RBD in a way that is affordable, feasible, durable and, overall, compatible with environmental protection. References Adreit, H.S., Andriantsimialona, D., Utami, D.W., Notteghem, J.L., Lebrun, M.H. and Tharreau, D. (2007) Microsatellite markers for population studies of the rice blast fungus Magnaporthe grisea. 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The application of fungicides to fruits after harvest to reduce decay has been increasingly curtailed due to the development of resistance in pathogens to many key fungicides, lack of replacement with better fungicides, negative public perception regarding the safety of pesticides and consequent restrictions on fungicide use. Biological control of postharvest diseases has emerged as an effective alternative and several products are available in the market. One of the major limitations with biological disease control is inconsistency in the efﬁcacy of the product. The limitations of biocontrol products can be addressed by enhancing biocontrol through genetic and environmental manipulations and integration with other alternative methods that, alone, do not provide adequate protection but, in combination with biocontrol, provide additive or synergistic effects. Introduction Approximately half of the population in the Third World does not have access to adequate food supplies. There are many reasons for this, one of which is food losses occurring in the postharvest and marketing system. A study on ‘Postharvest Food Losses in Developing Countries’ conducted by a committee of the US National Research Council concludes that, ‘postharvest losses are “enormous”’. The committee extrapolated from apparent loss patterns and expected production trends and projected postharvest food losses to be, at a minimum, 47,000,000 Mt of durable crops and 60,000,000 Mt of perishable crops. ‘The average minimum losses reported for roots and tubers and fruits and vegetables were 16 per cent and 21 per cent, respectively; many more “qualitative” references, not included here, indicate estimates of 40–50 per cent and above.’ The application of effective fungicides just prior to or shortly after harvest generally controls postharvest decay (Eckert and Ogawa, 1988). About 23m kg of fungicides is applied to fruits and vegetables annually and it is generally accepted that production and marketing would not be possible without their use (Ragsdale and Sisler, 1994). However, use of fungicides has been restricted due to their carcinogenicity, teratogenicity, residual toxicity and long degradation period causing environmental pollution (Unnikrishnan and Nath, 2002).  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 109 110 N. Sharma and P. Awasthi The Food Quality Protection Act (FQPA) in the USA, the Food and Environment Protection Act (FEPA) 1985 and Control of Substances Hazardous to Health (COSHH) Regulations 1988, made under the Health and Safety at Work Act, 1974, in the UK are the guiding forces in the regulation of pesticide use in their respective countries. Several countries have implemented their own speciﬁc policies to reduce pesticide use (Matteson, 1995). Similarly, the fruit industry worldwide has accepted the concept of integrated fruit production (IFP). IFP aims to produce high-quality fruit in harmony with the consumer and the environment. This implies minimum usage of chemicals, especially after harvest. Globally, greater restrictions on pesticide use in the developed nations have resulted in increasing trends for natural, non-chemical or organic approaches to disease control. Understandably, alternatives to chemical pesticides or products that allow reduced usage in terms of fewer or reduced rates of application are beginning to appear on the market in the form of biological control agents (BCA). The present chapter reviews the status of yeast as a biocontrol agent and the problems associated with its commercialization and registration. Cook and Baker (1983), in their book on biological control, cited only one example of the biocontrol of postharvest disease of strawberry fruit rot using Trichoderma sp. Subsequently, Wilson and Pusey (1985) presented their initial research on Bacillus subtilis to control brown rot on peaches, caused by Monilinia fructicola, and the organism was patented. A number of microorganisms (bacteria, yeasts and fungi), which effectively control postharvest pathogens, have been identiﬁed for the control of postharvest diseases and some of these have been patented and registered (El-Ghaouth and Wilson, 1997, 2002). In several studies, yeast strains (Aureobasidium pullulans, Candida oleophila, C. guilliermondii, C. sake, Cryptococcus laurentii, Debaryomyces hansenii, Metschnikowia pulcherrima, Pichia gulliermondii, Sporobolomyces roseus) are reported for biocontrol of postharvest fungal decays of fruits caused by Alternaria alternata, Botrytis cinerea, Geotrichum candidum, M. fructicola, Penicillium digitatum, P. italicum, P. expansum and Rhizopus stolonifer (Droby et al., 1989; Wisniewski et al., 1991; Sharma, 1992, 1993, 2000; Mehrotra et al., 1996, 1998; Sharma et al., 1997; Spadaro et al., 2002) . In the past 25 years, research on biological control of postharvest diseases has moved from laboratory to practical applications (Wisniewski and Wilson, 1992; Wilson and Wisniewski, 1994; Mari and Guizzardi, 1998; Droby et al., 2001; Janisiewiez and Korsten, 2002; Korsten, 2006). By early 2000, there were three postharvest biological products available in the market: Aspire™, a product developed from C. oleophila (limited to the USA and Israel); BioSave™, developed from P. syringae to control decay caused by P. italicum and P. digitatum (limited to the USA); and YieldPlus™ (limited to South Africa). Avogreen™, a commercial product of B. subtilis, was developed to control diseases caused by Cercospora spot and anthracnose of avocado. Isolation of Antagonist Often, carposphere, phylloplane, ﬂowers and, in a few cases, other matrixes have provided the major source for antagonists (Filonow et al., 1996; Sharma, 2003; Belve et al., 2006). Various strategies have been employed to isolate antagonists and these include isolation from natural cracks on the fruit surface; agar plates containing apple juice that were seeded with a rot pathogen (Wilson et al., 1993); freshly made wounds on apples in the orchard that were exposed to colonization by fruit-associated microbiota from 1 to 4 weeks before harvest (Janisiewiez, 1996); and from an apple juice culture resulting from seeding diluted apple juice with the orchard-colonized wounds and repeated reinoculation to fresh apple juice. Isolation of the antagonists can be improved by using fruit from unmanaged orchards (Falconi and Mendgen, 1994) where natural populations have not been disturbed by chemical usage and the pool of potential antagonists is greater than in a chemically managed orchard (Smolka, 1992). Postharvest Technology Natural microﬂora maintains a balance among the microbes normally present and inhibits the growth of newer arrivals. Sharma (2005) reported that undiluted fruit washings when plated on agar plates exhibited a dense population of yeast and bacteria and, on dilution, ﬁlamentous fungi of the pathogenic type were isolated. This suggests that bacteria and yeast, naturally present on the surface, may inhibit the growth of other microorganisms, including plant pathogenic fungi. Later, it was observed that the citrus fruits, when washed and stored, rotted faster than the unwashed fruits, suggesting that these bacteria and yeast provide protection to fruits against postharvest pathogens. Rather than in vitro screening of organisms in Petri plates, which favoured the identiﬁcation of antibiotic-producing organisms, a selection strategy was developed to identify suitable yeast antagonists (Wilson et al., 1993). The method involved placing washing ﬂuids obtained from the surface of the fruit into fruit wounds that subsequently were inoculated with a rot pathogen. Organisms were then isolated from the surface of wounds that did not develop infections. These were plated out and isolated. Pure cultures of potential antagonists were produced and then each organism was screened individually to assess its potential as a biocontrol agent. This method identiﬁed a number of antagonists that were studied more intensely and measured against the criteria set for suitability for commercial production, as outlined by Wilson and Wisniewski (1989) and Hofstein et al. (1994): ● ● ● ● ● ● ● ● genetically stable effective at low concentrations not fastidious in its nutrient requirements ability to survive adverse environmental conditions (including low temperature and controlled atmosphere storage) effective against a wide range of pathogens on a variety of fruits and vegetables amenable to production on an inexpensive growth medium amenable to a formulation with a long shelf life easy to dispense ● ● ● ● ● 111 does not produce metabolites that are deleterious to human health resistant to pesticides compatible with commercial processing procedures does not grow at 37°C and is not associated with infections in humans non-pathogenic to host commodity. Biocontrol Activity Most antagonistic yeasts are efﬁcient colonizers, even under adverse environmental conditions, as they utilize nutrients rapidly, produce extracellular materials that enhance their survival on fruit surfaces and restrict both colonization sites and ﬂow of germination caused to fungal propagules (Dugan and Roberts, 1995). In order to optimize disease control, it is important to understand the mode of action of the antagonists so that these attributes can be utilized to improve performance. The antagonist activity can be expressed in a number of ways. The most common is antibiosis (production of metabolites such as pyrrolnitrin or iturins), attributed mainly to bacterial antagonists (Smilanick and Dennis-Arrue, 1992). The antibiotic pyrrolnitrin, produced by Pseudomonas cepacia LT-4-12W (Janisiewiez and Roitman, 1988), reduced in vitro growth and conidia germination and controlled the pome fruit pathogens, P. expansum and B. cinerea, and citrus fruit pathogen, P. italicum. However, the signiﬁcance of the antibiotics in these biocontrol situations was not clear, since strain LT-4-12W still provided substantial control of blue mould decay on oranges inoculated with laboratoryderived mutants of P. italicum resistant to pyrrolnitrin. Spadaro et al. (2002), in studies on M. pulcherrima, found that in the in vitro antagonism studies on different substrates, the yeast could produce some metabolites toxic to the pathogen, as distinct from the application of culture ﬁltrates in vivo. In recent years, the use of antibiotic-producing bacteria has been abandoned in order to prevent the appearance of resistance in pathogen strains for humans or animals. 112 N. Sharma and P. Awasthi Competition for nutrients and/or space is the major mechanism involved for P. guilliermondii, C. laurentii, C. utilis, C. oleophila, D. hansenii and several other yeasts employed as bioagents (Chalutz and Wilson, 1990; Arras, 1996; Arras et al., 1997; Spadaro et al., 2002; He et al., 2003; Chan and Tian, 2005; Zhang et al., 2005). Janisiewiez et al. (2000) developed a non-destructive method using tissue culture plates having a defusing membrane at the lower end of cylindrical inserts for in vitro study of competition for nutrients separated from the competition for space. Living cells of the antagonist are necessary to guarantee fungal control. The ability to prevent infection by pathogen was lost when the antagonist cells were killed. It was also observed that competition for nutrients was not visible when a surplus of nutrients was available. Therefore, the nutritional environment available at the wound site may create a favourable microenvironment for antagonists to colonize, multiply and compete effectively (Zheng et al., 2004). The activity of an antagonist is dependent on the concentration of the antagonist: the higher the concentration, the more effective the control. The antagonist cell concentration of 106 – 108 CFU/ml or more of Candida spp., D. hansenii and Pantoea agglomerans provided satisfactory levels of control (Droby et al., 1989; McLaughlin et al., 1990). However, different isolates of M. pulcherrima at 106 CFU/ml were not found to provide satisfactory levels of control against B. cinerea and P. expansum (Spadaro et al., 2002). While early studies indicated that nutrient competition and the fast growth rate of antagonists played a major role in biocontrol activity, subsequent studies indicated a much more complex interaction, such as direct interaction with the pathogen (Wisniewski et al., 1991; Spadaro et al., 2002), induced resistance in host tissue (Wilson et al., 1994; Droby et al., 2002) or a gamut of interactions between the antagonist, pathogen and commodity. Pichia guilliermondii US-7 (Droby et al., 1989) and M. pulcherrima (Spadaro et al., 2002) exhibited nutrient competition along with direct parasitism against B. cinerea in apples. Pichia membranefaciens and C. albidus exhibited tenacious attachment with pathogen hyphae, along with secretion of extracellular lytic enzymes (Chan and Tian, 2005). Ultrastructural and cytochemical studies on yeast, C. saitoana, when co-cultivated with B. cinerea, showed cytological damage as papillae and protuberances in the cell wall and degeneration of the cytoplasm. It was also found to stimulate structural defence response in the host. Host cell walls were well preserved and displayed an intense and regular celluloselabelling pattern, as seen in transmission electron microscopy (El Ghaouth et al., 1998). Yeast cells are able to produce hydrolytic enzymes capable of attacking the cell walls of pathogens and extracellular polymers that appear to have antifungal activity. Yeast, P. anomala strain K, effective in the control of grey mould of apple, increased production of exo-b-1,3-glucanase threefold in the presence of cell wall preparations of B. cinerea in apple wounds. Higher b-1,3glucanase and chitinase activity was also detected in apple wounds treated with strains of another antagonist, A. pullulans, effective in controlling various decays on apple, table grape and other fruits (Ippolito et al., 2000; Castoria et al., 2001). Yeast, P. membranefaciens and C. albidus, show b-1,3-glucanase and exo-chitinase activity in the presence of cell wall preparations of R. stolonifer, M. fructicola and P. expansum (Chan and Tian, 2005). Yeasts like C. famata are reported to control green mould due to induction of phytoalexins, scoparone and scopolectin (Arras, 1996). However, the role of enzymes and phytoalexins in biocontrol activity warrants further investigation. Fajardo et al. (1998) reported differential induction of proteins in orange ﬂavedo by biologically based elicitors. More recently, molecular approaches to examine the mode of action have been studied on the biocontrol agent. A transformation system for C. oleophila yeast produced yeast lines with either higher or lower levels of a b-1,3-glucanase gene/enzyme expression compared to the wild type. Biocontrol activity did not differ between the different yeast lines, but the results did not Postharvest Technology rule out a role for this gene in biocontrol activity. It was also demonstrated that overexpression of a lytic peptide belonging to the defensin family of antimicrobial peptides in yeast could enhance biocontrol activity (Segal et al., 2002; Yehuda et al., 2003). Constraints in Product Development and Registration In the early years, several yeast antagonists that had commercial potential were misidentiﬁed, such as strain US-7 of C. guilliermondi, which was misidentiﬁed originally as D. hansenii. This caused some confusion in the patenting process and emphasized the need to have at least two conﬁrming identiﬁcations by reputable yeast taxonomic services. It also emphasized the weakness of using physiological tests as the basis for making taxonomic determinations (McLaughlin et al., 1990). Also, few isolates of C. guillliermondii were abandoned because they were found to be pathogenic to humans. Potential biocontrol agents often have some signiﬁcant limitations: sensitivity to adverse environmental conditions such as extreme dryness, heat and cold, limited shelf life, limited biocontrol efﬁcacy in situations where several pathogens are involved in decay development and an inability to control latent infections. For commercialization, several semi-commercial and commercial trials have to be conducted, for which large volumes of antagonist are required. The mass production of the bioagent by rapid, efﬁcient and inexpensive fermentation of the antagonist is a key issue. Therefore, it is fundamental to ﬁnd carbon and nitrogen sources that provide maximum biomass production at minimum cost, while maintaining biocontrol efﬁcacy. Cheap industrial waste materials such as cottonseed meal, corn steep liquor, partially digested peptone, yeast extract, dry brewer’s yeast, sucrose and molasses have been used as growth media for the multiplication of cells (Hofstein et al., 1994; Costa et al., 2001). Large-scale production of any yeast depends on the amount of technical information available on that speciﬁc strain, such 113 as osmotolerance, temperature, oxygen requirements, optimum pH and optimum growth rate. Growth rate of yeast is very high, but lower than that of bacteria; longer fermentation durations pose the risk of yeast cultures becoming contaminated. Yeast is also sensitive to low pH (below three), which is used generally as a measure to check bacterial contamination because pH above ﬁve is favourable for bacteria that may contaminate yeast culture. Aeration of fermentors, to fulﬁl the oxygen requirement for maximum output, can also be a source of contamination during the early phases of production and, to prevent such contamination, other technologies must be used. The contaminants should be identiﬁed at each stage of production and quantiﬁed in the end product. Yeast fermentation is an exothermic process; therefore, the fermentation temperature can never be below ambient and, since yeasts appear sensitive to high temperatures (above 28°C), a cooling system more efﬁcient than the evaporative system routinely used has to be employed. This, however, adds to the cost of production. A major obstacle to the commercialization of biocontrol products is the development of a shelf-stable product that retains bioactivity similar to that of fresh cells. Formulations can inﬂuence the survival and activity of biocontrol agents. An accurate formulation has a profound effect on the efﬁcacy of a biocontrol agent, including its shelf life, ability to grow and survive after application, effectiveness in disease control, ease of operation and application and the cost (Fravel et al., 1998). A biofungicide should be effective for at least 6 months, and preferably for 2 years (Pusey, 1994). This can be achieved by supplementing the yeast with protectants, carriers or additives. Alternatively, yeast can be conditioned during fermentation by using an emulsiﬁer. Drying the product and maintenance in a dry environment or suspension in oil are common approaches. Products are available as wettable powder, as frozen cell concentrated pellets or as liquid formulations. It was found that freeze-dried cells were signiﬁcantly less effective than fresh cells. 114 N. Sharma and P. Awasthi Certain freeze-drying protective agents and rehydration media enhanced the viability of the antagonist, P. agglomerans strain CPA-2, effective against blue mould and grey mould of pome fruits (Costa et al., 2000). Survival of cells of the antagonistic yeast, C. sake, was improved from 0.2% to 30–40%, by using freeze-drying protective media consisting of skim milk and other protectants, such as 10% lactose or glucose and 10% fructose or sucrose. The presence of trehalose in liquid formulations appeared to help preserve the viability of C. sake during storage. It is known that intracellular trehalose exerts a protective effect on yeast under extreme environmental conditions such as desiccation, freezing, osmotic stress and heat shock, and it also provides thermal stability to the cells (Abadias et al., 2001). The application of adjuvant can protect and stimulate the establishing of the antagonist on the host surface. The addition of xanthan gum to A. pullulans L47, applied to strawberries in the ﬁeld from bloom to fruit at the green stage, improved survival of the antagonist and increased biocontrol of storage rot caused by B. cinerea (Ippolito et al., 1998). Formulations may include wetters (humectants) to facilitate reabsorption of moisture from air. Wetters not only make water spray stay on plants but, like oil carriers, they also enable organisms to reach otherwise inaccessible places such as depressions, stomata and lenticels, thereby improving the chances of establishing antagonists for disease control. Oil carriers are expensive, but formulations containing oils can enhance the reliability of biological control agents (Jones and Burges, 1998). Research is needed to determine the value of each additive alone and also in the presence of other ingredients, as well as to ensure the requirements for ecological safety. One of the major limitations with biological disease control is the inconsistency in efﬁcacy that is often observed when useful antagonists reach the stage of large-scale testing, and which can arise from a variety of causes reﬂecting the biological nature of the control microorganism. Essentially, the organism must ﬁrst survive application and then retain activity in the environment of use throughout the period when active control is required, which may be several months for some pathogens. During this time, it must survive ﬂuctuations in the physical environment and the action of the indigenous and competitive microbiota. The use of appropriate inoculum production, formulation and application technologies, together with quality control checks, should also help in this process. Nevertheless, even if reliable BCAs can be produced, they must still be easy to use and cost-effective or they will either never reach the marketplace or not be used by growers. By early 2000, there were two yeastbased postharvest biological products available on the market: Aspire™ (C. oleophila I-182) and YieldPlus™ (El-Ghaouth and Wilson, 1997, 2002; Wilson and El-Ghaouth, 2002). The commercial development of Aspire by Ecogen-Israel Partnership Ltd, focused on the biocontrol of postharvest decays of citrus, mainly blue mould and green mould caused by P. italicum and P. digitatum, respectively, which invade through wounds after harvest. Throughout the course of developing Aspire™, considerable research went into ﬁnding methods to enhance the reliability and efﬁcacy of the product and other selected antagonists as well. As a result, second generation biocontrol products were developed using a combination of natural products along with a yeast antagonist to address the poor ability. Research efforts led to the development of two new products whose main components consisted of the yeast antagonist, C. saitoana, and either a derivative of chitosan (Biocoat) or lysozyme (Biocure) (El Ghaouth et al., 2000a). Both compounds have been tested worldwide and have shown strong eradicant activity. Both products contain additional additives, such as sodium bicarbonate, to enhance efﬁcacy and perform as well as the postharvest fungicides currently available. Another constraint concerns registration. Currently, there are no fungal biocontrol products registered and sold worldwide. Some products are available in several countries, while others are sold in their respective countries. This reﬂects the problems associated with registration requirements in Postharvest Technology different countries and includes concerns about releasing non-indigenous microorganisms. The legislation drafted essentially for chemical pesticides is not always applicable to biological pesticides and the requirements for the registration of biological pesticides are currently under discussion for appropriate review. The position of the biocontrol product in the market governs its future. For example, if the product enters the agrochemical market, it competes against synthetic fungicides that can kill pathogenic organisms, while yeast only-based products cannot do so and neither do they have systemic action. They act mainly as protectants that may also induce resistance in the hosts. The other option is to position the product in the ‘all green’ category in markets such as those of perishables, where no other option is available, thus eliminating any competition and fulﬁlling the principal objective of consumer and environmental safety. Integrated Control Since, biological agents alone are not capable of providing commercially acceptable levels of control, their integration with other control measures is expected to provide greater stability and effectiveness. It is also desirable that the use of antagonists must be compatible with current handling and storage practices which could otherwise cause a reduction in the effectiveness of antagonist strains. For biological control to be effective, use of antagonists must be compatible with other control measures. An effective biocontrol based on a mixture of several complementary and non-competitive antagonists has several advantages: apart from a wider spectrum of activity, they increase efﬁcacy, are more reliable and allow reduction in application times and treatment costs. They also permit the combination of different genetic characteristics, minimizing the need for genetic engineering. In a study on apples, a broader spectrum of pathogens was controlled and less total biomass of the antagonist was needed to control decay (Janisiewiez, 115 1996) when a mixture of antagonists was applied. The mixtures are either paired at random or after screening, for minimum mutual niche overlap. To determine further compatibility of the strains selected, it is important to conduct coexistence studies using De Wit displacement series in fruit wounds (Wilson and Lindow, 1994). The beneﬁts of this approach are clear, but its implementation requires approval from the industry. It also entails doubling of the cost. However, this can be overcome by using in the mixture at least one antagonist which has been commercialized. Some exogenous substances, such as chitosan, amino acids, antibiotics, calcium salts and carbohydrates, have been studied to enhance the biocontrol capability of antagonists against fungal pathogens. Calcium chloride improved biological control of the yeast, P. guilliermondii (Droby et al., 1997). Combining 0.2% glycolchitosan with the antagonist, C. saitoana, was more effective in controlling green mould of oranges and lemons, caused by P. digitatum, and grey and blue moulds of apples than either treatment alone (El- Ghaouth et al., 2000a,b). In a recent study by the authors, a combination of chitosan and the yeast, C. utilis, was found effective in controlling postharvest pathogens on tomato (Sharma et al., 2006). The studies also showed that several yeast genera were compatible with low concentrations of chitosan and the protection afforded by this combination was superior to the stand-alone treatments. GRAS (generally recognized as safe) substances such as sodium carbonate, sodium bicarbonate and ethanol reduced conidial germination of P. digitatum, the causal agent of green mould of citrus. Ethanol at 10%, in combination with ethanol-resistant S. cerevisiae strains 1440 and 1749, reduced the incidence of grey mould decay on apples from more than 90% to close to 0%, respectively, whereas either treatment alone did not reduce decay. The same concentration of ethanol reduced green mould of lemons to less than 5% (Smilanick et al., 1995, 1999). A. pullulans, in combination with calcium chloride or sodium bicarbonate, was found effective in controlling postharvest 116 N. Sharma and P. Awasthi pathogens on sweet cherries (Ippolito et al., 1998). Pre-storage hot air treatment of apples reduced or eliminated blue mould decay caused by P. expansum and grey mould decay (Fallik et al., 1995). Heat also improved biocontrol with heat-tolerant yeasts when applied to apples up to 24 h after inoculation with the pathogen. The heat treatment alone provided little residual protection, but the residual protection provided by Ca and the antagonist in combination enhanced the control by heat. When antagonists were applied to apple wounds before heat treatment, the heat reduced populations of P. syringae and increased populations of the two heat-tolerant yeasts more than tenfold. Conclusions Future lines of research should be directed to ﬁnd methods of enhancing the reliability and efﬁcacy of selected antagonists, and the ﬁeld is gaining momentum. It should aim at ﬁnding additives or physical control methods that will act synergistically with the antagonist. This involves combining the product with a low-level of postharvest fungicide or GRAS substances. It has been reported that physical treatments such as hot air, curing, hot water brushing and combinations of the above with pressure inﬁltration of calcium could also increase the efﬁcacy of antagonists. Using mixtures of antagonists, or combining antagonists with speciﬁc nutrients or sugar analogues, is also suggested as an approach to increase efﬁcacy. Genetic manipulation of antagonists is a ﬁeld in its infancy. Current efforts are focused on developing efﬁcient transformation procedures for yeast antagonists and inserting genes for tracking the antagonist in the environment rather than enhancing biocontrol (Yehuda et al., 2001). Other approaches could be: the insertion of the gene for amylase under the constitutive promoter in some BCAs to allow effective use of the fruit carposphere starch; biocontrol strains with a higher capability to exploit the nitrogen compounds present or with a higher transport or metabolism rate of the limiting factor can be developed, because nitrogen is often a limiting substance when the biocontrol mechanism of action is competition for nutrients; and use of mutants that use new substrates, not metabolized by the pathogen, to provide a nutritional advantage or attempt to obtain strains resistant to phenolic compounds (Bizeau et al., 1989). Early experiments in transformation for marker genes have been successful. Metschnikowia pulcherrima was transformed with the green ﬂuorescent protein gene (Nigro et al., 1999) and histidine auxotrophs of C. oleophila were transformed with HIS3, HIS4 and HIS5 genes (Chand-Goyal et al., 1999). In all cases, the transformed antagonists maintained their biocontrol capability and there were no detectable differences between the wild type and the transformants. All these studies were accomplished only to obtain variants of the antagonistic strains with a genetically stable marker. Jones and Prusky (2002) investigated the possibility of expressing a DNA sequence in S. cerevisiae to allow the production of a cecropin A-based antifungal peptide. Yeast transformants inhibited the growth of germinated Colletotrichum coccoides spores and inhibited decay developments caused by the pathogen in tomato fruit. The lack of activity toward non-target organisms by the peptide and the use of S. cerevisiae as a delivery system suggest that this method could provide a safe alternative for postharvest disease control. However, attempts to overexpress genes involved in biocontrol, for example, lytic enzymes, or engineering strains with desired biocontrol traits may soon yield positive results. 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Botanical Bulletin of Academia Sinica 45, 55–60. 10 Biological Control of Plant Diseases: An Overview and the Trichoderma System as Biocontrol Agents Abhishek Tripathi,1 Neeta Sharma2 and Nidhi Tripathi1 1Department of Bioscience and Biotechnology, Banasthali University, Banasthali Vidyapith, India; 2Mycology and Plant Pathology Division, Department of Botany, University of Lucknow, Lucknow, India Abstract Biocontrol is the reduction of inoculum density or disease-producing activities of a pathogen in its active or dormant state by one or more microorganisms, accomplished naturally. Research on biological control agents has utilized naturally occurring saprophytic soil fungi to compete with and/or destroy soilborne pathogens. Biological control has attracted attention from researchers for over 30 years, primarily because of the interest in developing more ‘environmentally friendly’ means of disease management in the absence of agricultural pesticides. Despite considerable effort in the area of biological control, few practical applications have become established in agriculture for the control of plant diseases. Common biocontrol agents include Trichoderma, Gliocladium, Aspergillus, Penicillium, Chaetomium, Dactylella, Glomus, etc. Biological control is achieved by competition, hyperparasitism, induced resisitance, hypovirulence, etc. Mycoparasitism and production of volatile and non-volatile antibiotics are important mechanisms operating in the case of Trichoderma, besides commercial uses and mass multiplication of the novel biocontrol agent. The future of biocontrol lies perhaps with the development of better application methods and the use of genetic engineering to increase the efﬁcacy of various wild strains. Introduction Empirical approaches to chemical disease control have been practised since ancient times, when concoctions consisting of salt brine, sulphur, lime, ashes and salts of copper, mercury and arsenic were used to combat disease. Reports of pesticide residues in food, soil, river and groundwater undermine consumers’ trust. Thus, the increasing concern, particularly in developed nations, is that modern methods of crop protection have an overall negative impact on the environment and on society. Pathogen resistance against certain classes of fungicides has further reduced the number of disease control measures available. In recent years, it has become evident, as a result of public opinion and environmental laws, that new and safer alternatives to traditional synthetic pesticides are both desirable and mandated. Research emphasis has therefore been on the development of alternative approaches to control the pathogens and pests of ornamental crops using biocontrol agents. There are considerably more success stories involving the control of insect pests. Garrett’s (1965) deﬁnition of biological control  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 121 122 A. Tripathi et al. of plant disease was, ‘any condition under which or practice whereby survival or activity of a pathogen is reduced through the agency of another living organism (except man himself), with the result that there is a reduction in the incidence of the disease caused by the pathogen’. Although biological control consists of diverse methods and approaches to suppress plant disease, in most cases antagonists to pathogens are added to the agroecosystem. Various approaches of biocontrol are directed at suppressing initial disease induced by a soilborne pathogen or the application of an avirulent isolate of the pathogen that ‘competes’ with the virulent pathogen on or in the host. Biological control employs living agents (usually antagonists or competitors of the causal agent) to control plant diseases. Effective biological controls take advantage of the natural competition of living organisms for limited resources or ecological niches. Thus, two organisms cannot occupy the same space at the same time, they cannot consume the same resource (e.g. food source) at the same time and, in some cases, one organism produces compounds that are inhibitory to the growth and development of the other organism. Certain microorganisms that normally compete for and live off debris and dead animal and plant cells in the soil environment have developed, through mutation, the ability to invade a host plant and escape the effects of antagonists. These invading organisms are referred to as pathogens. The lack of survival of the pathogen and the superior competitiveness of the antagonists relative to pathogens brings promise to the theory of using antagonists to control pathogens. Tubeuf (1914) coined the term ‘biological control’ in relation to plant pathogens, while Hartley (1921) ﬁrst attempted to control the root diseases of plants with introduced microorganisms. Cook and Baker (1983) deﬁned biological control as, ‘the reductions of the amount of inoculum or disease-producing activity of a pathogen accomplished by one or more organisms other than man’. Microorganisms, which are used in the management of plant diseases, are referred to as ‘biocontrol agents’. The important genera of fungi used as biocontrol agents are Trichoderma, Gliocladium, Aspergillus, Penicillium, Neurospora, Chaetomium, Dactylella, Arthrobotrys and Glomus, etc. According to Baker (1987), biological control is the decrease of pathogen activity accomplished by one or more organisms including the host plant but excluding humans. Harman (2000) deﬁned biological control as a critically needed component of plant disease management. Biocontrol agents are known as antagonists. The most important, well-studied antagonists against several plant pathogens are fungi like Ampelomyces sp., Aspergillus spp. (particularly A. niger and A. terreus), Chaetomium globosum, Coniothyrium minitans, Fusarium sp., Gliocladium virens, Penicillium citrinum, Peniophora gigantea, Trichoderma spp. (particularly T. harzianum and T. viride) and Sporodesmium sp.; and bacteria like Agrobacterium radiobacter strain K84, species of Bacillus, Enterobacter, Micromonospora, Pseudomonas and Streptomyces. Mechanisms of Biological Control of Plant Diseases Competition Competition occurs between microorganisms when space or nutrients (i.e. carbon, nitrogen and iron) are limiting and its role in the biocontrol of plant pathogens has been studied for many years, with special emphasis on bacterial biocontrol agents. An important attribute of a successful rhizosphere biocontrol agent would be the ability to remain at a high population density on the root surface, providing protection of the whole root for the duration of its life. Mycorrhizal fungi can also be considered to act as a sophisticated form of competition or cross-protection, decreasing the incidence of root disease. Fomes (Heterobasidion) annosum colonizes stumps of freshly cut pine and other conifers and spreads via root grafts to other healthy trees, where it causes root rot (refer to Chapter 26). Spraying freshly cut stumps with spore suspensions of Phlebia (Peniophora) gigantea will prevent Biological Control of Plant Diseases H. annosum from getting a foothold, and this is standard practice in the UK. Antibiosis Antibiosis is the inhibition of an organism by a metabolic product (such as an antibiotic) from another organism. Many organisms, especially soil fungi and Actinomycetes, produce antibiotic substances. The production of antibiotics by Actinomycetes, bacteria and fungi is demonstrated very simply in vivo. Numerous agar plate tests have been developed to detect volatile and non-volatile antibiotic products by putative biocontrol agents and to quantify their effects on pathogens. In general, however, the role of antibiotic production in biological control in vitro remains unproved. Three diseases can be controlled by antibiosis: Armillaria root rot by T. viride, Pythium and Rhizoctonia damping off and stem and root rot diseases by P. ﬂuorescens and crown gall by A. radiobacter. The most widely accepted commercial example is the control of crown gall using strain 84. Hyperparasitism and mycoparasitism Biological control can occur through direct parasitism. Parasitism involves the direct utilization of food of one organism by another organism. Hyperparasites are organisms parasitic on other parasites. Some have referred to this as ‘natural biocontrol’. A few examples of hyperparasitism include: Darluca (Sphaerellopsis) ﬁlum parasitizes rust fungi and species of Ampelomyces parasitize powdery mildews; Tuberculina maxima parasitizes the aecial stage of Cronartium ribicola, cause of white pine blister rust; T. viride, and a number of other species, are known to parasitize hyphae of R. solani. The most common example of mycoparasitism is that of Trichoderma sp., which attack a great variety of phytopathogenic fungi responsible for the most important diseases suffered by crops of major economic importance worldwide. 123 Hypovirulence Hypovirulence is a term used to describe reduced virulence found in some strains of pathogens. This phenomenon was ﬁrst observed in Cryphonectria (Endothia) parasitica (chestnut blight fungus) on European Castanea sativa in Italy, where naturally occurring hypovirulent strains were able to reduce the effect of virulent ones. These slower-growing hypovirulent strains contain a single cytoplasmic element of doublestranded RNA (dsRNA) similar to that found in mycoviruses, which is transmitted by anastomosis in compatible strains through natural virulent populations of C. parasitica. Hypovirulence has also been reported in many other pathogens, including R. solani, Gaeumannomyces graminis var. tritici and Ophiostoma ulmi, but the transmissible elements responsible for hypovirulence or reduced vigour of the fungi are subject to debate and may be due to dsRNAs, plasmids or viruses. Induced Resistance and Cross-Protection Induced resistance is a plant response to challenge by microorganisms or abiotic agents such that, following the inducing challenge, de novo resistance to pathogens is shown in normally susceptible plants. Both localized and systemic-induced resistance are non-speciﬁc and can act against a whole range of pathogens, but whereas localized resistance occurs in many plant species, systemic resistance is limited to some plants. Cross-protection differs from induced resistance in that, following inoculation with avirulent strains of pathogens or other microorganisms, both inducing microorganisms and challenge pathogens occur on or within the protected tissue. The most commonly reported examples of crossprotection involving fungi are probably those used against vascular wilts. Inoculation with non-pathogenic formae speciales of Fusarium and Verticillium species, or with other fungi or bacteria, has shown different levels of cross-protection. 124 A. Tripathi et al. Predation Predation has also been examined as a potential form of biocontrol. Nematode-trapping fungi and predaceous nematodes have been studied in detail as potential biological control agents, but ultimately have had little effect on the numbers of plant parasitic nematodes in the soil. Mycorrhizae Mycorrhizae are symbiotic (mutualistic) associations between fungi and plant roots. The increased surface area provided by mycorrhizal fungi allows for increased nutrient uptake, which indirectly beneﬁts disease management derived by healthier, more vigorous roots. Because of the generally beneﬁcial effect of mycorrhizae on plant growth and their common occurrence, many investigations have looked into the potential of root–fungus associations as potential biological control agents. Vesicular arbuscular mycorrhizal (VAM) fungi were recognized and described in the last few decades of the 19th century. The term ‘VAM’ was changed to ‘AM’ by Draft and Nicolson (1974) because some species did not form vesicles. AM fungi occur throughout the terrestrial ecosystem in almost all the herbaceous and woody plants, forming a symbiotic relationship with the roots (Gerdemann, 1968; Trappe and Fogel, 1977). This symbiotic association has been reported to play an important role in plant mineral nutrition (Gianinazzi and Gianinazzi-Pearson, 1986). It has been observed by several workers that these fungi facilitate the uptake of many nutrients (phosphorus, zinc, copper, sulphur, potassium, iron, calcium, etc.), resulting in increased biomass (Wani and Lee, 1992). Nutrient content of N, P and K, and also Fe, Mn and Cu, increased due to AM inoculation in papaya. Among all the AM species, G. mosseae was recorded as the most efﬁcient for nutrient uptake. Rajeshwari et al. (2001) reported that G. fasciculatum at low phosphorus level increased the root and shoot biomass. They also recorded that AM colonization was reduced at higher phosphorus level. Biocontrol of Airborne Diseases Many naturally occurring microorganisms have been used to control diseases on the aerial surfaces of plants. The most common bacterial species that have been used for the control of diseases in the phylloshpere include P. syringae, P. ﬂuorescens, P. cepacia, Erwinia herbicola and B. subtilis. Fungal genera that have been used for the control of airborne diseases include T. ampelomyces and the yeasts, Tilletiopsis and Sporobolomyces. Biocontrol agents normally must achieve a high population in the phyllosphere to control other strains, but colonization by the agent may be reduced by competition with the indigenous microﬂora. Integration of chemical pesticides and biocontrol agents has been reported with Trichoderma spp. and P. syringae. Biocontrol agents tolerant to speciﬁc pesticides could be constructed using molecular techniques. Resistance to the fungicide benomyl is conferred by a single amino acid substitution in one of the b-tubulins of T. viride, the corresponding gene thereby producing a biological control agent that could be applied simultaneously or in alternation with the fungicide. Biocontrol of Soilborne Diseases Chemical control of soilborne plant diseases is frequently ineffective because of the physical and chemical heterogeneity of the soil, which may prevent effective concentration of the chemicals from reaching the pathogen. Biological control agents colonize the rhizosphere, the site requiring protection and leave no toxic residues, as opposed to chemicals. Microorganisms have been used extensively for the biological control of soilborne plant diseases, as well as for promoting plant growth. Fluorescent Pseudomonads are the most frequently used bacteria for biological control and plant growth promotion, but Bacillus and Streptomyces Biological Control of Plant Diseases species have also been commonly used. Trichoderma, Gliocadium and Coniothyrium are the most commonly used fungal biocontrol agents. Perhaps the most successful biocontrol agent of a soilborne pathogen is A. radiobactor strain K84, used against crown gall disease caused by A. tumefaciens. Molecular techniques have also facilitated the introduction of beneﬁcial traits into rhizosphere competent organisms to produce potential biocontrol agents. Chitin and b-(1,3)-glucan are the two major structural components of many plant pathogenic fungi, except Oomycetes, which contain cellulose in their cell wall and no appreciable levels of chitin. Biological control of some soilborne fungal diseases has been correlated with chitinase production. Bacteria producing chitinases or glucanases exhibit antagonism in vitro against fungi. A recombinant Escherichia coli expressing the chiA gene from S. marcescens was effective in reducing disease incidence caused by Screrotium rolfsii and R. solani. In other studies, chitinase genes from S. marcescens have been 125 expressed in Pseudomonas spp. and the plant symbiont, Rhizobium meliloti. The modiﬁed Pseudomonas strain controlled the pathogens, F. oxysporum f. sp. rodelens and G. graminis var. tritici. Commercial Biocontrol Agents The following is a list of commercially available products formulated for the biocontrol of plant pathogens and/or plant growth promotion involving the induction of plant host defence. The list originated in 2000 through the efforts of Dr Deborah Fravel, USDA-ARS, and is now being updated by the APS Biological Control Committee (Table 10.1). The Trichoderma System as Biocontrol Agents Trichoderma spp. are free-living, saprophytic fungi that exhibit a high rate of interactions Table 10.1. Fungi, bacteria, activators and their available commercial products. Commercial products Fungi Ampelomyces quisqualis Candida oleophila Coniothyrium minitans Fusarium oxysporum Gliocladium sp. Myrothecium verrucaria Paecilomyces lilacinus Phlebia gigantea Pythium oligandrum Trichoderma sp. Bacteria Agrobacterium radiobacter Bacillus sp. Burkholderia cepacia Pseudomonas sp. Streptomyces sp. Activators of host defence Bacteria Bacterial protein Synthetic chemical AQ10 Aspire Contans, Intercept WG, KONI Biofox C, Fusaclean Primastorp, SoilGard DiTera Paecil Rotstop Polyversum Bio Fungus, Binab T, Root Pro, RootShield/PlantShield, T-22G, T-22 Planter Box, Trichodex, Trichopel, Trieco Galltrol, Nogall BioYield, Companion, EcoGuard, HiStick N/T, Kodiak, Rhizo Plus, Serenade, Subtilex, YieldShield Deny, Intercept BioJect Spot-Less, Bio-save, BlightBan, Cedomon Actinovate, Mycostop Actinovate, BioYield, YieldShield Messenger Actigard 126 A. Tripathi et al. with root, soil and foliar environments. The antagonistic nature of fungi from the genus Trichoderma was demonstrated more than 70 years ago. Furthermore, excellent progress has been made towards the improvement of Trichoderma sp. as a biological control agent in the past few years. Many Trichoderma isolates have been used as biocontrol agents against soilborne pathogens (Weindling, 1934). Trichoderma is a ubiquitous genus present in almost all types of habitat fungal antagonists. It comprises 3% of the total fungal population in forests and 1.5% of the total fungal population in other soils. It also exhibits the property of competition with fellow plant pathogenic fungi for key exudates from seeds that stimulate the germination of propagules of plant pathogenic fungi in soil, and also with soil microorganisms for nutrients and space. Trichoderma spp. act against a range of economically important aerial and soilborne plant pathogens. They have been used in the ﬁeld and greenhouse against silver leaf on plum, peach and nectarine; Dutch elm disease on elms, honey fungus (A. mellea) on a range of tree species and against rots on a wide range of crops, caused by Fusarium, Rhizoctonia, Pythium and Sclerotium (Table 10.2). Lacicowa and Pieta (1994) reported that Trichoderma spp. and Gliocladium sp. gave signiﬁcant control against soilborne pathogenic fungi of pea, which was better than that obtained with the use of chemicals. Spiers et al. (2004) described the mode of action of Trichoderma sp. against plant pathogens. Recently, Herrera-Estrella and Chet (2004) discussed the role of Trichoderma spp. as a biological control agent; the expression of mycoparasitism related genes (MRGs); antibiosis; the role of MRGs in biological control and strain improvement; competition; induced resistance; plant growth promotion; and Trichoderma spp. as a source of genes for crop improvement. The biocontrol action is due largely to the inherent nature of inhibition or degradation of pectinases and other enzymes, which are deemed essential for phytopathogenic fungi in order to cause pathogenesis in plants. These direct effects on other fungi are remarkable yet complex and, until now, were attributed to being the basis for the action exerted by Trichoderma sp. on plant growth and development. Mechanism of Action of Trichoderma Several modes of action have been proposed to explain the suppression of plant pathogens by Trichoderma spp. These include mycoparasitism, antibiosis, competition, siderophore production, induction of systemic resistance, growth promotion, etc. (Dennis and Webster, 1971; Upadhyay and Mukhopadhyay, 1986; Chet, 1987). Table 10.2. Trichoderma as biocontrol agents and their target pathogens which cause diseases in various host plants. Biocontrol agent Pathogens Host crop Trichoderma spp. T. harzianum Pythium sp. Fusarium oxysporum Fusarium sp. Pythium sp. Rhizoctonia solani Sclerotinia sclerotiorum Sclerotium rolfsii Gaeumannomyces sp. Pythium sp. R. solani Bean, pea, cucumber Cucumber, cotton, wheat, muskmelon, tomato, ginger Lentil, cotton Pea, radish, cucumber, tomato Pea, radish, snapbean Cucumber, Mentha sp. Sugarbeet, groundnut, chickpea, Mentha sp. Wheat White mustard Potato T. viride Biological Control of Plant Diseases Direct action of biocontrol agent Trichoderma Mycoparasitism Mycoparasitism is the phenomenon in which fungal parasites attack other fungi. It is divided into necrotrophic (destructive) and biotropic (balanced) parasitism (Barnett and Binder, 1973). Trichoderma spp. are grouped in necrotrophic mycoparsites. Velikanov et al. (1994) noticed hyperparasitism with different strains of T. viride, T. harzianum and G. virens, which were tested against ﬁve phytopathogenic fungi, namely F. oxysporum, F. solani, Pythium sp., R. solani and S. sclerotiorum causing root rot of pea. Trichoderma recognizes signals from the host fungus, triggering coiling and host penetration. Remote sensing is due at least partially to the sequential expression of cell wall degrading enzymes. Different strains can follow different patterns of induction, but the fungi apparently always produce low levels of an extracellular exochitinase. The possible role of agglutinins in the recognition process determining fungal speciﬁcity has been examined recently. Barak et al. (1985) proposed that lectins of plant pathogenic fungi might play a role in recognition. Inbar and Chet (1992) proved the role of lectins in recognition during mycoparasitism using a biometric system. Secretion of lytic enzymes, including b-1,3-glucanase(s), proteinase(s), chitinases and lipases, enables Trichoderma spp. to degrade the host cell wall, thereby reducing the incidence of disease (Harman, 2001). Ordentlinch et al. (1990) reported that there was no correlation between in vivo and separated in vitro dual culture or enzyme assays. Involvement of chitinase and b-1,3glucanase in Trichoderma-mediated biological control was also reported by Harman (2001). Involvement of b-1,6-glucanases and b-1,4-glucanases may also play an important role in mycoparasitism (Thrane et al., 1997). T. harzianum-mediated mycoparasitism may involve 20 separate genes and gene products; most of these gene products are synergistic with one another (Lorito, 1998). It is considered that mycoparasitism is one of the main mechanisms involved in 127 the antagonism of Trichoderma as a biocontrol agent. The process apparently includes: 1. Chemotropic growth of Trichoderma; 2. Recognition of the host by the mycoparasite; 3. Secretion of extracellular enzymes; 4. Penetration of the hyphae; and 5. Lysis of the host. Antibiosis The high percentage of effectiveness of the biocontrol ability of Trichoderma is conferred most likely by more than one exclusive mechanism. Another known mechanism of biocontrol is antibiosis, which is the release of antibiotics and other metabolites that are harmful to the pathogen and inhibit their growth. Many such substances have been isolated from Trichoderma sp., namely gliotoxin and glyoviridin from T. viride (Sharma and Dohroo, 1991), viridin, alkyl pyrones, isonitriles, polyketides, diketopiperazines and some steroids (Upadhyay and Mukhopadhyay, 1986). Many Trichoderma spp. are reported to produce volatile and nonvolatile antibiotics, chloroform soluble antibiotics, including trichodermin, and peptide antibiotics active against a range of plant pathogenic fungi (Dennis and Webster, 1971). Indeed some isolates of Trichoderma excrete growth-inhibitory substances. In fact, it seems advantageous for a biocontrol agent to suppress a plant pathogen using multiple mechanisms. Competition This mode of action implies the competition among microorganisms for space and nutrients when these factors are limiting in nature. It is considered a ‘classical’ mechanism of biocontrol. The mechanism is considered involved when no evidence of either mycoparasitism or antibiosis is found in a particular interaction. Since Trichoderma is an omnipresent fungus and is found in agricultural and natural soils throughout the world, it is enough proof of it being an excellent competitor for space and nutritional resources. Excellent competitiveness for space and nutrition is supposed to be 128 A. Tripathi et al. useful for biological control in the absence of mycoparasitism or antibiosis (Cook and Baker, 1983). Elad (2000) reported that when conidia of T-39 were sprayed on leaves, germination of conidia of B. cinerea was slowed down, because the pathogenic conidia required external nutrients for germination and infection. Indirect action of biocontrol agents In addition to the ability of Trichoderma spp. to attack or inhibit the growth of plant pathogens directly, recent discoveries indicate that they can also induce systemic and localized resistance to a variety of plant pathogens. Biochemical elicitors of disease resistance and induced systemic resistance Induced systemic resistance (ISR) is another phenomenon of biocontrol exhibited by the plant to combat the harmful effects of the pathogen. It implies the elicitation of resistance or plant response against the microorganism or abiotic agent, such that following the inducing challenge posed to the plant, de novo resistance to pathogens is shown in normally susceptible plants. Localized and systemic induced resistance occurs in all or most plants in response to attack by pathogenic microorganisms, physical damage due to insects or other factors, treatment with various chemical inducers and the presence of non-pathogenic rhizobacteria. Speciﬁc strains of fungi in the genus Trichoderma colonize and penetrate plant root tissues and initiate a series of morphological and biochemical changes in the plant, which are considered to be part of the plant defence response. Finally, it leads to ISR in the entire plant. The capability of T. harzianum to promote increased growth response was veriﬁed both in greenhouse experiments and in the hydroponic system. A 30% increase in seedling emergence was observed and these plants exhibited a 95% increase in root area. Similarly, an increase in P and Fe concentration was observed in Trichoderma inoculated plants. In recent times, there has been tremendous progress related to pathways of resistance and much has been done to elucidate them. In many instances, salicylic acid or jasmonic acid, together with ethylene or nitrous oxide, induce a cascade of events that lead to the production of a variety of metabolites and proteins with diverse functions. Different pathways are induced by different challenges, although there seems to be crosstalk or competition between pathways. There has been a great leap in explaining the ISR pathway activated by rhizobacteria; the best part is that it is the closest analogue of induced resistance activated by Trichoderma. The rhizobacteria-induced systemic resistance (RISR) pathway phenotypically resembles systemic acquired resistance (SAR) systems in plants. Heil (2001) deﬁned ISR as the set of changes by which plants respond to an initial infection or elicitor treatment in becoming systemically resistant against pathogen attack. Several workers demonstrated that Trichoderma spp. could also affect the host plant, which shows an induced resistance-type response. Chang et al. (1986) reported hastened ﬂowering, increased number of blooms in Chrysanthemum and an increase in the height and weights of other plants as a result of T. harzianum inoculation in steamed soil. Trichoderma viride-coated seeds of broad bean resulted in increased fresh and dry weight of shoots, roots and nodules (Yehia et al., 1985). Pea seeds treated with apple pomace-based Trichoderma inoculant extracts resulted in increased emergence, rapid plant growth, increased seedling vigour and phenolics content. The increase in overall phenolic content may contribute to improved ligniﬁcation and antioxidant response (Zheng and Shetty, 2000). Altomore et al. (1999) reported for the ﬁrst time the ability of a Trichoderma strain (T-22) to solubilize insoluble or sparingly soluble minerals by three possible mechanisms, namely acidiﬁcation, production of chelating agents and redox activity. Further, they reported the solubilization of Fe2O3, MnO2, Zn and rock phosphate by the Biological Control of Plant Diseases cell-free culture ﬁltrate of T-22. Trichoderma strains are also supposed to induce the production of hormone-like metabolites on release of nutrients from soil or organic matter (Kleifeld and Chet, 1992). Chemicals Produced by Trichoderma What has been stated above is the induced resistance exhibited by some plants that is a result of some microorganism, in this case, Trichoderma. In this context, it has been found that Trichoderma produces three classes of compounds to exert its effect and induce resistance in plants. These include: Proteins with enzymatic or other functions With regards to the ﬁrst class of biochemical elicitors of Trichoderma, it is stated that much before the discovery of the induction of resistance by Trichoderma, a small 22-kDa xylanase protein was shown to induce ethylene production and plant defence. Working in the direction of Trichoderma, it has been found very recently that a series of proteins and peptides that are active in inducing terpenoid phytoalexin biosynthesis and peroxidase activity in cotton are produced by strains of T. virens. Avr homologues Another class is the protein product of Avr genes, which have been identiﬁed in a variety of fungal and bacterial plant pathogens. These are usually seen functioning as raceor pathovar-speciﬁc elicitors, possessing the capability of inducing hypersensitive responses and other defence-related reactions in plant cultivars that contain the corresponding resistance gene. Proteome analysis of T-22 identiﬁed proteins that are homologues of Avr4 and Avr9 from Cladosporium fulvum; T. atroviride strain P1 also produces similar proteins. 129 Oligosaccharides and low molecular weight compounds Another ﬁnding in this sphere has been the transformation of Trichoderma mutants with reporter based on green ﬂuorescent protein or speciﬁc enzymatic activities (glucose oxidase) under the control of biocontrolrelated promoters. One of the advantages of this discovery has been the possibility that biomolecules released by the action of Trichoderma secreted cell wall degrading enzymes on the cell walls of fungal pathogens and plants can be isolated. These molecules function as inducers of the antagonistic gene-expression cascade in Trichoderma and some also function as elicitors of plant defence mechanisms. Plant Growth Promotion Fungal as well as bacterial biocontrol agents are reported and known to induce growth of various crops and also increase crop yield. Trichoderma spp., and other beneﬁcial root-colonizing microorganisms, also enhance plant growth and productivity. Mukhopadhyay (1996) has reported increased growth of several crop plants following seed treatment with T. harzianum and T. virens. The reason attributed to this effect of Trichoderma and other microbes on plants has been explained based on the following arguments. 1. Suppression of harmful root microﬂora, including those not a direct causal organism of disease. 2. Production or activation of growthstimulating factors. 3. Increased nutrient uptake through solubilization and sequestering of nutrients. It is a well-established fact that microorganisms closely associated with the roots of a plant can inﬂuence plant growth and development directly. Although the ability of species of Trichoderma spp. to promote or inhibit plant growth directly has been noted for many years (Ozbay and Newman, 2004), efforts to deﬁne and exploit these inﬂuences 130 A. Tripathi et al. have met with limited success. Many workers have reported plant growth promotion by different strains of Trichoderma spp. Chang et al. (1986) observed plant growth promotion resulting in enhanced germination, more rapid ﬂowering, increased ﬂowering and increased height and fresh weight in pepper, periwinkle, Chrysanthemum and several others after treatment of the soil with peat/bran inoculum or conidial suspension of T. harzianum. Solubilization and Sequestration of Inorganic Plant Nutrients It is a common natural occurrence that plant nutrients undergo a complex, intricately woven conversion from soluble to insoluble forms when in the soil; this is a precursor to the ease of access and absorption by roots. It is here that microorganisms may inﬂuence these transitions (Altomare et al., 1999). The most commonly and extensively studied nutrients are iron and manganese. Trichoderma sp. has been reported to produce some compounds called siderophores (Sen, 2000). Iron chelated with these siderophores is in the unavailable and bound form for plant pathogens and so they do not have access to iron. On the contrary, plant roots are capable of absorbing iron in this form, so these are accessible to the plant. This is one of the mechanisms that operate for the growth of plants and the supply of nutrients to them. Trichoderma sp. increases the uptake and concentration of a variety of nutrients (copper, phosphorus, iron, manganese and sodium) in roots of hydroponic culture, even under axenic conditions. This increased uptake indicates an improvement in plant active uptake mechanisms. Pesticide Susceptibility Another aspect and quality of Trichoderma sp. lies in the fact that it possesses innate and natural resistance against most agricultural chemicals, including fungicides. The capability differs with strain. Some lines have been selected or modiﬁed to be resistant to speciﬁc agricultural chemicals. Mass Multiplication of Trichoderma The most critical obstacles to the application of biological control fungi as an effective means of disease management are the lack of knowledge of methods for mass culturing and a proper delivery system, which is needed to augment the soil directly with fungal antagonists (Papavizas, 1985; Singh et al., 2002, 2004; Dissevelt and Ravensberg, 2004). Solid media for the experimental production of Trichoderma sp. and Gliocladium sp., two of the most common fungal antagonists, have been used frequently in laboratory and greenhouse studies (Bateman, 2004). Some workers have tried composted hardwood bark as a substrate for the largescale production of biocontrol fungi (Nelson and Hoitink, 1983). Sundheim (1977) used bark pellets as a medium for mass production of Trichoderma and Gliocladium sp. to control Phomopsis sclerotioides in cucumber. A variety of media have been used by various researchers for the production of Trichoderma sp. in stationary ﬂasks, shakers (Jin et al., 1991) and liquid fermenters (Jin et al., 1996). Backman and Rodriguez-Kabana (1975) used diatomaceous earth granules along with molasses for developing a formulation of biocontrol agents for application in soil. Hadar et al. (1979) used wheat bran formulations for mass-multiplying biocontrol agents for ﬁeld application. Papavizas et al. (1984) developed a liquid fermentation technology for mass production of fungal antagonists by employing a combination of molasses and brewer’s yeast. Sivan et al. (1984) developed a formulation of T. harzianum on wheat bran and peat. Mukhopadhyay et al. (1986) used sorghum grains to prepare the powdered formulations of fungal antagonists. Tapioca rind, cow dung, biogas slurry, farmyard manure, paddy chaff, rice bran, groundnut shell, sugarcane bagasse, sheep manure, chickpea husk, maize cob, etc., are some of the substrates used for mass Biological Control of Plant Diseases multiplication of T. harzianum and T. viride (Kousalya and Jeyarajan, 1990). Conway et al. (1996) used oat seeds for mass culturing of T. harzianum isolate OK-86. Alginate pellets were used for formulating a biomass of G. virens and T. hamatum and various food bases like wheat bran, maize cobs, groundnut hulls, soy ﬁbre, castor pomace, cocoa hulls and chitin were used. They found that the pellets with G. virens and all the food bases with bran, soy ﬁbres, castor, pomace or chitin resulted in stands similar to those of the control, except cocoa hull meal signiﬁcantly reduced damping-off of Zinnia caused by R. solani and P. ultimum. Kumar and Marimuthu (1997) tested the effect of decomposed coconut coir pith (DCCP) added to normal nursery media on the survival of T. viride. The pure DCCP gave efﬁcient sporulation of T. viride population. Lewis et al. (1998) used commercially manufactured cellulose granules (Biodac) in a mixture with a sticker and fermenterproduced biomass of Trichoderma sp. and G. virens to produce a formulation in which chlamydospores in the biomass were activated with dilute acid. Tiwari et al. (2004) suggested that among the eight substrates, namely grains of Sorghum vulgare [S. bicolor], wheat, Pennisetum typhoides [P. glaucum], S. vulgare cv. M.P. Chari and Sorghum sp., a locally available millet; wheat bran; rice bran; and sugarcane bagasse were evaluated for the mass propagation of T. viride. Sorghum sp., a locally available millet, resulted in the greatest spore concentration, spore viability and total biomass of the fungal antagonist. The greatest spore concentration (8 × 109) was observed after 15 days of incubation at 27 ± 1°C. The spores of T. viride remained viable for 6 months at 5°C. 131 would be beneﬁcial to a larger degree than individual components. A primary obstacle in the commercial use of Trichoderma spp. for both disease control and growth enhancement is the mass production and delivery methods of its formation to the plants (Papavizas, 1985; Mukhopadhyay, 1996). The problem lies in the fact that biocontrol products represent living systems. A large number of growth media are reported to be suitable for the genus Trichoderma, but most of these are either food grains or are expensive. For solid-state fermentation substrates like sorghum grain, wheat grains, wheat bran, tea leaf waste, coffee husk, sawdust, etc., have been used (Gogoi and Roy, 1996; Mishra, 1998). A liquid fermentation method consisting of molasses, wheat bran and yeast is proposed for largescale production of Trichoderma (Montealegre et al., 1993). Bioefﬁcacy of T. harzianum produced by solid fermentation, which contains only conidia, was found more effective than when produced by liquid fermentation, where a mixture of chlamydospores, hyphal fragments and conidia were present. Conidia of Trichoderma in pyrophyllite survived better than alone at between –5 and 30°C. A temperature range from –5 to 5°C was found most suitable for an improved shelf life (Mukherjee, 1991). Mukherjee reported that shelf life of T. virens was almost constant on coated chickpea seeds at 5°C and, at room temperature, it was decreased by 12%. Chlamydospore-based formulations exhibited longer shelf life than conidiabased formulations (Mishra et al., 2001). Basic Components of Biocontrol Systems There are three basic components of biocontrol systems. These are as follows: Commercial Use of Trichoderma Commercialized systems for the biological control of plant diseases are few. It has been stressed that microbes cannot be used in isolation and exceptional results expected. On the contrary, a biocontrol system or consortia needs to be developed, which Biocontrol strain The ﬁrst step towards successful biocontrol is to obtain or produce a highly effective biocontrol strain or other material (Table 10.3). For instance, the development of the T-22 132 A. Tripathi et al. Table 10.3. Inexpensive production and formulation of the biocontrol agent using various base materials. Base material Biocontrol agent Formulation References Blackgram shell, shelled maize cob, coir pith, peat, gypsum, barley grains Coffee fruit skin + biogas slurry Trichoderma viride, T. harzianum T. harzianum Powder Coffee husk T. harzianum, T. viride, T. virens T. harzianum, T. viride, T. virens T. harzianum, T. viride, T. virens T. viride T. viride T. harzianum, T. viride T. harzianum, T. virens Kumar and Marimuthu, 1997 Sawant and Sawant, 1996 Bhai et al., 1994 Coffee berry husk Fruit skin and berry mucilage Groundnut shell Mustard oil cake Soil Sorghum grain Sugarcane straw Wheat bran Rice husk, maize cob powder, spent tea leaves, wheat bran, citrus fruit pulp T. harzianum, T. viride, T. reesei, T. koningii T. virens T. harzianum (MTCC 3843) strain of T. harzianum by Harman and fellow researchers was the result of a decade and more of hard work. Still, its commercial product, Root Shield, picked up pace in the late 20th century (Harman, 2000). Besides the usual properties of a biocontrol agent, the strain must also possess the following: (i) to be able to compete and persist in the environment in which it must operate and (ii) ideally, to be able to colonize and proliferate on existing and newly formed plant parts well after application. Sundaram (1996) developed fusants of two isolates of T. harzianum (Th-1 and Th-2), among them some showed morphological characters immediately between Th-1 and Th-3. When T. harzianum (Th-3) was fused with T. virens, many fusants were developed and few exhibited improved biocontrol activity (Ghosh, 1996) (Table 10.4). Ease of delivery and application Some delivery methods for Trichoderma are listed in Table 10.5. Pellets Pellets Pellets Pellets Powder Pellets Powder Powder Sawant and Sawant, 1989 Sawant and Sawant, 1989 Pellets Singh, 2002 Upadhyay and Mukhopadhyay, 1986; Mishra, 1998 Singh et al., 2004 Powder Powder Singh et al., 2002 Tripathi, 1998 Compatibility Testing of Trichoderma The success of a biocontrol agent depends on its compatibility with other disease management systems. This requires holistic testing of biocontrol agents (BCA) in combination with other disease management practices in a system approach. Once the BCA is found to be compatible, it can be integrated successfully with the disease management modules for each cropping system. Csinos et al. (1983) evaluated the compatibility of Trichoderma spp. with fungicides for the management of S. rolfsii in groundnut. T. harzianum, Rhizobium and carbendazim were integrated successfully for the management of stem rot of groundnuts caused by S. rolfsii. A combination of either Trichoderma or Gliocladium with fungicides like carboxin or metalaxyl protected crop plants against soilborne pathogens and was emphasized by several workers (Sawant and Mukhopadhyay, 1990; Mukhopadhyay et al., 1992). The alternation of BCA with fungicides was found to be more effective than mixtures. Biological Control of Plant Diseases 133 Table 10.4. Commercial products of Trichoderma currently in the open market or under registration. Product Biocontrol agent Effective against Manufacturer/distributor Antifungus Trichoderma sp. Various fungi Bas-derma Binab T Various fungi Control of wound decay and wood rot Bioderma T. viride Basarass T. harzianum (ATCC 20476) and T. polysporum (ATCC 20475) T. harzianum/T. viride Grondortsmettigen De Cuester n.v., Belgium Biocontrol Res. Lab., India Bio-innovation AB, UK Biofungus Trichoderma sp. Bio-trek 22G T. harzianum Sclerotinia, Phytophthora, Rhizoctonia solani, Pythium spp., Fusarium, Verticillium Various fungi Ecoﬁt T. viride Various fungi Root pro, Root Protato T. harzianum Root shield, Plant shield, T-22 Planter Box RUTOPIA T. harzianum Rifai strain KRL-AG(T-22) Trichoderma sp. R. solani, Pythium spp., Fusarium spp. and Sclerotium rolfsii Pythium spp., R. solani, Fusarium spp. SoilGard (formerly GlioGard) Supresivit Trichoderma sp. T. harzianum T-22 G, T-22 HB Trichoderma 2000 Trichodex, Trichophel Trichophel, Trichoject, Trichodowels, Trichoseal Tri-control Trieco T. harzianum strain KRL-AG2 Trichoderma spp. Trichoderma sp. T. viride TY Tusal Trichoderma spp. Trichoderma spp. T. harzianum T. harzianum and T. viride Various fungi Organic Soil Amendment Turfgrass Biostimulant Damping-off diseases caused by Pythium and Rhizoctonia spp. Various fungi Various fungi R. solani, S. rolfsii, Pythium spp., Fusarium spp. Botrytis of vegetables and grapevines Armillaria, Botryosphaeria, Chondrosternum, Fusarium, Nectria, Phytophthora, Pythium, Rhizoctonia Various fungi Rhizoctonia spp., Pythium spp., Fusarium spp., root rot, seedling rot, collar rot, red rot, damping-off Fusarium wilt Various fungi Damping-off diseases caused by Pythium, Phoma and Rhizoctonia species, rhizomania disease of sugarbeet and drop of lettuce Biotech International Ltd., India Grondortsmettigen De Cuester n.v., Belgium Bioworks, Inc. of Geneva, NY Hoechst Schering Agro Evo Ltd., India Efal Agr, Israel Bioworks Inc., USA NaEx Corp/Poulenger USA, Inc USA Borregaard and Reitzel, Czech Republic THT Inc., USA Myocontrol Ltd., Israel Makhteshim Chemical Works Ltd., USA Agrimm Technologies Ltd., New Zealand Jeypee Biotechs, India Ecosense Labs Pvt. Ltd., Mumbai, India Myocontrol, Israel Spain 134 A. Tripathi et al. Table 10.5. Mass production and delivery methods of Trichoderma. Biocontrol agent Mass production Trichoderma viride Commercially produced pellets Applied directly to the soil along (BINAB T SEPPIC). Also produced with food base on wheat bran: sawdust and tap water (3:14). Have been produced on a variety of growth media (autoclaved rye, barley and sunﬂower seeds) As in T. viride; also produced on Backman and Rodriguez-Kabana molasses and enriched clay applied it @ 140 kg/ha after 70 granules as food base days of planting T. harzianum Delivery method Integration of T. harzianum with a sublethal dose of methyl bromide (300 kg/ha) and soil solarization yielded maximum control of Fusarium crown and root rot of tomato caused by F. oxysporum f. sp. radicis-lycopersici (Sivan and Chet, 1993). In order to get the maximum efﬁciency from Trichoderma, it is important that it should be applied properly. It is effective as a seed treatment with or without fungicides. The basic reason why this is used is its multifaceted nature and broad range. It colonizes roots, increases root mass and improves plant health, and consequently provides yield increases, which chemical fungicides applied at reasonable rates cannot do. It can also be used in conjugation with other microbes, which thereby increases its efﬁciency. The two-pronged advantage would be a reduction in the use of pesticides and limiting root-attacking diseases, plus protection of transplants in the ﬁeld by virtue of its ability to colonize roots. Besides this, powdered formulations can be made and applied to the seed directly, and then the seeds are sown. This would reduce the amount of biocontrol agent used, as well as protect the plants from pathogen attack. Further, plant growth would also improve. References Altomare, C., Norvell, W.A., Björkman, T. and Harman, G.E. 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(2000) Enhancement of pea (Pisum sativum) seedling vigor and associated phenolic content by extracts of apple pomace fermented with Trichoderma spp. Crop Protection 36(1–2), 79–84. 11 Physiological Specialization of Ustilaginales (Smut) of Genera Bromus, Zea and Triticum in Argentina Marta M. Astiz Gassó1 and María del C. Molina1,2 1Instituto Fitotécnico de Santa Catalina (IFSC) and 2Consejo Nacional de Investigaciones Cientíﬁcas y Tecnológicas (CONICET), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Llavallol, Buenos Aires, Argentina Abstract The objective of this project was to determine the existence of physiological forms of Ustilaginales in Bromus, Zea and Triticum types in Argentina. Studies were carried out on the physiological specialization of Ustilago bullata Berk on Bromus spp., Zea seedlings’ reaction to inoculation with U. maydis (D.C.) Corda and physiological specialization of Tilletia laevis Wallr. (common bunt) on Triticum spp. The smut was collected in different agricultural and cattle-raising regions in the country, using Ustilaginales taxonomic keys for smut identiﬁcation and classiﬁcation. The experiments were carried out in greenhouses and in ﬁelds at the Instituto Fitotecnico de Santa Catalina (FCAyF-UNLP). For U. bullata and T. laevis, the techniques used were as follows: inoculation by sprinkling of teliospores on host seeds and inoculation by hypodermic syringe with suspension of U. maydis sporidia on plantlets of Z. mays and related wild species. As a result of said studies, it was determined that: (i) different physiological forms exist in each of the kinds of smut analysed; (ii) genetic variability exists in the hosts which have genes that express different degrees of resistance to the disease; and (iii) genetic improvement is the most efﬁcient and least environmentally harmful method. Introduction Smuts are pathogens of plants that belong to Phylum Basidiomycota, Class Ustilaginomycetes, Order Ustilaginales. Smut has the characteristic of forming greyish-black powdery masses of teliospores (basidiospores) on different organs such as the seeds, stems, leaves, ﬂowers and fruit of the hosts. Approximately 1400 species of smut are known, which attack around 75 families of Angiospermae; the most familiar diseases are those affecting Monocotyledoneae, especially cere138 als, where the pathogens produce important economic losses (Fischer and Holton, 1957; Hirschhorn, 1986; Snetselaar and Mims, 1992). Until the 20th century, they were considered, worldwide, one of the most serious causes of loss of grain and/or seeds, similar to the effects produced by rust. In Argentina, between 1934 and 1995, Hirschhorn and collaborators carried out several studies on Ustilaginales covering the taxonomic classiﬁcation of the species, geographical distribution, germination types and histopathology and cytology of the different  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Physiological Specialization of Ustilaginales (Smut) species of smut (Hirschhorn, 1986). Currently, control of these diseases is by means of agrochemicals and, on a smaller scale, by obtaining species with resistant genes through improvement programmes and studies on variability of these pathogens. The objective of this project was to determine the existence of physiological forms of Ustilaginales in Bromus, Zea and Triticum in Argentina: 1. Physiological specialization of U. bullata Berk on Bromus spp. 2. Zea seedlings’ reaction to inoculation with U. maydis (D.C.) Corda. 3. Physiological specialization of T. laevis Wall. (common bunt) on Triticum spp. Physiological Specialization of Ustilago bullata Berk on Bromus spp. Head smut (U. bullata Berk) is a pathogen which affects the growth of various grass species, especially within the genus Bromus. The disease is initiated when fungal hyphae penetrate seedlings; the attack develops from the inﬂorescences at the expense of the ovaries, forming a typical sorus. Severe infection affects limbs and glumes, reducing seed and forage production. In the USA, Fischer and Holton (1957) and Hirschhorn (1977, 1986) veriﬁed experimentally the existence of genes for resistance, physiological forms and the ability to cross-breed U. bullata and U. striiformis. Kreizinger et al. (1947) recorded the different reactions of U. bullata on Bromus which grew in the mountains and Bromus which grew on the plains; these experiences indicated that resistant Bromus varieties and lines could be obtained by artiﬁcial infection under controlled conditions and in the ﬁeld. Also, 13 physiological forms of the pathogen could be studied (Meinrs and Fischer, 1953). In New Zealand, Falloon (1976, 1979a,b) carried out studies on the effect of U. bullata infection on B. catharticus. Also, Falloon and Hume (1988) reported the effects of the pathogen on B. willdenowii productivity and endurance in the ﬁeld. In Argentina, Hirschhorn (1977) studied teliospore morphological variations 139 in samples of B. catharticus, B. mollis, Hordeum jubatum and H. compresum. The author also determined that Bromus head smut in Argentina was represented by U. bullata, U. bullata cv. macrospora (Hirschhorn, 1977, 1986). Astiz Gassó (1983, 1985, 1994) reported the presence of genes for resistance to the pathogen and in vitro U. bullata teliospore formation, a phenomenon unrecorded for this pathogen and uncommon in other smuts. The objective of this work was to determine the existence of physiological forms in U. bullata populations on several Bromus species. In this experiment, we used seeds of B. catharticus. B. parodii, B. brevis, B. auleticus and B. inermis cv. gombaszpuzta were provided by the Instituto Fitotécnico de Santa Catalina, FCAyF and Department of Genetics, and the Experimental Estación of Pergamino (INTA). The seeds were deinfested with a 2% formaldehyde solution for 20 min and then washed in sterile water three times. For identiﬁcation of the pathogen, spores from each isolate harvested from plants naturally infected in the ﬁeld were examined microscopically (Table 11.1). Viability of teliospores was tested by plating them in PDA medium 2% (Fischer and Holton, 1957). The seeds were infested with teliospores (1.8 × 10 3g teliospore/g seed), placed in sulphite paper envelopes and shaken well, so that spores would stick to the seed. Precautions were taken to avoid contamination with the different isolates of the pathogen. Thirty live seeds per isolate in three replications were inoculated during 4 consecutive years. An uninoculated sample was also included during the study. Inoculated samples were sown in experimental plots 1.5 m × 0.40 m in three rows Table 11.1. Ustilago bullata isolates collected in different localities in Argentina. Locality Province Pergamino Tres Arroyos Llavallol Gowland General Roca Check Buenos Aires Buenos Aires Buenos Aires Buenos Aires Río Negro Mixture 140 M.M. Astiz Gassó and M. del C. Molina with a distance of 0.20 m between them. The experimental design used was a randomized complete block. Evaluations in the ﬁeld were conducted by head countings, recording the percentage of infection based on the number of infected and healthy heads. Then, the average infection for the 4 years was calculated. The level of resistance/susceptibility was determined using a disease rating scale (Table 11.2). Isolates showed an 80–90% teliospore germination, approximately 20–25 h after they were cultivated on PDA. The teliospore germination rate increased with temperature from 20 to 25°C, with signiﬁcant amongpopulation differences. Boguena et al. (2007) also obtained similar results when they examined the effect of temperature from teliospore germination. Table 11.3 shows the reaction of the Bromus species tested with the different U. bullata isolates. Bromus catharticus was susceptible to all isolates including the mixture and similar results were reported for Astiz Gassó and Aulicino (1999); B. parodii showed similar reactions, Table 11.2. bullata. Disease rating scale for Ustilago Reaction Infection (%) Resistant (R) Moderately resistant (MR) Moderately susceptible (MS) Susceptible (S) 0–5 6–10 11–30 31–100 Table 11.3. Argentina. but the levels of infection were lower than B. catharticus; B. brevis gave a resistant reaction to isolate Gowland, a moderately resistant reaction to Pergamino, Llavallol and the mixture, a moderately susceptible reaction to isolate Tres Arroyos and a susceptible reaction to General Roca. Similar results were reported previously by Astiz Gassó (1983). B. auleticus and B. inermis cv. gombaszpuzta were resistant to all isolates and the uninoculated check did not show any infection. Four physiological forms in the populations of U. bullata are shown in Fig. 11.1: (i) Tres Arroyos; (ii) Pergamino and Llavallol; (iii) Gowland; and (iv) General Roca. The species B. brevis would be the differential host. Reaction to Inoculation with Ustilago maydis (D.C.) Corda on Zea seedlings Ustilago maydis is a smut that promotes the development of galls in Zea, the relation with the host being necessary to fulﬁl its life cycle. Damage produced in plants by the presence of corn stunt is: chlorosis, seedling death and tumours in leaves, stems, ears and tassels. At ﬁrst, it was considered that U. maydis attacked Z. mays and Z. mexicana, but it was later veriﬁed that it also attacked Z. perennis, Z. diploperennis, Z. parviglumis, Z. luxurians and their hybrids with the grown species (Hirschhorn, 1986; Duran, 1987). Reaction of Bromus species to different Ustilago bullata collected in different localities in Ustilago bullata isolates HOSTS Bromus catharticus B. parodii B. brevis B. auleticus B. inermis cv. gombaszpuzta Nor-inoculated check Pergamino Tres Arroyos Gowland Llavallol General Roca Mixture S S MR R R S S MS R R S S R R R S S MR R R S S S R R S S MR R R 0 0 0 0 0 0 Physiological Specialization of Ustilaginales (Smut) 141 100 90 80 Infection (%) 70 60 50 40 Bromus catharticus Bromus parodii Bromus brevis Bromus auleticus Bromus inermis cv Non-inoculated check 30 20 10 0 PERGAMINO TRES ARROYOS GOWLAND LLAVALLOL GENERAL ROCA MIXTURE Isolates Fig. 11.1. Reaction of Bromus spp. to U. bullata isolates. Until 1964, corn stunt did not any have incidence at the Instituto Fitotécnico de Santa Catalina, but in that year, a Z. perennis from Jalisco (México) was introduced and later on Z. mexicana, Z. parviglumis, Z. luxurians and Z. diploperennis were also grown and hybridized to Z. mays. As the hybrids are grown in the ﬁeld as well as in the greenhouse, vegetative plants are available throughout the year (Astiz Gassó and Molina, 1996). The pathogen multiplies on these plants with the corresponding increase in the number of spores disseminated by air and in the soil. Losses from corn smut range from 1% to up to 10% of all Zea species and hybrids are also attacked, depending on the environmental conditions favouring pathogen development; sweet corn may show losses approaching 100% from corn smut in localized areas (Callow and Ling, 1973; Hirschhorn, 1986; Banuett, 1995; Astiz Gassó and Molina, 1999). In this chapter, the results from analysing the response of Z. mays, Z. perennis and Z. diploperennis seedlings when they are inoculated with six populations of U. maydis are presented. This was done with the purpose of determining resistance of the species and/or inbreds to U. maydis. The host materials used were the population ‘Colorado Klein’, the inbreds SC66, B73, E624A688 of Z. mays, as well as clones of Z. perennis and Z. diploperennis. Over a time period of 2 years, 1296 plants were inoculated with different strains of U. maydis isolated from the province of Buenos Aires (Santa Catalina, Balcarce, Necochea and 25 de Mayo), the province of Entre Ríos (Paraná) and the province of Córdoba (Río Cuarto). These strains were cultivated in a liquid medium of PDB 2% on a shaker for 18–24 h running at 25°C ± 2. The pathogen was inoculated by puncturing the base of the seedling with a hypodermic syringe and the sporidial suspension with concentrations 105–106 sporidia/ml was then forced up into the leaf whorl (Callow and Ling, 1973; Snetselaar and Mims, 1992, 1993; Banuett, 1995; Edmunds, 1998; du Toit and Pataky, 1999). In many previous works, this method was very successful in producing disease galls in seedlings (Astiz Gassó and Molina, 1999). 142 M.M. Astiz Gassó and M. del C. Molina The trial involved three replications and a tester (non-treated plants). The plants were evaluated using a reaction scale to determine the mean percentage of infection with U. maydis (Table 11.4). The ﬁrst symptoms in seedlings were observed 4–6 days after inoculation and gall development occurred 7–8 days after the treatment (Fig. 11.2). The behaviour of the host when inoculated with six populations of U. maydis was analysed in Fig. 11.3. The hosts that reacted forming galls (grade 4) were cv. Colorado Klein: Necochea (8.34%) and Balcarce (2.78%); B73: Río Cuarto (14.15%), 25 de Mayo (11.11%), Santa Catalina (5.84%) and Balcarce (1.04%); E642A688: 25 de Mayo (8.33%) and Santa Catalina (3.34%); SC66: Río Cuarto (4.55%); Z. perennis: Santa Catalina (1.67%) and Z. diploperennis: 25 de Mayo (13.89%), Paraná (2.78) and Santa Catalina (1.67%). Table 11.4. Reaction scale in hosts. Behaviour Host reaction 0 = Immune 1 = Resistant 2 = Medium resistant No reaction Partial chlorosis Accent chlorosis and/or presence of stripe or anthocyanin stain Necrosis and reduction of growth in plant Formation of tumours 3 = Medium susceptibility 4 = Susceptibility (a) (b) (c) (d) (e) (f) Fig. 11.2. Reaction of hosts after inoculations with U. maydis. (a) No reaction, immune; (b) partial chlorosis; (c–d) accent chlorosis and/or presence of stripe or anthocyanin stain; (e) necrosis and reduction of growth in plant; (f) formation of tumours (galls). Physiological Specialization of Ustilaginales (Smut) 143 % reaction % reaction 60.00 50.00 40.00 30.00 20.00 10.00 0.00 Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta. Necochea Balcarce de Mayo Parana Cuarto Catalina (b) Isolates Isolates 50.00 % reaction 60.00 % reaction 20.00 0.00 Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta. Necochea Balcarce de Mayo Parana Cuarto Catalina (a) 40.00 40.00 20.00 0.00 30.00 20.00 10.00 0.00 Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta. Necochea Balcarce de Mayo Parana Cuarto Catalina (c) 40.00 Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta. Necochea Balcarce de Mayo Parana Cuarto Catalina (d) Isolates Isolates 100.00 80.00 % reaction % reaction 80.00 60.00 40.00 20.00 40.00 20.00 0.00 0.00 Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta. Necochea Balcarce de Mayo Parana Cuarto Catalina (e) 60.00 (f) Isolates Grade 0 Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta. Necochea Balcarce de Mayo Parana Cuarto Catalina Grade 1 Isolates Grade 2 Grade 3 Grade 4 Fig. 11.3. Reaction of Zea mays (lines and populations), Zea perennis and Zea diploperennis to six strains of U. maydis isolates: (a) Colorado Klein (Z. mays); (b) Lines E642A688 (Z. mays); (c) Line SC66 (Z. mays); (d) Line B73; (e) Z. perennis; and (f) Z. diploperennis. Physiological Specialization of Tilletia laevis Wallr. (Common Bunt) on Triticum spp. in Argentina Common bunt of wheat is caused by T. tritici and T. laevis; infection takes place in the coleoptile when teliospores are found on the coleoptile surface and/or the ground (Fischer and Holton, 1957; Hirschhorn, 1986; Wilcoxson and Saari, 1996). Chemical control is achieved through seed treatments; however, the disease is aggravated due to inefﬁciency in the method of fungicide application and the widespread use of susceptible wheat cultivars. In common bunt, the spores survive in the soil for long periods and can cause infection of seedlings. The most effective control method is by genetic resistance to the pathogen and by establishing the variability or physiological specialization of Tilletia species. Historically, pathogenic races that are virulent to resistant cultivars have appeared, so new germplasm is screened continually for resistance. Investigations to determine disease resistance were incorporated into breeding programmes (Meinrs and Fischer, 1953; Kendirck, 1961; Metzger and Hoffmann, 1978; Gaudet, 1990; Johnsson, 1991; Gaudet et al., 1994; Wilcoxson and Saari, 1996). In Argentina, Hirschhorn and collaborators studied the morphology, taxonomy, symptomatology, spore germination, basidial cytology and geographical distribution of pathogens, T. tritici and T. laevis, to common bunt of wheat (Hirschhorn, 1986; Astiz Gassó, 1992; Astiz Gassó and Hirschhorn, 1994). The presence of 12 T. foetida (= T. laevis) 144 M.M. Astiz Gassó and M. del C. Molina physiological forms and cultivar wheat differentials for identiﬁcation of T. foetida were reported by Astiz Gassó (1992, 1997a,b) and Astiz Gassó and Hirschhorn (1994). The objective of this work was to establish the physiological forms of T. laevis and to study the reaction of commercial wheat cultivars to the pathogen in Argentina. In this experiment, we used ten hexaploid bread wheat cultivars with different levels of resistance and two tetraploid cultivars considered resistant. Seeds were deinfested with a formaldehyde solution (3:1) and washed in sterile water. Pathogens from 25 localities in the Argentine wheat belt were tried. Wheat cultivars were inoculated with 0.5 g of teliospore/100 g of seed. Experimental ﬁeld plots consisted of three rows 2 m long per cultivar/pathogen isolate. Field evaluations were carried out by head countings and the percentage of infection was Table 11.5. based on the number of infected and healthy heads. Results were transformed through the Arcosen and the average for 6 years of testing was calculated. Data were subjected to ANOVA (Statistix, 2008). Where signiﬁcant differences were detected, treatment means were separated using HSD Tukey test (P < 0.05). Our ﬁeld research to date indicates that T. laevis shows several physiological forms: Tandil, Rio Cuarto, Villa María, Cabildo, Castelar and Casilda. The rest of the 19 populations of common bunt showed homogeneous behaviour, so it could be considered as one physiological form (Table 11.5). Tetraploid cultivar, Buck Cristal, proved the presence of resistant genes. The hexaploid wheat cultivars, Buck Ñapuca and Buck Yapeyú, were moderately resistant to pathogen incompatibility to different isolates (Table 11.6). The rest of the hexaploid Means of infections of 25 T. laevis populations. Tilletia laevis populations Province Mean 1. Tandil 2. Río Cuarto 3. Bordenave Col.1 4. Sta Rosa 5. Venado Tuerto 6. Tres Arroyos Col.1 7. Lincoln 8. Tres Arroyos Col.2 9. Laboulaye 10. Rafaela 11. Pergamino 12. San Francisco 13. Villa María 14. Salliquelo 15. Marcos Juarez 16. Necochea 17. Cabildo 18. Bordenave Col.2 19. Cañada de Gomez 20. Bragado 21. Río Tercero 22. Paraná 23. Castelar 24. Casilda 25. Bolivar Buenos Aires Córdoba Buenos Aires La Pampa Santa Fé Buenos Aires Buenos Aires Buenos Aires Córdoba Santa Fé Buenos Aires Córdoba Córdoba Buenos Aires Córdoba Buenos Aires Buenos Aires Buenos Aires Santa Fé Buenos Aires Córdoba Entre Ríos Buenos Aires Santa Fé Buenos Aires 21.50 a 19.83 ab 17.12 abc 16.60 abcd 16.45 abcd 15.70 abcde 15.58 abcde 15.38 abcde 14.48 abcde 14.10 abcde 14.01 abcde 13.70 abcde 12.92 bcde 12.61 bcde 11.89 bcde 11.87 bcde 10.91 cde 10.73 cde 10.30 cde 19.49 cde 19.07 cde 18.32 cde 18.28 de 17.29 e 17.17 e Note: Means followed by different letters within column indicate signiﬁcant differences according to Tukey’s test (P < 0.05). Physiological Specialization of Ustilaginales (Smut) Table 11.6. Means of infection of common bunt in wheat cultivars. Hosts Mean Buck Charrua Buck Ombú Buck Catriel Buck Bagual Buck Fogón Buck Guaraní Buck Ñapuca Buck Yapeyu Buck Cristal 19.97 a 18.78 a 17.07 ab 16.58 ab 13.01 bcd 11.21 bcd 18.88 cd 18.72 e 12.85 f Note: Means followed by the same letter with a column indicate cultivars that are homogenous according to Tukey’s test (P < 0.05). wheat was moderately susceptible. Also, the interaction among wheat cultivar populations of T. laevis was signiﬁcantly high and the interaction among pathogen population replications was signiﬁcantly high according to Tukey’s test (P < 0.05). Conclusions From this analysis, four physiological forms of U. bullata were found in the isolates studied: Tres Arroyos, Pergamino and 145 L1avallol, Gowland and General Roca. Bromus brevis is the differential host for the fungus populations and shows genetic resistance to the Gowland isolate. Bromus auleticus and B. inermis cv. gombaszpuzta were resistant to all the fungus isolates. This was the ﬁrst report in Argentina determining the physiological forms of smut U. bullata of Bromus spp. It can be concluded that the wild species and the grown species of the genus Zea reacted in different ways (tolerant and/or resistant to moderately susceptible), depending on the geographic origin of U. maydis populations. These results might be considered when selecting germplasm to obtain new forage plants from interespeciﬁc hybrids of the genus Zea. The wheat cultivars evaluated would also be used as differentials for identiﬁcation of T. laevis races. Six physiological forms were detected among the used populations of T. laevis. This is the ﬁrst report in Argentina determining the physiological forms of smut T. laevis of Triticum spp. The most effective methods to control the disease are genetic resistance and establishing the variability of the smut populations. Determination of the physiological forms of U. bullata, U. maydis and T. laevis and genetic improvement is the most efﬁcient and least environmentally harmful method. References Astiz Gassó, M.M. (1983) Búsqueda de fuentes de resistencia en Bromus spp. a Ustilago bullata Berk. V Jornadas Fitosanitarias Argentinas. Resúmenes, 21 pp. Astiz Gassó, M.M. (1985) Formación de clamidosporas ‘in vitro’ de Ustilago bullata Berk. XII Jornadas Argentinas de Micología. Resúmenes, 40 pp. Astiz Gassó, M.M. (1992) Estudios sobre especialización ﬁsiológica de las caries del trigo. VIII Jornadas Fitosanitarias Argentinas. Paraná provincia de Entre Ríos, Argentina. Resúmenes, 8 pp. Astiz Gassó, M.M. (1994) Specialization physiological forms in Ustilago bullata Berk. of Bromus spp. In: Fuentes-Dávilas, G. (ed.) Proceedings del IXth Biennial Workshop on the Smut Fungi. CIMMYT, El Batán D.F., México, pp. 74–80. Astiz Gassó, M.M. (1997a) Comportamiento de cultivares y líneas de trigo a las caries (Tilletia foetida). Revista de Fitopatología ALF 33(3), 16–17. Astiz Gassó, M.M. (1997b) Variabilidad patógena de poblaciones de Tilletia foetida en Triticum spp. en Argentina. Revista de Fitopatología ALF 33(3), 18. Abstract. Astiz Gassó, M.M. and Aulicino, M.B. (1999) Selección para resistencia al carbón de la panoja (Ustillago bullata Berk) líneas y poblaciones de Bromus catharticus Vhal de la provincia de Buenos Aires. Actas 29º Congreso Argentino de Genética. III Jornadas Chileno-Argentino de Genética. Resúmenes, 362 pp. 146 M.M. Astiz Gassó and M. del C. Molina Astiz Gassó, M.M. and Hirschhorn, E. (1994) Physiologic specialization of Tilletia foetida Wallr (common bunt) in Triticum spp. in Argentina. In: Fuentes-Dávilas, G. (ed.) Proceedings del IXth Biennial Workshop on the Smut Fungi. CIMMYT, El Batán D.F., México, pp. 90–97. Astiz Gassó, M.M. and Molina, M.C. (1996) Estudios preliminares para determinar el grado de resistencia a Ustilago maydis DC. Corda en especies cultivadas y silvestres del Gro Zea. Proceedings Xth Biennial Workshop on the Smut Fungi. University of Calgary, Calgary, Alberta, Canada, pp. 57–62. Astiz Gassó, M.M. and Molina, M.C. (1999) Zea seedling reaction to inoculation with Ustilago maydis (DC) Corda. Maize Genetics Cooperation Newsleter 73, 58–60. Banuett, F. (1995) Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize. Annual Review of Genetics 29, 179–208. Boguena, T., Meyer, S.E. and Nelson, D. (2007) Low temperature during infection limits Ustilago bullata (Ustilaginaceae, Ustilaginales) disease incidence on Bromus tectorum (Poaceae, Cyperales). Biocontrol Science and Technology 17, 33–52. Callow, J.A. and Ling, I.T. (1973) Histology of neoplasms and chlorotic lesions in maize seedlings following the infection of sporidia of Ustilago maydis (DC) Corda. Physiological Plant Pathology 3, 489–494. Duran, R. (1987) Ustilaginales of México. Taxonomy, Symptomatology, Spore Germination and Basidial Cytology. Washington State University, Pullman, Washington, 331 pp. Edmunds, L.K. (1998) Use of sporidial hypodermic infection to test sorghum for head smut resistance. Plant Disease Report 47, 903–913. Falloon, R.E. (1976) Effect of infection by Ustilago bullata on vegetative growth of Bromus catharticus. New Zealand Journal of Agricultural Research 19, 249–254. Falloon, R.E. (1979a) Description and illustration of Ustilago bullata growing in culture. Transactions of the British Mycological Society 73, 223–227. Falloon, R.E. (1979b) Further studies on the effects of infection by Ustilago bullata on vegetative growth of Bromus catharticus. New Zealand Journal of Agricultural Research 22, 621–626. Falloon, R.E. and Hume, D.E. (1988) Productivity and persistance of prairie grass (Bromus willdenowii Kunth) 1. Effects of the head smut fungus Ustilago bullata Berk. Grass and Forage Science 43, 179–184. Fischer, G.W. and Holton, C.S. (1957) Biology and Control of the Smut Fungi. Ronald Press, New York, 622 pp. Gaudet, D.A. (1990) Culm height and susceptibility of winter and spring wheat cultivars to common bunt (Tilletia tritici and T. laevis). Proceedings of the Seventh Biennial Workshop on the Smut Fungi. University of Maryland, Frederick, Maryland. Gaudet, D.A., Puchalski, B.L. and Kozub, G.C. (1994) Reaction of CIMMYT and Candian red spring wheat cultivars to common bunt (Tilletia tritici and T. laevis). In: Fuentes-Dávilas, G. (ed.) Proceedings del IXth Biennial Workshop on the Smut Fungi. CIMMYT, El Batán, México, pp. 59–60. Hirschhorn. E. (1977) Novedades sobre el carbón que ataca Bromus spp. en Argentina. Boletín de la Sociedad Argentina de Botánica 18, 56–64. Hirschhorn, E. (1986) Las Ustilaginales de la Flora Argentina. Edit Comisión de Investigaciones Cientíﬁca de la provincia de Buenos Aires. Publicación Especial. CIC, 530 pp. Johnsson, L. (1991) Climate factors inﬂuencing attack of common bunt (Tilletia caries (D.C.) Tul) in winter wheat in 1940–1988 in Sweden. Journal of Plant Diseases and Protection 99(1), 21–28. Kendirck, E.L. (1961) Race groups of Tilletia caries and Tilletia foetida for varietal resistance testing. Phytopathology 51, 537–540. Kreizinger, E.J., Fischer, G.W. and Law, A.G. (1947) Reactions of mountain brome and Canada wild-rye strains to head smut (Ustilago bullata). Journal of Agricultural Research 75, 105–111. Meinrs, J.P. and Fischer, G.W. (1953) Further studies of host specialization in the head smut of grasses, Ustilago bullata. Phytopathogy 43, 200–203. Metzger, R.J. and Hoffmann, J.A. (1978) New races of common bunt useful to determine resistance of wheat to dwarf bunt. Crop Science 18, 49–51. Snetselaar, K.M. and Mims, C.W. (1992) Sporidial fusion and infection of maize seedlings by the smut fungus Ustilago maydis. Mycologia 84, 193–203. Snetselaar, K.M. and Mims, C.W. (1993) Infection of maize stigmas by Ustilago maydis: light and electron microscopy. Phytopathology 83, 843–850. Statistix for Windows (2008) Analytical Software,Tallahassee, Florida. Toit, L.J. du and Pataky, J.K. (1999) Variation associated with channel inoculation for common smut of sweet corn. Plant Disease 83, 727–732. Wilcoxson, R.D. and Saari, E.E. (eds) (1996) Bunt and Smut Diseases of Wheat. Concepts and Methods of Disease Management. CIMMYT, México, 66 pp. Part IV Endophytes in Plant Disease Control This page intentionally left blank 12 Status and Progress of Research in Endophytes from Agricultural Crops in Argentina Silvina Larrán and Cecilia Mónaco Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, La Plata, Argentina Abstract Plants harbour a heterogeneous population of endogenous microorganisms, comprising both pathogens and non-pathogens, including fungi, bacteria, actinomycetes, etc. Their association has substantial impact on plant health and ﬁtness. Endophytes reside inside healthy plant tissues without producing any disease symptoms. They are helpful in modifying biochemicals produced by plants and may add to their protection from insect herbivores, fungal pathogens and even grazing by animals. However, the ecological role of these endophytes is not yet fully understood. This chapter reports on endophytic fungi present in beet and tomato leaves. Isolation and analysis of endophytic microorganisms of soybean and wheat are also described. It is advocated that endophytes may have a deﬁnite role in the biological control of Drechslera tritici-repentis, responsible for tan spot disease in wheat. Introduction Before beginning, the term ‘endophyte’ must be deﬁned. Literally, an endophyte is an organism which lives inside a plant, ‘endo’ meaning within and ‘phyte’ is derived from the Greek word ‘phyton’, meaning plant. There are several deﬁnitions of endophytes, such as ‘endophyte’ is an all-encompassing topographical term that includes all organisms that are living in plant tissues during a more or less long period of their life, colonizing symptomlessly the living internal tissues of their hosts (Petrini, 1991). Such infections are termed ‘endophytic’, particularly when the association is believed to be mutualistic or at least non-pathogenic, or ‘latent infections’, where a latent pathogen is involved (Cabral et al., 1993). Therefore, the term endophyte has been used lately in a broad sense to include any fungi isolated from symptomless plant tissues, but the concepts of endophytic colonization and latent infection by fungi are clearly different. Endophytic colonization or infection cannot be considered as causing disease, since a plant disease is an interaction between the host, parasite, vector and the environment over time, which results in the production of disease signs and/or symptoms. Endophytic fungi may be described as mutualistic (Clay, 1991). Latent infecting fungi are parasitic but cannot be considered mutualistic. Latent infection is the state in which a host is infected with a pathogen, but does not show symptoms and persists until signs or symptoms are prompted to appear by environmental or nutritional conditions or by the state of  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 149 150 S. Larrán and C. Mónaco maturity of the host or pathogen (Sinclair and Cerkauskas, 1996). Petrini’s deﬁnition of endophytes (1991) encompasses not only mutualistic and neutral symbionts, but also those pathogens known to live latently within their hosts. Therefore, Wilson (1995) has expanded the ‘endophyte’ deﬁnition to include internal bacteria that live inside plant tissues without causing disease. A wide range of bacterial genera has been isolated from healthy plant species of agricultural and horticultural crops (Chanway, 1996, 1998; Sturz et al., 1998). Endophyte associations may range from intimate contact where the fungus inhabits the intercellular spaces and xylem vessels in the plant, to more or less superﬁcial colonization of peripheral, often dying or dead tissues (Petrini, 1996). They may colonize single cells (Stone et al., 1994) or tissues (Schulz et al., 1999). The endophytes of aerial parts of plants could be assembled in two different groups: the fungal endophytes of grasses and nongrass endophytes (fungi and bacteria). Grass endophytes are a particular type of systemic symbiosis, these are fungi of the family Clavicipitaceae, which grow between host cells in vegetative tissues, ovules and seeds and are seed transmitted vertically (Stone and Petrini, 1997). Most studies of endophytes have dealt with grasses due to their economic importance to livestock (Clay, 1988, 1991). The close association of an endophyte (Neotyphodium coenophialum (Morgan-Jones & Gams) Glenn, Bacon & Hanlin = Acremonium coenophialum) and tall fescue (Festuca arundinacea L.) has been widely studied. As a result of the association between host and fungus, alkaloids are produced. These are responsible for fescue toxicosis in livestock (Bacon et al., 1977). Since the initial work of Bacon et al. (1977), numerous researchers have come to understand further the relationship between fungal endophytes of grasses and animal toxicosis. On the other hand, it has been well documented that grass endophytes provide their host with a number of beneﬁts that increase host ﬁtness. The most intensely studied symbioses are tall fescue with N. coenophialum and perennial ryegrass with N. lolii (Latch, Samuels & Christensen) Glenn, Bacon & Hanlin. In tall fescue, N. coenophialum causes enhanced tillering and root growth, increases drought tolerance (Arechavaleta et al., 1989) and protects against certain nematodes (Kimmons et al., 1990), fungal pathogens (Gwinn and Gavin, 1992) and insect herbivores (Rowan and Latch, 1994). The protective nature of endophytes is due to the presence of alkaloids, whereas these alkaloids are responsible for poisoning domestic animals. Ergovaline is associated with various maladies often observed among cattle that graze N. coenophialum-infected tall fescue and collectively called ‘tall fescue toxicosis’. Likewise, lolitrem B is associated with the malady ‘ryegrass staggers’, most commonly observed in sheep grazing perennial rye grass in New Zealand (Schardl and Phillips, 1997). On the other hand, symptomless endophytes of plants other than grasses have been known for more than 80 years (Lewis, 1924; Carroll and Carroll, 1978; Fisher et al., 1992; Menendez et al., 1995; Faeth and Hammon, 1997; Gasoni and Stegman, 1997; Fröhlich et al., 2000; Larran et al., 2007). Endophytes are found in all plants and are extremely abundant and very diverse. Endophytes of a non-grass host represent a broad range of genera. Taxonomically, the endophytic fungi recovered from plants belong mainly to the phylum Ascomycota and Basidiomycota (fungi) and some Oomycetes (phylum Oomycota, Chromista) have been isolated as endophytes (Sinclair and Cerkauskas, 1996), along with members of phylum Ascomycota and their conidial form or anamorphic form lacking a sexual state. The strategy of endophytes is commonly characterized by early occupation of living host tissue, ensuring possession of the nutritional resource (Dingle and McGee, 2003). Host colonization by these fungi is frequently localized in foliage, roots, stems and bark and they are transmitted horizontally via spores. Frequently, colonization is more often non-systemic. These endophytic infections are often presumed to form mutualistic association with their hosts in a manner similar to the endophytes in grasses (Stone and Petrini, 1997). The plant tissues act as host Research in Endophytes from Agricultural Crops in Argentina for complex fungal communities. In the past few years, several works have provided evidence for the development of a highly speciﬁc endophytic assemblage for a given host (Bertoni and Cabral, 1988; Petrini and Fisher, 1988; Sieber et al., 1988, 1991; McInroy and Kloepper, 1991; Pereira et al., 1999; Larran et al., 2000, 2001, 2002a,b). Organ speciﬁcity, probably the result of adaptation by some endophytes to the particular microecological and physiological conditions present in a given organ, has been demonstrated in several studies (Fisher et al., 1991; Petrini et al., 1992). Whereas a large number of species can be isolated from a given host, in general, only a few species are present in signiﬁcant amounts (Petrini et al., 1992). The ecological roles of endophytes are not yet clariﬁed in all associations. Only the interaction of Neotyphodium/grass has been studied in depth, but less is known about other endophytic associations (Clay, 1990). The endophytes may provide a rapidly evolving defence mechanism against herbivory (Carroll, 1988, 1991; Findlay et al., 1995) and many are potential producers of secondary metabolites and enzymes that will probably ﬁnd diverse applications in the most diverse ﬁelds of biology (Petrini et al., 1992; Schulz et al., 1995; Istifadah and McGee, 2006; Istifadah et al., 2006). Several studies have demonstrated auxin and cytokinin production (Pugh, 1972; Bacon and De Battista, 1991) and antibiotic compounds (Clark et al., 1989; Brunner and Petrini, 1992). Competition for infection site, their capacity to produce secondary metabolites and their potential to stimulate defence reactions may contribute to antagonism by the endophytes against pathogens living in the same tissues (Dingle and McGee, 2003; Istifadah and McGee, 2006). Also, several authors have proposed that endophytes could be used as vectors of genes to be introduced artiﬁcially in the population of the host, due to natural genomes showing useful characteristics and attributes that could be selected. For example, endophytes used as vectors of genetic information could also be of particular interest for the development of mycoherbicides (Petrini et al., 1992). The knowledge of endophyte 151 distribution, biodiversity and biochemical characteristics could be important in improving plant ﬁtness. Moreover, they could play an important role in the interactions present in an ecological agriculture. In the past few years, research on endophytes has been carried out at the CIDEFI Research Centre in the city of La Plata, Buenos Aires, Argentina. It is thought that endophytes could be used as biocontrol agents. In Argentina nowadays, biological control is an attractive option for the management of some plant diseases. A considerable amount of knowledge on endophytes has been accumulated. Preliminary studies have focused mainly on determining the biodiversity of endophytes on economically important plants. Likewise, species composition from different organs has been investigated. Finally, research will be undertaken to test the antagonistic interactions between endophytes and plant pathogens. Signiﬁcant research is summarized in this chapter. Endophytic Fungi in Beet (Beta vulgaris var. esculenta L.) Leaves The aim of this investigation was in order to document the species composition of endophytic fungi of healthy cultivated beet leaves; to determine their infection frequencies and to verify possible qualitative and quantitative changes of species isolated during the growing season (Larran et al., 2000). Samples were collected from healthy beet leaves of plants cultivated in the experimental ﬁeld of the Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata (UNLP), Buenos Aires, Argentina. The plants were sampled three times during the growing season. Leaves were cut, surfacesterilized and then leaf disks were incubated on 2% potato dextrose agar (PDA) for 8 days. Nested ANOVA and Tukey tests were applied to evaluate the differences in infection frequencies for different fungi. Data were transformed according to y = arcsin R2 (P/100). Microscopic examinations were made from leaf disks previously surface-sterilized and 152 S. Larrán and C. Mónaco then incubated in a humid chamber for 48 h. The disks were cleared and stained. Hyphae were the principal fungal structures observed (Fig. 12.1). They could be observed emerging through the stomata or growing intercellularly under the cuticle and could be followed between the layers of cells. No visible disruption or impairment of the plant cells by the fungi was noted. The endophytes isolated from healthy beet leaves are shown in Table 12.1. Fungi colonized 100% of the leaves sampled. Twelve taxa of endophytic fungi were isolated and identiﬁed. Yeast, Alternaria alternata, Pleospora herbarum, Stemphylium sp. and Epicoccum nigrum were the most frequently isolated fungi. The frequency of A. alternata and P. herbarum increased signiﬁcantly in time, whereas yeast decreased along the growth stages. There were no relevant quantitative changes in the frequency of colonization by other species. The diversity of isolated fungi species decreased from the ﬁrst to the last sampling. Fig. 12.1. Hyphae emerging from stomata. Endophytic Fungi in the Leaves of Lycopersicon esculentum Mill. We have selected tomato plants for this investigation because both greenhouse and ﬁeld production in La Plata horticultural area are economically important (Larran et al., 2001). Tomato production is used mainly for fresh consumption, as well as being a source of many value-added products. The investigation reports the endophyte frequencies from healthy tomato leaves (cultivar Tommy) cultivated in the ﬁeld of the Facultad de Ciencias Agrarias y Forestales, UNLP, Buenos Aires, Argentina. Samples were collected for 2 years to determine possible qualitative and quantitative changes of species. Data were analysed by ANOVA for factorial experiments. Differences between means were separated with Tukey’s test (P ≤ 0.05). Likewise, different surface-sterilized techniques were evaluated previously and the technique selected was used. The diversity of isolated endophytes is shown in Table 12.2. Research in Endophytes from Agricultural Crops in Argentina 153 Table 12.1. Mean density of colonization (%) of endophytic fungi from beet leaves at three different time intervals during the growing season. Sampling dates Endophytes 1 12.5a** 1.0 1.0 1.0 5.3 2.0 1.0 0 1.0 7.3 6.1 18.0 0 11 Alternaria alternata (Fr.) Keissler Chaetomium sp. Cladosporium spp. Colletotrichum dematium (Pers.) Grove Epicoccum nigrum Link. Glomerella cingulata (Stonem.) Spaulding & Schrenk Penicillium spp. Phoma betae Fr. Phomopsis sp. Pleospora herbarum (Pers. ex Fr.) Rabenh. Stemphylium sp. Yeast Sterile mycelia Total number of endophytes Total segments sampled: 300 2 3 23.0** 0 1.0 0 3.0 1.0 4.0 1.0 0 9.0 9.0 10.0 1 10 31.0** 0 1.0 0 3.0 0 1.0 0 0 11.0 8.0 7.0 0 7 Note: aMean of ten replications. Numbers followed by ** differ statistically according Tukey’s test (P ≤ 0.05). Table 12.2. Mean frequencies (%) of endophytic fungi isolated from tomato leaves in 1998 and 1999. Mean frequencies (%) Endophytes 1998 1999 Alternaria alternata (Fr.) Keissler Arthrinium sp. Bipolaris cynodontis (Marig.) Shoem. Chaetomiun globosum Kunze ex Fries Cladosporium sp. Colletotrichum coccodes (Wallr.) Hughes C. gloeosporioides (Penz.) Sacc. Epicoccum nigrum Link. Cryptococcus sp. Nigrospora sphaerica (Sacc.) Mason Penicillium spp. Phomopsis sp. Ulocladium alternariae (Cooke) Simmons Stemphylium botryosum (Pers.ex Fr.) Rabenh. Rhodotorula sp. 8.75 3.78 0 2.50 3.75 2.50 13.75* 0 0 2.50 2.50 3.75 2.50 1.25 0 25.8* 0 1.44* 0 5.48 0 0 1.59* 1.87* 0 2.55 0 0 0 2.25* Note: Means followed by * differ signiﬁcantly according to Turkey’s test (P ≤ 0.05). Total segments sampled at each growth stage: 75. Different endophytic species were isolated in 1998 and 1999, although some of them were isolated in both years. This could be due to the different climatic conditions registered, as several authors observed that various climatic conditions – site moisture, rainfall and wind exposure – yielded distinct endophyte assemblages (Chapela, 154 S. Larrán and C. Mónaco 1989; Petrini et al., 1992). Alternaria alternata was the fungus isolated most frequently from tomato leaves in 1999, but it was the second most common species in 1998. In contrast, C. gloeosporioides was the fungus isolated most frequently in 1998, but it was not found in 1999. Species of other genera, such as Cladosporium and Penicillium, were isolated in both years. These two genera have been described as endophytes from other plants as well (Fisher et al., 1992; Cabral et al., 1993). Endophytic Fungi in Healthy Soybean Leaves Soybean (Glycine max (L.) Merr.) in Argentina is one of the most important crops, not only by its production but also because of the volume exported, and it is planted on about 16.5 m ha. A study (Larran et al., 2002b) was undertaken to document the diversity of endophytic fungi of healthy cultivated soy- bean leaves and their infection frequency and to verify possible qualitative and quantitative changes of species isolated at two growth stages: R2–R3 and R4–R5 (according to Fehr et al., 1971). Fifty asymptomatic plants were randomly sampled at each growth stage from a segregating population (F3 generation) cultivated at the experimental ﬁeld of the Facultad de Ciencias Agrarias y Forestales, UNLP, Buenos Aires, Argentina. Samples were surface-sterilized and incubated over 9 days. The student t-test and percentage differences test were used to evaluate differences in infection frequencies for various fungi. The results are shown in Table 12.3. Twelve genera of endophytic fungi were isolated and identiﬁed from healthy soybean leaves. In general, in both growth stages, the same species were isolated and most of them did not show signiﬁcant differences in their infection frequencies, except for Phomopsis sp., P. longicolla and Cladosporium sp. The endophytic fungi isolated more frequently from healthy leaves of soybean were Table 12.3. Mean percentage frequencies of endophytic fungi and their variations from soybean leaves at R2–R3 and R4–R5 stages (total segments sampled: 591). Frequencies (%) Endophytes Alternaria alternata (Fr.) Keissler A. tenuissima (Kunze ex Pers.) Wiltshire Bipolaris sorokiniana (Sacc.) Shoem. Cladosporium sp. Colletotrichum sp. Curvularia lunata (Wakker) Boedijni Epicoccum nigrum Link. Glomerella cingulata (Stoneman) Spauld. & Schrenk G. glycines Lehm. & Wolf Nigrospora sphaerica (Sacc.) Mason Penicillium sp. Phomopsis longicolla Hobbs P. sojae Lehman Phomopsis sp. Pleospora herbarum (Pers. ex Fr.) Rabenh. Stemphylium sp. R2–R3 stagea R4–R5 stage Variation (%) 78.48b 0 0.94 0 1.28 0 1.23 17.20 68.79 1.60 0 2.06 0 0.40 1.93 14.04 –12.34 – –100.00 – –100.00 – +56.90 –18.40 NS NS NS * NS NS NS NS 0.51 1.33 0 1.99 3.48 2.89 0.66 3.32 0.40 2.00 1.86 0 3.86 5.50 0.99 2.99 –21.60 +50.40 – –100.00 +10.90 +90.30 +50.00 –9.90 NS NS NS * NS ** NS NS Note: aBased on the plant stages designated by Fehr et al. (1971). bThe infection frequency was calculated as the number of subsamples infected by a given fungus divided by the total number of subsamples incubated. *Signiﬁcant difference (P < 0.05); **highly signiﬁcant difference (P < 0.01); NS, no signiﬁcant difference. Research in Endophytes from Agricultural Crops in Argentina A. alternata and G. cingulata. Most of the fungi isolated in this work are cited as soybean pathogens in different parts of the world (Farr et al., 1989). Because it is known that most fungal pathogens of soybean have an asymptomatic or latent period after infection or colonization, these fungi could be either avirulent or hypovirulent, or virulent but in a latent phase. Pathogenicity tests would be needed to investigate this hypothesis. Soybean leaves are hosts to an abundance of endophytic fungi, but only A. alternata is the dominant species. Further studies will be carried out to evaluate the potential use of endophytes from soybean leaves in biological control. Isolation and Analysis of Endophytic Microorganisms in Wheat Leaves The presence of endophytic fungi in healthy wheat crops has been demonstrated previously in other countries of the world. The present investigation was undertaken in order to document the spectrum of endophytes of healthy leaves from three wheat cultivars and to determine their infection frequencies at three growth stages in Argentina (Larran et al., 2002a). Wheat cultivars, Buck Ombú, Klein Centauro and Klein Dragón, were grown in the experimental ﬁeld of the Facultad de Ciencias Agrarias y Forestales, UNLP, Buenos Aires, Argentina. Ten asymptomatic plants of each cultivar were randomly sampled at three deﬁned growth stages: second node detectable, medium milk and soft dough stages (32, 75 and 85, according to Zadoks et al., 1974). Samples were surface-sterilized and incubated on 2% PDA and, after 9 days, identiﬁcations were made. Data were analysed by ANOVA for factorial experiments. Differences between means were separated by LSD (P ≤ 0.05). From the 450 wheat leaf segments incubated, 3 bacterial isolates and 130 fungal isolates were obtained (Table 12.4). From all the isolates, 19 fungal species were identiﬁed. There were signiﬁcant differences between microorganisms, stages of growth and stages × microorganism interactions. Differences between cultivars, stages × cultivars, 155 microorganisms × cultivars and the triple interaction were not signiﬁcant. The frequency of the microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested. Rhodotorula rubra, A. alternata, C. herbarum and E. nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. Most fungal endophyte isolates from wheat leaves have been described as endophytes of wheat and others plants (Sieber et al., 1988; Petrini et al., 1992; Gindrat and Pezet, 1994). A variation in the number of taxa isolated was recorded along the growing season of wheat. A change in species composition from the three growth stages was observed; however, no differences were noted between cultivars. Further studies were needed to analyse endophyte composition and variation from other organs and cultivars. Therefore, the following study was undertaken. Endophytic Fungi from Wheat (Triticum aestivum L.) In this work, ﬁve wheat cultivars (Buck Poncho, B. pronto, Klein Cobre, K. Dragón and Pro INTA Federal) were grown in the experimental ﬁeld of the Facultad de Ciencias Agrarias y Forestales, UNLP, Buenos Aires, Argentina. The purpose of this investigation was to document the diversity of endophytes from different cultivars and to determine their infection frequencies from different plant organ (leaves, stems, glumes and grains) (Larran et al., 2007). Samples were collected at ﬁve growth stages from crop emergence to harvest (GS 2, GS 8, GS 10.5, GS 11.1 and GS 11.4) (Large, 1954), with the aim of verifying possible qualitative and quantitative changes of the species isolated. Pieces of tissues were surface-sterilized and incubated on 2% PDA over 9 days. An ANOVA including organs, microorganisms, cultivars and growth stages as a source of variation was carried out but, due to differences between organs, an ANOVA was performed considering each organ separately. Differences between means were separated by LSD 156 S. Larrán and C. Mónaco Table 12.4. Frequencies of endophytes isolated from wheat leaves of three cultivars at three growth stages. Samplings Endophytes Gs. 35* Gs. 75 Gs. 85 Average Alternaria alternata (Fr.) Keissler Alternaria sp. I Alternaria sp. II Arthrinium sp. Aspergillus sp. Bipolaris sp. B. cynodontis (Marig.) Shoem. B. sorokiniana (Sacc.) Shoem. Chaetomium globosum Kunze ex Fries Cladosporium herbarum (Pers.: Fr.) Link. Cryptococcus sp. Epicoccum nigrum Link. Fusarium sp. Penicillium sp. Phoma sp. Phomopsis sp. Pleospora herbarum (Fr.) Raben. Rhodotorula rubra Harrison Stemphylium sp. SM I SM II Bacillus sp. Average of growth stages 0a 0a 0a 1.33 a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0.67 a 0.67 a 0a 0a 0a 0a 0a 0a 0.12 aa 0.67 ab 0a 0a 0a 0a 2.67 abc 3.33 bc 0.67 ab 0a 3.33 bc 4.0 c 5.33 c 2 abc 0a 0a 0a 0a 9.33 d 0.67 ab 0.67 ab 0a 0.67 ab 1.52 b 14.0 d 0.67 ab 2.0 abc 0a 0.67 ab 2.0 abcd 2.0 abcd 0.67 ab 1.33 abc 7.33 f 1.33 abc 4.67 de 0a 0.67 ab 0a 0a 4 cde 6.67 ef 3.33 bcd 0a 1.33 abc 1.33 abc 2.45 c 4.89 de 0.22 a 0.67 a 0.44 a 0.22 a 1.56 ab 1.78 abc 0.44 a 0.44 a 3.56 cd 1.78 abc 3.33 bcd 0.67 a 0.22 a 1.33 a 0.22 a 0.22 a 5.33 e 1.33 a 0.44 a 0.22 a 0.67 a Gs. 85: soft dough stage. *Growth stages according to Zadoks et al. (1974). Data are the mean of 150 leaf pieces (5 pieces × 10 replications × 3 cultivars)/growth stage. Means followed by same letter in the same column are not statistically different according to LSD (P ≤ 0.05). aFor the average of growth stages means followed by the same letter in the same row are not statistically different (P ≤ 0.05). Gs.35: second node detectable. Gs.75: medium milk. (P ≤ 0.05). A total of 1750 plant segments were processed from wheat tissues and 33 microbes were recovered. Three bacteria, 27 fungal taxa and 3 non-sporulating mycelia, assigned as ‘sterile mycelia’, were registered (Tables 12.5 and 12.6). A. alternata, C. herbarum, E. nigrum, Cryptococcus sp., R. rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency in all the tissues and organs analysed. As is shown, the bacterial isolates (Serratia sp., Bacillus sp. and unidentiﬁed yellow bacteria) were registered with high frequencies. The results of this statistical analysis showed that organs, microorganisms and interaction of organs × microorganisms were signiﬁcant. On the other hand, as results of ANOVA from each organ, we obtained that the number of taxa isolated was greater in the leaves than in the other organs analysed. Respectively, 25, 17, 12 and 15 were the number of taxa recovered from leaves, stems, glumes and grains. Few species were dominant from grains, whereas they had the highest percentages of isolates from the total samples analysed. Likewise, a variation occurs in the species composition of endophytes isolated from different organs and growth stages. No signiﬁcant differences between cultivars were obtained, except when the glumes were analysed. Whereas Bacillus sp. was isolated from stems and grains, Serratia sp. and yellow bacteria were recovered from all organs analysed. Although most of the microorganisms followed a similar pattern in the four organs, Research in Endophytes from Agricultural Crops in Argentina Table 12.5. Frequency (means) of microorganisms isolated from leaves, stems, glumes and grains on ﬁve wheat cultivars. Endophytes Alternaria alternata (Fr.) Keissler A. infectoria species group Arthrinium sp. Bacillus sp. Bipolaris sorokiniana (Sacc.) Shoem. B. spicifera (Bainier) Subramanian Bipolaris sp. Candida albicans (C.P. Robin) Berkhout Cephalosporium sp. Chaetomium globosum Kunze ex Fries Cladosporium herbarum (Pers.:Fr.) Link. Cryptococcus sp. Cochliobolus spicifer Nelson Curvularia lunata (Wakker) Boedijni Epicoccum nigrum Link. Fusarium oxysporum Schlechtend.: Fr. F. graminearum Schwabe Helicocephalum sp. Nigrospora sp. Penicillium sp. Phoma sp. Pleospora herbarum (Fr.) Raben. Rhodotorula rubra Harrison Septoria tritici Roberge in Desmaz. Serratia sp. Stachybotrys sp. Stemphylium botryosum Wallr. Trichoderma hamatum (Bonord.) Bainier Ulocladium sp. SM 1 SM 2 SM 3 Yellow bacteria Organs Leaves Stems Glumes Grains Cultivars Klein Dragon Buck Pronto Klein Cobre Buck Poncho Pro INTA Federal Means (all organs) and growth stages 8.48 e* 0.56 a 0.58 ab 1.26 ab 0.73 ab 0.00 a 0.26 a 0.04 a 0.06 a 0.19 a 6.55 d 2.14 b 0.14 a 0.01 a 4.38 c 0.53 a 1.01 ab 0.00 a 0.04 a 1.16 ab 0.00 a 0.00 a 1.27 ab 0.00 a 8.95 e 0.00 a 0.09 a 0.17 a 0.04 a 0.00 a 0.00 a 0.38 a 4.33 c 0.94 a 1.27 a 0.98 a 2.03 b 1.54 a 1.37 a 1.33 a 1.39 a 0.91 a Note: *Means followed by the same letter in the same column within the same treatment are not statistically different according LSD (P ≤ 0.05). SM, sterile mycelia. 157 158 S. Larrán and C. Mónaco Table 12.6. Means of the frequencies of microorganisms, cultivars and growth stages for each organ (leaves, stems, glumes and grains) of ﬁve wheat cultivars. Endophytes Leaves Stems Glumes Grains Alternaria alternata (Fr.) Keissler A. infectoria species-group Arthrinium sp. Bacillus sp. Bipolaris sorokiniana (Sacc.) Shoem. B. spicifera (Bainier) Subramanian Bipolaris sp. Candida albicans (C.P. Robin) Berkhout Cephalosporium sp. Chaetomium globosum Kunze ex Fries Cladosporium herbarum (Pers.: Fr.) Link. Cryptococcus sp. Cochliobolus spicifer Nelson Curvularia lunata (Wakker) Boed. Boedijni Epicoccum nigrum Link. Fusarium oxysporum Schlechtend.: Fr. F. graminearum Schwabe Helicocephalum sp. Nigrospora sp. Penicillium sp. Phoma sp. Pleospora herbarum (Fr.) Raben. Rhodotorula rubra Harrison Septoria tritici Roberge in Desmaz. Serratia sp. Stachybotrys sp. Stemphylium botryosum Wallr. Trichoderma hamatum (Bonord.) Bainier Ulocladium sp. SM 1 SM 2 SM 3 Yellow bacteria Cultivars Klein Dragon Buck Pronto Klein Cobre Buck Poncho Pro INTA Federal Growth stages 2 8 10.5 11.1 11.4 4.8 e* 1.4 abc 1.2 abc 0.0 a 1.0 abc 0.2 a 0.4 a 0.0 a 0.0 a 0.2 a 1.4 abc 2.4 cd 0.0 a 0.4 a 3.0 d 2.2 cd 1.0 abc 0.0 a 0.4 a 2.0 bcd 0.2 a 0.2 a 2.4 cd 0.2 a 3.3 d 0.2 a 0.2 a 0.6 ab 0.0 a 0.2 a 0.0 a 0.0 a 3.0 d 2.4 efg 0.0 a 0.16 ab 3.68 gh 0.0 a 0.0 a 0.48 abc 0.0 a 0.48 abc 0.0 a 1.28 abcde 4.8 h 0.0 a 0.0 a 2.88 fg 0.16 ab 2.88 fg 0.16 ab 0.0 a 2.08 def 0.0 a 0.0 a 3.04 fg 0.0 a 13.6 i 0.0 a 0.0 a 0.64 abcd 0.0 a 0.0 a 0.0 a 1.76 cdef 1.6 bcdef 9.33 e 0.26 ab 0.00 a 0.00 a 0.53 abc 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 2.13 cd 1.60 abc 0.00 a 0.26 ab 1.86 bc 0.00 a 0.00 a 0.00 a 0.00 a 0.80 abc 0.00 a 0.00 a 0.26 ab 0.00 a 12.53 f 0.00 a 0.00 a 0.26 ab 0.00 a 0.00 a 0.00 a 0.00 a 3.73 d 17.6 d 0.8 a 1.2 a 1.6 a 1.6 a 0.0 a 0.4 a 0.4 a 0.0 a 0.8 a 21.6 e 0.0 a 0.4 a 0.0 a 10.8 c 0.0 a 0.8 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 6.8 b 0.0 a 0.4 a 0.0 a 0.4 a 0.0 a 0.0 a 0.0 a 9.6 bc 0.90 a 1.09 a 0.83 a 1.12 a 0.97 a 1.21 a 1.01 a 1.40 a 1.26 a 1.48 a 1.53 c 0.89 ab 1.31 bc 1.05 abc 0.48 a 2.73 a 2.73 a 2.18 a 2.36 a 1.39 a 0.73 a 0.65 a 0.80 a 1.76 b 0.85 a 2.16 b 1.02 a 1.26 a 1.09 a 0.24 a 1.87 c 0.94 b 1.72 a 2.83 b Note: *Means followed by the same letter in the same column within the same treatment are not statistically different according to LSD (P ≤ 0.05). SM, sterile mycelia. Research in Endophytes from Agricultural Crops in Argentina there were some, A. alternata for example, with higher values in grains and glumes than in leaves and stems. The spectrum of species isolated ranges from potential saprobes over taxa that probably are present as natural symbionts to known pathogens (Fisher et al., 1992). Whereas A. alternata, C. herbarum and E. nigrum are species commonly abundant in the phylloplane and are considered primary saprobes and minor pathogens, others like B. sorokiniana, C. lunata and F. graminearum are economically important pathogens of wheat (Zillinsky, 1984). Due to the fact that some of these endophytes adapted to a given organ may beneﬁt the host against pathogens, further studies were undertaken. A Biological Control Approach to Infection of Drechslera tritici-repentis in Wheat The investigation was carried out to study the interactions between some endophytes isolated from healthy wheat plants and Drechslera tritici-repentis and to determine its possible signiﬁcance in the biological control of tan spot (Larran et al., unpublished). Endophytes isolated previously from wheat cultivars in Buenos Aires Province, Argentina, were selected for the assay. They were: A. alternata, Bacillus sp., C. globosum, C. herbarum, E. nigrum, Penicillium sp., R. rubra, Trichoderma hamatum and P. lilacinus. Mycelial and conidial morphological alterations and inhibition of colony growth of D. tritici-repentis were registered under in vitro conditions. Likewise, greenhouse experiments were also carried out. The results obtained from all tests have demonstrated 159 that endophytes may have a role as biocontrol agents against D. tritici-repentis. Conclusions The study of endophytes began with the aim of studying their biodiversity and distribution from different hosts. We conﬁrmed that endophytes were present in all the hosts evaluated. Then, we found that endophytes colonized distinct ecological niches and could suggest their organ speciﬁcity according to several authors (Sieber, 1988; Fisher et al., 1991). On the other hand, in our studies, we have isolated a large number of species from healthy tissues of beet, tomato, soybean and wheat but only few species were dominant, in agreement with Petrini et al. (1992). Distinct endophyte assemblages were obtained from healthy tomato leaves in 1998 and 1999, which could be explained because of the different climatic conditions prevailing in both years. Endophytes could be adapted to their hosts and be antagonists for their pathogens and, depending on their antagonistic capacity, they would be able to displace, reduce, suppress or induce resistance against them. 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Cabello1,3 1Instituto de Botánica ‘Spegazzini’; 2CONICET (Consejo Nacional de Investigaciones Cientiﬁcas y Technicas), Universidad Nacional de La Plata, La Plata, Argentina; 3CICBA (Comision de Investigaciones Cientiﬁcas de la Provincia de Buenos Aires), Argentina Abstract In this chapter we discuss the effects of tillage and no-tillage systems on the characteristics of the arbuscular mycorrhizal fungi (AMF) propagule bank in soils. These fungi, which belong to the phylum Glomeromycota, are of great interest in agriculture. AMF are often assumed to be solely beneﬁcial; however, in certain environmental conditions, growth depressions related to AMF have been observed. In soils under no-tillage, an intact hyphal network is present, whereas under conventional tillage, this network can be damaged and AMF spores may remain as propagule sources. Some direct effects of tillage on AMF propagules are: (i) disruption of the hyphal network; (ii) dilution of the propagule-rich topsoil; and (iii) accelerated root decomposition. Spore counts in soils should be considered as useful indicators for AMF activity in situ; however, the presence of spores does not always imply recent activity of AMF and mechanical disturbance may change their spatial distribution in the soil proﬁle. Therefore, the information about spore numbers in agricultural systems needs to be analysed cautiously. The different environmental conditions and direct effects related with tillage and no-tillage on AMF communities generate shifts not only in the composition of the AMF soil propagule bank, but also in its diversity. If the differential use of the various types of propagules by the Glomeromycota families, as many authors suggest, is conﬁrmed, the lack of disruption of the hyphal network in no-tillage can help to explain the differences in Glomeromycota diversity that are found in ﬁeld experiments. Importance of AMF in Agriculture Arbuscular mycorrhizae (AM) show symbioses between plant roots and fungi belonging to the phylum Glomeromycota (Schübler et al., 2001). These fungi are obligate biotrophs and form associations with most plant species (Trappe, 1987). AM associations are the most frequent symbioses in nature because of their broad association with plants and 162 their cosmopolitan distribution (Harley and Smith, 1983). They have been found from the Antartic Peninsula to the tropics (Huante et al., 1993; Cabello et al., 1994). The wide host range of these fungi and their ability to grow in different environments are the reason why arbuscular mycorrhizal fungi (AMF) are usually considered ‘generalists’ with low host speciﬁcity (Smith and Read, 1997). Studies have conﬁrmed that mycorrhizal  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Effect of Tillage Systems fungi colonize most agricultural plants and that they can have a substantial impact on crop productivity (Johnson, 1993). The interaction between the fungus and its host plant consists mainly in nutrient transfer: the plant provides the fungus with carbon compounds, while the fungus delivers nutrients to the plant. The increased nutrient uptake from the soil, particularly of phosphorus and nitrogen, is the main beneﬁt attributed to mycorrhizal symbioses (Smith and Read, 1997; Govindarajulu et al., 2005). Other beneﬁts may include enhancement of resistance to root parasites (Borowicz, 2001), improvement of drought tolerance (Augé, 2001) and reduction of the impact of environmental stresses such as salinity (RuizLozano et al., 1996). AMF also have an important role in the improvement of soil stability, which can possibly diminish erosion (Rillig et al., 2002). AM fungi are often assumed to be solely beneﬁcial, since they are widely thought to function as mutualists. However, their effects on host growth often depend on environmental conditions such as nutrient availability and soil moisture (Peng et al., 1993; Al-Karaki et al., 1998; Graham and Abbott, 2000; Valentine et al., 2001). As AMF draws C from the host, the overall effect on host growth depends on the cost–beneﬁt relationship of the symbiosis (Johnson et al., 1997; Grimoldi et al., 2005). Consequently, in fertile soils, growth patterns of mycorrhizal plants often do not differ signiﬁcantly from those of non-mycorrhizal ones (Newsham et al., 1995) and even growth depressions related to AMF have been observed in many plant species (Johnson et al., 1997; Allen et al., 2003). In such plant–AMF interactions, only the fungal symbiont has a net beneﬁt, and this has sometimes been interpreted as parasitism (Johnson et al., 1997). AM fungi are grouped into genera that encompass more than 150 species described to date and the effects that they have on their host plants, or ‘effectivity’, differ greatly between fungal strains or species (Miller et al., 1985; Modjo and Hendrix, 1986). Since a single root can be colonized simultaneously by various Glomeromycota species, AMF root colonization is mediated by interspeciﬁc 163 fungal interactions, such as competition, antagonism and dominance (Allen et al., 2003). Because of the importance of AMF in agrosystems, their study is relevant both for the manipulation of indigenous AMF in the ﬁeld through appropriate agricultural practices and for the development of a successful inoculation. Agricultural Practices and Mycorrhizae Agricultural practices for annual crops, such as crop rotations, tillage, sowing, fertilization, pest, weed and disease control, and harvest, generate changes that affect the microbial communities in the rhizosphere. Conventional tillage is characterized by the use of disc or mouldboard ploughs, followed by harrowing for seedbed preparation. In no-tillage, seeds are drilled directly into the soil with an appropriate planting machine (Crovetto, 1992). No-tillage systems are characterized by the accumulation of crop residues on the soil surface, leading to greater carbon, nitrogen and surface water, compared to conventional tillage (Doran and Linn, 1994). Several changes in soil properties have been reported with no-tillage management systems: improved aggregate stability, moisture availability with residue retention, changes in the distribution of organic matter residues down the soil proﬁle, for example, a more even distribution of organic matter in cultivated soil as compared to that in non-tilled soil, where residues are concentrated on the surface (Alvarez et al., 1998). One of the problems that may occur in no-tillage is the nutritional deﬁciency because of the reduced mineralization of the soil organic matter (Fox and Bandel, 1986). In the case of AMF, the lack of soil physical disturbance in no-tillage might wrongly suggest that soils with annual crops under this system may be similar to those of natural grasslands. However, agroecosystems have particular characteristics which inﬂuence AMF activity. Natural ecosystems present various plant species hosting AMF, 164 S. Schalamuk and M.N. Cabello at different phenological stages. Annual crops, however, inherently represent a change for AMF, because of the reduction in host biodiversity. In addition, cropped systems show two clearly different periods: a period with high density of host plants of the same species growing simultaneously and, after harvesting, the fallow period with no host or, in some cases, scarce presence of spontaneous vegetation (i.e. weeds). As obligate symbionts, Glomeromycota relies on the plant host for the supply of C assimilates required for its growth, maintenance and functioning. Therefore, dynamics and biodiversity are clearly affected by agricultural practices (Kurle and Pﬂeger, 1994). Signiﬁcance of the AMF Propagule Bank on Root Colonization Effect of tillage Colonization of roots by AM fungi can arise from three sources of inoculum: spores, colonized root fragments and hyphae. The propagules in soils therefore may be called a ‘propagule bank’ that is ‘waiting’ for suitable conditions to germinate, grow and eventually colonize new plant roots (Öpik, 2004; Schalamuk, 2005). Most of the host plant beneﬁts obtained by AM symbiosis, mainly phosphorus acquisition, depend on the early colonization of roots. The rapid colonization is related to AMF propagule density and composition, i.e. the so-called propagule bank. A graph of the percentage of the root length colonized against time has a sigmoid form showing three phases: lag phase, linear phase and a plateau (Sieverding, 1991). A higher AMF propagule density often reduces the length of the lag phase and thereby accelerates the process of mycorrhizal colonization (Smith and Read, 1997). Numerous studies have shown that mycorrhizal colonization is affected negatively by tillage (Douds et al., 1995; McGonigle and Miller; 1996a; Kabir et al., 1998; Mozafar et al., 2000). Soil disturbance reduces AMF propagule density since tillage of soil breaks up the AM fungi hyphal network and consequently lowers mycorrhizal colonization (McGonigle and Miller, 1996a). At the ﬁnal crop stages, the AMF colonization levels in no-tillage and conventional tillage often do not differ signiﬁcantly; however, at the early stages, crop plants under no-tillage often show higher mycorrhizal colonization (Schalamuk et al., 2004). As already mentioned, in no-tillage systems, the reduced mineralization of the soil organic matter often generates plant nutritional deﬁciencies. Nevertheless, a higher nutrient concentration related to a rapid AMF colonization has been observed under no-tillage systems (McGonigle and Miller, 1996a; Mozafar et al., 2000; Schalamuk et al., 2004). By using the method of Plenchette et al. (1989), we have previously found higher levels of mycorrhizal soil infectivity in no-tillage systems (Schalamuk et al., 2004). As already pointed out, colonization of roots by AM fungi can arise from different sources of inoculum. Colonized root fragments (Rives et al., 1980), spores (Gould and Liberta, 1981; Jasper et al., 1987, 1988) and hyphae (Jasper et al., 1989) lose their ability to initiate colonization with soil disturbance, which can be related to physical damage to the propagules by tillage and/or unfavourable conditions for germination or colonization after disturbance (Stahl et al., 1988; Bellgard, 1993). Mycorrhizal soil infectivity (MSI) (Plenchette et al., 1989) compares the ability of different soils to induce colonization in plants and depends on the activity of all the propagule types in soil. It is difﬁcult to distinguish the relative contributions of the different types of propagules to the colonization of root systems (Smith and Read, 1997), and mycorrhizal infectivity does not provide information about the relevance of each propagule type in any particular ﬁeld situation. Although a number of different propagule types exist in the soil, they may not be equally effective at producing new infection units (Klironomos and Hart, 2002). In many habitats, the hyphal network in the soil, together with root fragments, is probably the main means by which plants become colonized, even when signiﬁcant spore populations are also present (Hepper, 1981; Effect of Tillage Systems Tommerup and Abbott, 1981; Birch, 1986; Jasper et al., 1992). Studies have shown that AMF extraradical hyphae are affected severely by soil disturbance at tillage (Fairchild and Miller, 1990; McGonigle and Miller, 1996b; Kabir et al., 1997; Wright and Upadhyaya, 1998). Jasper et al. (1989) have stated that due to the importance of the AMF hyphal network as inoculum in undisturbed soil, a lower infectivity of soil propagules after the disturbance usually can be determined by the damage on the network, rather than on spores and colonized root fragments. Another effect of tillage on the AMF propagule bank, which occurs simultaneously with the disruption of the hyphal network, is the dilution of the topsoil rich in propagules, with the poorest part in the subsurface (Sieverding, 1991). Clearly, mechanical soil mixing affects all types of AMF propagules. As a conclusion, it is suggested that tillage affects all types of AMF propagules directly, to a greater or lesser extent, through different mechanisms acting together: (i) disruption of the hyphal network; (ii) dilution of the propagule-rich topsoil; and (iii) accelerated root decomposition. Through all these direct effects, tillage may reduce soil mycorrhizal infectivity and thereby AM root colonization at the early stages of crop growth. Effects of Tillage and Cropping on AMF Spore Densities in Soils AMF spores are formed by differentiation of vegetative hyphae in soil or roots and appear to be long-term survival structures. In agricultural systems with annual crops, other propagule types (i.e. hyphae inside and outside the roots) seem to be more important to start colonization in particular conditions. Nevertheless, spore counts in soils should be considered as useful indicators for AMF activity in situ. Several studies have found higher spore numbers in no-tillage than in conventional tillage (Crovetto, 1985; Kabir et al., 1998; Jansa et al., 2002; Schalamuk et al., 2003). In agroecosystems with annual crops, the number of spores generally 165 increases during the growing cycle (Cabello, 1987) and sporulation is frequently linked to host phenology in the ﬁeld (e.g. maximum spore production occurs near the middle or the end of a growing season) (Morton et al., 2004). At the early stages of the crop, higher spore densities are usually found in untilled soils, in comparison with conventional systems, whereas at the more advanced phenological stages, differences between tillage systems are reduced (Schalamuk et al., 2003). It is well known that spores can survive in soils for several years (Sieverding, 1991). Thus, spore counts reﬂect both the sporulation and the action of many factors that affect their survival and accumulation in the soil. Consequently, spore density is a result of a complex balance and, while sporulation is probably related to the recent activity of the AMF, spore counts in the soil include structures formed at different times. Spore production depends on carbon supply from the host to the fungus (Furlan and Fortin, 1977; Daft and El Giahmi, 1978). Douds et al. (1993) have indicated that the production of fungal AM spores can decrease when soils are tilled. Increases in spore numbers have been associated with root growth (Hayman, 1970) and/or with host maturity or senescence (Hayman, 1970; Koske and Halvorson, 1981; Giovannetti, 1985; Gemma et al., 1989; Troeh and Loynachan, 2003). Agricultural practices generate disturbances that affect AMF colonization and, in turn, spore formation in soils (Kurle and Pﬂeger, 1994). Therefore, tillage, either through changes in mycorrhizal colonization or through indirect effects, such as changes in the soil environment and plant growth, largely affect AMF spore production in soils. The survival of a spore depends on its morphological traits, determined mainly by the species of Glomeromycota to which it belongs, as well as on the characteristics of the soil environment. Spore survival is an important factor determining the variations in AMF spore counts in soils; however, information about spore survival is scarce as compared to that about sporulation (Lee and Koske, 1994a). In natural ecosystems, decreases in spore numbers have been 166 S. Schalamuk and M.N. Cabello attributed mainly to their germination, the activity of macro and micro fauna and their destruction by other soil fungi and parasites (Gerdemann and Trappe, 1974; McIlveen and Cole, 1976; Ross and Ruttencutter, 1977; Ross and Daniels, 1982; Rabatin and Stinner, 1985, 1988). AMF spores are commonly infected either by other fungi (Daniels and Menge, 1980; Lee and Koske, 1994a; Rousseau et al., 1996) or by actinomycetes (Lee and Koske, 1994b), and environmental conditions have a strong inﬂuence on these processes (Janos, 1980; Koske, 1988). In agricultural systems, another effect that directly reduces spore counts is the dilution of the topsoil rich in spores with the part in the subsurface poorer in propagules (Crovetto, 1985; Sieverding, 1991). For all these reasons, spore survival and accumulation may have a great inﬂuence on spore counts, and the largest spore numbers in notillage at the early stages may be the result of either higher or faster sporulation and/or the presence of residual spores produced during the fallow or the previous crop. As the presence of spores does not always imply recent activity of AMF, and mechanical disturbance may change their spatial distribution in the soil proﬁle, the information about spore numbers in agricultural systems is useful, but needs to be analysed cautiously. AMF Propagule Bank and Biodiversity As already pointed out, tillage may alter the AMF propagule bank in several ways and the lack of disturbance in continuous notillage systems can generate accumulative effects. Therefore, in soils under no-tillage, an intact hyphal network can be present, whereas under conventional tillage, this network can be damaged and AMF spores may remain as propagule sources. Little information exists on the effect of tillage systems on Glomeromycota diversity (Jansa et al., 2002; Schalamuk et al., 2006). Several studies have shown that Glomeromycota taxa may vary in their colonization strategies and that these variations can be associated with the utilization of different propagule types by AMF families (i.e. Acaulosporaceae, Gigasporaceae and Glomeraceae) (Tommerup and Abbott, 1981; Biermann and Linderman, 1983; INVAM, 1993; Braunberger et al., 1996; Brundrett et al., 1999; Klironomos and Hart, 2002; Hart and Reader, 2002, 2004). Jansa et al. (2002), in an intensively used agricultural soil under long-term reduced tillage management, found that the presence of certain AMF species, especially those that did not belong to Glomus spp., had a tendency to increase. However, we have found that the contribution of species belonging to the Glomeraceae family increases in notillage plots, to the detriment of Acaulosporaceae and Gigasporaceae (Schalamuk et al., 2006). In that experiment, the greatest contribution of Glomeraceae species in no-tillage indicated a lower equitability in the distribution among the families of Glomeromycota, and thereby a lower diversity, in comparison with conventional tillage. These ﬁndings differ from those of Jansa et al. (2002). Nevertheless, it is important to point out that mycorrhizal communities are sitespeciﬁc and that each AMF species can be affected in several ways by different agricultural management practices; therefore, generalization is difﬁcult. De Souza (2005), based on life history strategy studies, suggested that members of the Gigasporaceae family were ‘K’ strategists in contrast to single spore-producing ‘Glomus’ species. Hart and Reader (2004) found that the Gigasporaceae family was less sensitive to soil disturbance than the Glomeraceae. The basis for this difference between both families is due probably to differences in their colonization strategies. AM fungi in the Gigasporaceae colonize primarily from spores, whereas those belonging to the Glomeraceae can colonize from hyphae (Tommerup and Abbot, 1981; Biermann and Lindermann, 1983). Hyphae are more sensitive to soil disturbance than spores and thus subsequent colonization of additional roots is affected more. Tillage or the lack of disturbance in continuous no-tillage determine different Effect of Tillage Systems environmental conditions and direct effects on AMF communities, and thereby shifts in the composition of the AMF soil propagule bank. Consequently, if the differential use of the various types of propagules by the Glomeromycota families, as many authors suggest, is conﬁrmed, the lack of disruption of the hyphal network in no-tillage for a period of several years can help to explain the higher proportions of Glomeraceae that have been found previously in the system (Schalamuk et al., 2006). 167 Conclusions Tillage and continuous no-tillage systems change the composition of the AMF propagule banks in the soil, whereas mechanical soil mixing affects all types of AMF propagules. Continuous no-tillage systems favour the presence of an intact hyphal network in soils. Possible differences in colonization strategies among Glomeromycota taxa might have a great inﬂuence on the impacts of tillage on AMF diversity. References Allen, E.B., Swenson, W., Querejeta, J.I., Egerton-Waburton, L.M. and Treseder, K.K. (2003) Ecology of mycorrhizae: a conceptual framework for complex interactions among plants and fungi. 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Scientia Horticulturae 88, 177–189. Wright, S.F. and Upadhyaya, A. (1998) A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant and Soil 198, 97–107. 14 Mechanism of Action in Arbuscular Mycorrhizal Symbionts to Control Fungal Diseases Arun Arya, Chitra Arya and Renu Misra Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India Abstract Currently, the world over, especially in developing countries, maintenance of soil fertility and control of plant diseases have become crucial issues in meeting the biomass needs for food, fodder and fuel, as well as preserving a clean environment. An ideal fertile soil is characterized not only by optimum physical properties and chemical constituents conducive for plant growth, but also by microbiological processes that are maintained in equilibrium. More than 90% of land plants are estimated to form arbuscular mycorrhizal (AM) associations with soilborne fungi in the phylum Glomeromycota. They have a wide host range, yet certain host and fungal combinations are more effective from either the perspective of the fungus, i.e. greater spore/hyphae production, or from that of the host, i.e. enhanced growth, nutrient acquisition or pathogen resistance. Besides improving uptake of phosphorus, AM fungi improve plant health through improved resistance to various biotic and abiotic stresses. Of particular importance is the bioprotection conferred to plants against many soilborne pathogens, such as species of Aphanomyces, Cylindrocladium, Fusarium, Macrophomina, Phytophthora, Pythium, Rhizoctonia, Sclerotium, Thielaviopsis and Verticillium, as well as various nematodes by AM fungal colonization of the plant roots. Achieving the effective and sustainable control of plant diseases remains a formidable challenge for all agricultural systems. Despite the continued release of resistant cultivars and pesticides, pathogens still cause crop damages and losses that exceed 12% worldwide. Studies have shown that root rot in wheat caused by S. rolfsii was prevented by the inoculation of Glomus fasciculatum. Reduced quantum of lesioned roots was found in take-all diseases caused by Gaeumannomyces graminis tritici due to G. deserticola in wheat. The association of G. radiatum with apple has been studied in the USA. It was found that soilborne fungi, Cylindrocarpon, Pythium and the parasitic nematode, Pratylenchus spp., were common with replant diseases of apple. In this disease, young trees are stunted and develop fewer branches than healthy trees. The exact mechanisms by which AM fungal colonization confers the protective effect are not completely understood, but a greater understanding of these beneﬁcial interactions is necessary for the exploitation of AM fungi in organic and/or sustainable farming systems. The mechanisms employed by AM fungi indirectly to suppress plant pathogens include enhanced nutrition to plants; morphological changes in the root; increased ligniﬁcation; changes in the chemical composition of the plant tissues like antifungal chitinases, isoﬂavonoids, etc.; alleviation of abiotic stress and changes in the microbial composition in the mycorrhizosphere. Bioprotection within AM fungal-colonized plants is the outcome of complex interactions between plants, pathogens and AM fungi. In this chapter, the different diseases of cereals, pulses, fruits and vegetables and the potential mechanisms by which AM fungi contribute to bioprotection against plant soilborne pathogens are discussed.  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 171 172 A. Arya et al. Introduction Arbuscular mycorrhizal (AM) symbiosis is the most commonly occurring underground symbiosis in plants. It can be found in a large majority of terrestrial plants (Newman and Reddell, 1987) and in almost a quarter of a million plant species. It is as normal for the roots of plants to be mycorrhizal as it is for the leaves to photosynthesize (Mosse, 1986). The AM fungi are included in the phylum Zygomycota, order Glomales (Redecker et al., 2000), but recently they have been classiﬁed into the phylum Glomeromycota (Schussler et al., 2001). The phylum is divided into 4 orders, 8 families, 10 genera and 150 species; the common genera are Aculospora, Gigaspora, Glomus and Scutellospora (Schussler, 2005). They are characterized by the presence of extra-radical mycelium, branched haustoria-like structures within the cortical cells, termed arbuscules. These are the main sites of nutrient transfer between the two symbiotic partners (Hock and Verma, 1995; Smith and Read, 1997). AM fungi colonize plant roots and penetrate the surrounding soil, extending the root depletion zone and the root system. They supply water and mineral nutrients from the soil to the plant, while AM beneﬁt from carbon compounds provided by the host plant (Smith and Read, 1997). AM fungi are associated with improved growth of host plant species due to increased nutrient uptake, production of growth-promoting substances, tolerance to drought, salinity and synergistic interactions with other beneﬁcial microorganisms (Sreenivasa and Bagyaraj, 1989). The beneﬁcial role of AM fungi in plant biomass production is associated with their capacity to reduce or prevent the development of plant disease (Manoharachary, 2004). The protective ability of mycorrhizae is generally observed against soilborne diseases and is often related to the nature of the host plant, mycorrhizal symbionts, plant pathogens and the condition of the soil (Tello et al., 1987). AM fungi are helpful in controlling disease; however, Ross (1972) reported increased development of Phytophthora root rot in soybean. The known interaction may include a number of mechanisms such as exclusion of pathogens, ligniﬁcation of plant cell wall and change in phosphorus nutrition, leading to exudation by roots and the formation of inhibitory low molecular weight compounds. The mycorrhizal fungi can produce certain compounds that inhibit or kill the pathogenic fungi. Interaction of AM Fungi with Fungal Pathogens Cereal crops Achieving the effective and sustainable control of plant disease remains a formidable challenge for all agricultural systems. Despite the continued release of resistant cultivars and pesticides, pathogens still cause crop damages and losses that exceed 12% worldwide (Johar, 2005). Root rot in wheat caused by S. rolfsii was prevented by inoculation of G. fasciculatum (Harlapur et al., 1990). Graham and Menge (1982) reported reduced quantum of lesioned roots in take-all disease caused by G. graminis tritici due to G. deserticola in wheat. It was found that root dry weight of paddy was not affected by R. solani in mycorrhizal plants, but the pathogen caused 29% loss in root dry weight in nonmycorrhizal plants (Khadge et al., 1990). Also, the pathogen multiplied less in mycorrhizal plants. Cochliobolus sativus negated the effect of VAM inoculation in locally adapted WI 2291 cultivar of barley, whereas in the absence of the pathogen, AM inoculation increased grain yield from 31.9 g to 46.6 g in phosphorus fertilized plants but did not have fertilized plants (Grey et al., 1989). Contrary results were obtained by Schonbeck and Dehne (1979), who observed increase in disease due to Erysiphe graminis and Helminthosporium sativum in barley. The severity of common root caused by Bipolaris sorokiniana in barley was reduced by three species of Glomus (Boyethko and Tewari, 1990). Mechanism of Action to Control Fungal Diseases Pulses and oil crops Gigaspora calospora exerted an inhibitory effect on the development of pigeon pea blight caused by P. drechsleri f. sp. cajani (Bisht et al., 1985). Similarly, in Tamil Nadu Agricultural University, India, studies showed that another AM fungus, G. etunicatum, induced tolerance to cowpea (Vigna unguiculata) against Macrophomina root rot. Disease incidence was 16% in inoculated plants as against 33% in uninoculated plants (Ramraj et al., 1988). Rosendahl (1985) observed a decrease in disease incidence in peas due to Aphanomyces euteiches. Similar results were observed for soybean (Zambolin and Schenck, 1983) and groundnut (Abdalla and Abdel-Fattah, 2000) due to F. solani. Krishna and Bagyaraj (1983) observed a reduction in disease due to M. phaseolina in soybean. Studies conducted at the University of Bayreuth, Germany, showed that in leachates of AM rhizospheric soil of Zea mays and Trifolium subterraneum, fewer sporangia and zoospores were produced by P. cinnamomi as compared to non-AM plants, suggesting that sporangium-induced microorganisms declined or sporangium inhibitors increased (Meyer and Linderman, 1983). Pandey and Upadhyay (2000) studied the effect of microbial populations on the development of pigeon pea in Pusa, Bihar, India. Screening for resident antagonists was carried out and the mode of mycoparasitism was studied. Dual inoculation with AM endophyte (G. mosseae) and M. phaseolina restricted the progression of the pathogen signiﬁcantly in the roots of mungbean (V. radiata). Disease incidence was reduced from 77.9% in pathogen inoculated to 13.3% in AM + pathogen inoculated plants (Jalali et al., 1990). G. fasciculatum reduced the number of sclerotia produced by S. rolfsii in groundnuts (Arachis hypogaea) (Krishna and Bagyaraj, 1983). Horticultural crops The early wilt symptoms caused by F. oxysporum on tomato appeared 8–10 days 173 earlier in mycorrhizal plants. However, 2 months later, disease severity was reduced signiﬁcantly in these plants. Between the two species tested, G. etunicatum was more effective than G. mosseae (Sharma and Johri, 2002). Brassica oleracea infected with AM fungus had lower infection by R. solani; higher moisture content (25%) enhanced disease incidence (Iqbal et al., 1988). Studies conducted at the University of Jordan, Jordan, showed that the mycorrhizal plants of tomato inoculated with F. oxysporum had signiﬁcantly higher root and shoot weights and plant heights than plants inoculated with F. oxysporum only (Al-Momany and Al-Radded, 1988). Only the presence of G. intraradices resulted in a signiﬁcant decrease in the population of F. oxysporum and root necrosis (Caron et al., 1986). Early infestation of G. fasciculatum enhanced tomato plant growth and reduced Fusarium wilt (Manian et al., 2006). They also observed that the percentage disease index was less in mycorrhizal than in non-mycorrhizal tomato plants when inoculated with Alternaria solani. The presence of G. mosseae decreased both weight reduction and root necrosis in tomato caused by P. nicotianae var. parasitica (Trotta et al., 1996). In vitro experiments in which Ri T-DNA transformed roots of alfalfa were inoculated with AM fungi showed normal mycorrhizal formation by G. intraradices and hypersensitivity-like response to G. margarita. Colonized cells became necrotic and HPLC studies indicated concentration of phenolics and isoﬂavonoids in these roots. The data strongly support the existence of a degree of speciﬁcity between AM fungi and the host (Douds et al., 1998). Onion pink rot caused by Pyrenochaeta terrestris and tomato root rot caused by T. basicola are controlled by mycorrhizal fungi (Vidhyasekaran, 2004). Inoculation of G. mosseae in tomato and eggplant seedlings controlled the incidence of Verticillium wilt caused by V. dahliae in Greece (Karagiannidis et al., 2002). Trotta et al. (1996) studied the interaction between the soilborne root pathogen P. nicotinae var. parasitica and the arbuscular mycorrhizal 174 A. Arya et al. fungus G. mosseae in tomato plants. Treatment with Phytophthora resulted in a visible reduction in plant weight and in a widespread root necrosis in plants without mycorrhiza. The presence of AM fungus decreased both weight reduction and root necrosis. The percentage reduction of root necrosis ranged between 63 and 89%. Utkhede et al. (1992) studied the effect of G. mosseae on replant disease of apple. It was found by Graham and Egel (1988) in Florida, USA, that G. intraradices did not increase the resistance or tolerance of sweet orange seedlings to Phytophthora root rot unless mycorrhizae conferred a phosphorus nutritional advantage over the nonmycorrhizal plants. Citrus root rot caused by P. parasitica and T. basicola can be controlled by AM fungi (Vidhyasekaran, 2004). Prior root colonization by mycorrhizal fungi, G. margarita or G. macrocaropum, reduced the damage caused by P. parasitica in two citrus root stocks, Carrigo citrage and Sour orange (Schenck et al., 1977). To ensure good mycorrhizal establishment in citrus roots, plants were exposed for 110 days to mycorrhizal fungi before challenging them with the pathogen. In phalsa (Grewia subinaequalis), better root growth and feeding sites of nematodes during the rainy season promoted better colonization of AM fungi (Hasan and Khan, 2006). Cash crops Studies conducted at the Rajasthan Agriculture University, India, showed that Cuminum cyminum in association with G. calospora, G. fasciculatum, G. mosseae and Acaulospora laevis enhanced nutrient uptake and reduced wilt severity due to F. oxysporum f. sp. cumini (Champawat, 1991). In Germany, G. etunicatum reduced leaf blight in rubber plants caused by Microcycles ulei (Feldmann et al., 1990). G. monosporum inoculated tobacco plants showed better tolerance against T. basicola (Giovannetti et al., 1991). Sivaprasad et al. (2006) controlled foot rot of black pepper by inoculation of G. monosporum. Two other species, G. etunicatum and G. mosseae, exhibited a medium level of resistance to the disesases. Rhizome rot of ginger caused by P. aphanidermatum was controlled by G. mosseae and G. fasciculatum (Sivaprasad et al., 2006). Field application of a commercially available formulation of AM marketed as Josh by Cadila Pharmaceuticals, Agro Division, was tried for the management of charcoal stump rot disease caused by Ustulina zonata (Chakraborty et al., 2005). Commercial production of the medicinal plants in arid and semi-arid areas of the Thar Desert is affected mostly by the soilborne plant pathogens ready to attack any seedlings transplanted into the ﬁeld. Mycorrhizal symbiosis resulted in signiﬁcant disease severity in Chlorophytum borivillianum, Convolvulus microphyllous and Withania somnifera (Vyas, 2005). Role of AM fungi in forestry Studies conducted at the Northern Forest Research Centre, Canada, showed that Fusarium wilt disease severity in Albizia procera and Dalbergia sissoo was reduced signiﬁcantly when inoculated with mycorrhizal fungi (Chakravarty and Mishra, 1986). The effect of AM fungi, Pseudomonas and Rhizobium, was observed on the rate of photosynthesis and colonization in D. sissoo (Bisht et al., 2006). The rate of photosynthesis was signiﬁcantly higher in plants inoculated with AM consortium. Arya and Chaterjee (1995–1996) found better plant biomass and good growth of neem seedlings after inoculation of G. fasciculatum. Arya (2006) recorded a change in soil mycoﬂora after inoculation of AM fungus in neem seedlings. Fungi like Aspergillus fumigatus, A. nidulans, A. ochraecous and F. pallidoroseum were not recorded after 3 months. A signiﬁcant increase in dry weight of Santalum (Krishnamurthy et al., 1998) and Tamarindus (Bagyaraj and Reena, 1990) seedlings has been observed after inoculation of AM fungi. In ectomycorrhizae, the presence of a mantle around the root prevents the entry of pathogens, while in endomycorrhizae, the better nutrient uptake makes the plant more resistant to various pathogens. Mechanism of Action to Control Fungal Diseases Fungi are harmful agents to humans but mycorrhizal fungi are indispensable for luxuriant growth of forest trees. Contrary to popular belief, the luxuriance of rainforest is not because the rainforest soil is more fertile (as torrential rains over millennia leach out soluble minerals), but because the roots associate with fungi, whose spreading hyphae increase the area of absorption of scarce nutrients and transport this to the plant in return for photosynthetically ﬁxed carbon (Maheshwari, 2005). In Ghana and the Mopri Forest Reserve of Cote d’Ivoire, Terminalia ivorensis plantations are susceptible to dieback, the cause of which is unknown; poor mycorrhizal infection may be a contributory factor (Wilson et al., 1994). Signalling Pathway in Mycorrhiza The signalling pathway to activate the mycorrhiza-speciﬁc phosphate transporter has its origin in the PL (phospholipid) PC (phosphatidylcholine), imager component of membranes of plants and probably, also of the AM fungus. However, PC is not active in itself. It gains activity only after treatment with PLA2 and PC from plants, fungus or both remains to be explored further. Several PLA2s have been identiﬁed in plants and all are secretory proteins. Their regulation and substrate speciﬁcity are unknown. This might hint at extracellular production of the LPC (lyso-phosphatidylcholine) signal might be generated more speciﬁcally in the arbuscules containing cells. LPCs are highly mobile within the intact cells and LPC is therefore a good candidate for a cytoplasmic messenger that transduces signals to activate downstream processes and gene expression in the nucleus (Drissner et al., 2007). Bioprotectant Nature of AM Fungi Plant diseases can be controlled by manipulation of indigenous microbes or by introducing antagonists to reduce the diseaseproducing propagules (Linderman, 1992). AM fungi and their associated interactions 175 with plants reduce the damage caused by plant pathogens (Harrier and Watson, 2004). These interactions have been documented for many plant species. With the increasing cost of inorganic fertilizers and the environmental and public health hazards associated with pesticides and pathogens resistant to chemical pesticides, AM fungi may provide a more suitable and environmentally acceptable alternative for sustainable agriculture (Table 14.1). Mechanism of Disease Control Any one or more mechanisms may be operative in plants, imparting them with resistance against pathogens. 1. 2. 3. Physical alteration in plant body. Physiological changes. Biochemical mechanisms Physical alteration in plant body According to some scientists, AM affects soilborne plant pathogens on the basis of physical alterations. Ligniﬁcation of cell wall and production of other polysaccharides has been reported, which prevents penetration of mycorrhizal plants by F. oxysporum (Dehne and Schonbeck, 1979) and Phoma terrestris (Becker, 1976). Mycorrhizal inoculation improves plant growth. Arya (2006) found better growth of neem seedlings after inoculation with three isolates of G. fasciculatum. It has also been suggested that a stronger vascular system of the mycorrhizal plants is likely to increase the ﬂow of nutrients, impart greater mechanical strength and diminish the effect of vascular pathogens (Schonbeck, 1979). A few electron opaque structures resembling the deposits were found in some cells and intercellular spaces of non-infected mycorrhizal carrot roots, but were absent in infected, non-mycorrhizal carrot roots. Restriction of pathogen growth, together with an increase in hyphal alteration and accumulation of new plant products in mycorrhizal roots, but absent in 176 A. Arya et al. Table 14.1. Effects of AM fungi on fungal diseases of certain crops. Crop AM fungi Pathogen Reference Tomato Glomus intraradices Fusarium oxysporum f. sp. lycopersici F. oxysporum Caron et al., 1986; Akkopru and Demir, 2005 Al-Momany and Al-Raddad, 1988 Bhagawati et al., 2000 Pozo et al., 2002 Berta et al., 2005 Declerck et al., 2002 Declerck et al., 2002 Yao et al., 2002 Rosendahl and Rosendahl, 1990 Hao et al., 2005 Al-Momany and Al-Raddad, 1988 Ozgonen and Erkilic, 2007 Torres-Barragan et al., 1996 Rosendahl, 1985 Devi and Goswami, 1992 Sundaresan et al., 1993 Siddiqui and Singh, 2004 Akhtar and Siddiqui, 2006 Akhtar and Siddiqui, 2007 Liu, 1995 G. mosseae Banana Cucumber Pepper Onion Pea Cowpea Chickpea Cotton G. etunicatum G. mosseae G. intraradices Glomus sp. G. proliferum G. etunicatum G. etunicatum Glomus sp. G. etunicatum G. mosseae Glomus sp. G. fasciculatum G. fasciculatum G. fasciculatum G. fasciculatum G. intraradices G. fasciculatum G. mossae G. vesiformae F. oxysporum f. sp. lycopersici Phytophthora parasitica Rhizoctonia solani Cylindrocladium Spathiphylli R. solani Pythium ultimum F. oxysporum f. sp. cucumerinum F. oxysporum P. capsici Sclerotium cepivorum Aphanomyces euteiches Macrophomina phaseolina F. oxysporum F. oxysporum f. sp. ciceris M. phaseolina M. phaseolina V. dahliae non-mycorrhizal roots, shows that mycorrhizal infection is responsible at least in part for the plant defence system which provides protection against pathogen attack (Benhamon et al., 1994). Physiological changes AM fungi can interact directly with the pathogens through phenomen like antagonism, antibiosis or predation. The studies conducted so far suggest that they affect the host–pathogen relationship indirectly through physiological alteration or by competing for space or host resources. Through increased P nutrition, AM fungi enhance root growth, expand the absorptive capacity of the root system for nutrients and water and affect cellular processes in roots (Hussey and Roncadori, 1982; Reid et al., 1984; Smith and Gianinazzi, 1988). In addition to phosphorus, AM fungi are known to enhance uptake of Ca, Cu, S and Zn (Gerdemann, 1968; Sharma, 1990). Glomus monosporum was found effective against P. capsici in black pepper (Sivaprasad et al., 2006). The authors found resistance due to improved nutrient uptake. Host susceptibility to infection by the pathogen and tolerance to disease is inﬂuenced by the nutritional status of the host and the fertility status of the soil (Wallace, 1973). For example, nematodedamaged plants frequently show deﬁciencies of B, N, Fe, Mg and Zn (Good, 1968). High levels of P fertilization in the absence of AM fungi can interact with minor elements, creating a deﬁciency situation which predisposes plants to root knot nematodes (Smith et al., 1986). AM fungi may, therefore, also increase host tolerance to pathogens by increasing uptake of essential nutrients other than P which are otherwise deﬁcient in non-mycorrhizal plants. Production of Mechanism of Action to Control Fungal Diseases siderophore can suppress root pathogens (Sharma and Johri, 2002). Higher levels of amino acids, especially arginine, in combination with root exudates of the mycorrhizal plant have been reported to reduce chlamydospore production of T. basicola (Baltruschat and Schoenbeck, 1975). Increased levels of phenylalanine and serine have been observed in tomato roots inoculated with G. fasciculatum. High concentrations of orthodihydroxy (O-D) phenols in mycorrhizal roots suppressed the growth of S. rolfsii (Goodman et al., 1967; Krishna and Bagyaraj, 1983). The presence of HCN precursors has been observed in rubber plant infected with G. etunicatum (Lieberei and Feldmann, 1990). Biochemical mechanisms The production of phytoalexins in AMcontaining plants has been demonstrated conclusively. Enhanced accumulation of glyceollin I, a highly antifungal phytoalexin, has been reported in the roots of mycorrhizal soybeans (Morandi et al., 1984). According to Sharma and Johri (2002), it is not clearly understood how AM fungi induce the production of phytoalexins and elicitors. It may be possible that mycorrhizal fungi perturb root tissues so that the plant elicitors are liberated. Cell damage, which is closely associated with the production of isoﬂavanoids in legumes (Bailey, 1982), has been observed rarely in mycorrhizal soybean roots. The concentration of coumestrol increased in mycorrhizal roots (25 µg/g) and was much greater than that of glyceollin I (Morandi et al., 1984); coumestrol inhibits the growth of bacteria and nematodes. According to Chakraborty et al. (2005), induction of disease resistance in pea plants against charcoal stump rot was associated with the accumulation of defence enzymes, followed by stimulation of antifungal phenolics. Roots colonized by an AM fungi exhibit high chitinolytic activities. These enzymes can be effective against other fungal pathogens under the direct inﬂuence of mycorrhizal fungi and root tissues become more 177 resistant to pathogenic attack. Since the ﬁrst report of mycorrhiza-related chitinase in tobacco (Dumas-Gaudot et al., 1992), additional ones have been demonstrated in various plant species. Lambias and Mehdy (1996) evaluated the expression of mycorrhizaspeciﬁc chitinases and ß-1,3-glucanases in soybean root infected with G. intraradices. The efﬁcacy of six AM species, A. morrawae, G. margarita, G. fasciculatum, G. macrocarpum, S. calospora and Sclerocystis rubiformis obtained from rhizosphere of C. microphyllus was evaluated for enhancement of PRO (peroxidase), PPO (polyphenol oxidase) effect, with S. calospora being the most promising of all the fungi. Good results were observed with G. fasciculatum in W. somnifera. AM fungi ensure protection against certain soilborne pathogens (Diop, 1996). An AM fungus inﬂuences microbial populations and improves soil texture by the secretion of mucilaginous compounds (Strullu et al., 1991). Vesicles are lipid-ﬁlled and are initiated after the formation of arbuscules, but live longer after the senescence of arbuscules (Diouf et al., 2003). In Medicago truncatula, at an early stage of arbuscule development by G. versiforme, bright diffuse ﬂorescence is seen around the arbuscular branches following antitubulin labelling. At later stages of development, short microtubules are closely associated with plasma membrane surrounding the labyrinthine surface of the arbuscule. γ Tubulin has been shown to be associated with the nuclear envelope and perifungal membrane in tobacco arbuscular mycorrhizas (Genre and Bonfante, 1999). Mycorrhizosphere changes in populations of antagonists to speciﬁc pathogens depend on having those antagonists present in background soil. If antagonists are absent and deleterious microbes are present in signiﬁcant numbers and enhanced by AM, the incidence of disease can be increased (Sharma et al., 2002). Conclusions The use of AM fungi as a biofertilizer is the only alternative for successful farming. 178 A. Arya et al. With this approach, we can obtain maximum return, not only for a season or a year, but also over centuries. Mycorrhizal– disease interaction has been studied in different plants by various workers. In many cases, a better growth of plant is reported after inoculations of an exotic AM fungi. A low level of fungal aggressiveness and a weak plant reaction are no doubt two key factors that help in the establishment of a successful symbiotic relationship between the two organisms. Increase in peroxide activity which is localized in plant vacuoles and the cell wall (Schloss et al., 1987) in AM-infected root is one example of a mechanism of plant resistance to microorganisms. Likewise, production of phytoalexins (glyceolin I, daidzein and coumestrol), chitinase, ß-1,3glucanases and b1 (PR) protein are important components of AM-induced changes leading to the resistance of the host to other pathogens. The systemic effect of AM fungi to Phytophthora infection in tomato has been demonstrated by Pozo et al. (2002). Spores of AM fungi are reported from different soils in the country. These symbionts improve plant growth. 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Kharwar Mycopathology and Microbial Technology Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, India Abstract Endophytes are the microorganisms that reside inside healthy plant tissues without causing any detectable disease symptoms to the host. Often, each and every plant harbours either one or a battery of endophytic microorganisms. The study of endophytes is now on a voyage of interest, not only because of their role in ﬁlling the divide between discovered and undiscovered microbial diversity, but also due to their harbouring a great potential to produce novel natural products. Other than soil, higher plants also act as an alternative resource to isolate potential microorganisms. Natural compounds ranging from crop protection to human welfare have been isolated from this alternative source of endophytes. Several anticancer, antibiotic, antimycotic, antiviral, antioxidant, nematicide, insecticide and immunosuppressive compounds have been reported from endophytes, such as cytochalasines, ambuic acid, oocydin, jesterone, cryptocandin, lolitrem B, and 3-hydroxypropionic acid and taxol, etc. Many of them produce some toxic alkaloids and protect their hosts from herbivores. They also improve the growth and yield of crops under various stressed conditions. Endophytic fungi have been emerging as a new tool in genetic engineering, the pharmaceutical industry and in crop protection as well. In this chapter, the ability and role of endophytic fungi to ward off pests and environmental stresses on plants is discussed. Introduction The use of agrochemicals as a single control measure in the ﬁeld to protect crops from their pests has been generating resistance in these pests, and also represents a high risk to ﬁeld workers and consumers. Most of these chemicals are non-biodegradable and are responsible for polluting the environment. The control of phytopathogens has relied mostly on chemical control agents such as methyl bromide (Jarvis, 1993) but after the Montreal Protocol (1991), the manufacturing of and trade in methyl bromide was phased out in 2005. The utilization of biological materials is an alternative and safe way to protect plants from phytopathogens. The control of plant pathogens by phylogenetically diverse microorganisms acting as natural antagonists has been demonstrated repeatedly over the past 100 years. The antifungal ability of Trichoderma sp. has been well known since the 1930s and extensive efforts have been made since then to use them seriously for plant disease control (Harman, 1996). Although the term ‘endophyte’ was used much earlier in 1866 by German scientist,  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 183 184 S.K. Gond et al. Heinrich Anton De Bary, the presence of endophytes in plant was only recorded in 1904 when Freeman (1904) described an entire plant’s endophyte life history in the seeds of darnel (Lolium perenne sub sp. temulentum = L. temulentum). This is a topographical term and includes bacteria, fungi, actinomycetes and algae which spend their whole life, or a period of their life cycle, inside healthy plant tissue without causing any disease symptoms. Among all endophytes, after bacteria, fungi are dominant in higher plants (Figs. 15.1 and 15.2). It seems that other microbial forms almost certainly exist in plants as endophytes such as mycoplasmas (pleuro pneumonia-like organisms – PPLO), rickettsia and archaebacteria; however, no evidence of them has yet been observed. On the basis of their nature, endophytes may be categorized in three groups: (i) pathogens of another host that are non-pathogenic in their endophytic relationship; (ii) non-pathogenic microbes; and (iii) pathogens that have been rendered nonpathogenic but still capable of colonization by selection methods or genetic alteration Fig. 15.1. (Backman and Sikora, 2008). Endophytes are recorded from lower plant to higher plant hosts (Stone et al., 2000). Each and every plant is a reservoir of one or a suite of endophytes. In angiosperms, Poaceae members are studied more for their endophytes. Endophytic fungi are now attracting great interest from researchers as an alternative source in controlling plant and human pathogens. Some of the earlier workers before the 1970s documented the endophytic fungi residing inside the plant, exploring the biodiversity of hidden fungi. The period of 1981 to 1985 can be considered a historical one in the study of endophytes, as plant protection against herbivore insects was demonstrated by endophytic microorganisms. Webber (1981) demonstrated for the ﬁrst time the role of endophytic Phomopsis oblonga in the protection of elm trees against the beetle Physocnemum brevilineum. This report generated interest in the role of endophytes in plant protection. Now, their beneﬁcial role to plants as well as to humans is being considered. In this regard, a large number of antimicrobial compounds have been isolated Endophytic fungal mycelia and spores within plant tissue stained with aniline-blue. Role of Fungal Endophytes Fig. 15.2. 185 Leaf pieces in Petri plate (21 days old) showing emergence of endophytic fungal mycelia. from these endophytic microorganisms (Strobel, 2002, 2003; Zhang et al., 2006; Kharwar et al., 2009). Endophytic fungi are now recognized as a new tool in the production of antimicrobials and pharmaceutical compounds. In the search for bioactive compounds, several endophytic fungi have been reported from the medicinal plants of North India (Gond et al., 2007; Verma et al., 2007; Kharwar et al., 2008). Antimicrobials and their Activities Produced from Endophytes Antifungal activity of endophytes Fungi are major causal organisms of various diseases in plants. Many synthetic fungicides are available on the market, but they are giving resistance to pathogens and are also assisting in increasing the hazards to human health. Data show that 52.3% of endophytic fungal fermentation broths display growth inhibition to at least one pathogenic fungus, such as Neurospora sp., Trichoderma sp. and Fusarium sp. (Huang et al., 2001). In a similar study, fermentation broths of 9 (4.8%) out of 187 endophytic fungi isolated from mainly woody plants were highly active against Phytophthora infestans in tomato plants (Park et al., 2005). Induced resistance against Fusarium wilt by endophytic F. oxysporum was generated in tomato plants (Duijff et al., 1998). Sclerotinia sclerotiorum is a common root, crown and stem rot causing pathogen to several hosts such as cabbage, common bean, citrus, celery, coriander, melon, squash, soybean, tomato, lettuce, cucumber, etc. Cyclosporine is characterized as a major antifungal substance against S. sclerotiorum from the fermentation broth of endophytic F. oxysporum (Rodriguez et al., 2006). Out of 510 isolates of endophytic fungi, 64 isolates gave antifungal activities against Candida albicans, C. glabrata, C. krusei, Cryptococcus neoformans, Aspergillus fumigatus, A. ﬂavus, Rhizopus oryzae, Trichophyton rubrum and Microsporum canis (Anke et al., 2003). Narisawa et al. (2000) found that the root endophytic hyphomycete, Heteroconium chaetospira, suppressed Verticillium sp. in 186 S.K. Gond et al. Chinese cabbage in the ﬁeld. Verticillium wilt is one of the most destructive diseases of aubergine. Eleven out of 123 isolates of endophytic fungi, especially H. chaetospira, Phialocephala fortinii, Fusarium, Penicillium, Trichoderma and Mycelium radicis atrovirens ( MRA), after being inoculated on to axenically reared aubergine seedlings, almost completely suppressed the pathogenic effects of a post-inoculated, virulent strain of V. dahliae (Narisawa et al., 2002). Out of 39 endophytes of Artemisia annua, 21 showed in vitro antifungal activity against a number of fungal pathogens (Liu et al., 2001). The extracts of endophytic Alternaria sp., isolated from medicinal plants of the Western Ghats of India, inhibited the growth of C. albicans (Raviraja et al., 2006). Colletotrichum gloeosporioides was isolated as an endophyte from healthy leaves of Cryptocarya mandioccana, giving antifungal activity against phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum (Inacio et al., 2006). Fungal endophytes Chaetomium and Phoma sp., isolated from asymptomatic leaf of wheat, reduced the number and the area of pustules of Puccinia recondita f. sp. tritici. A study showed 40%, 65% and 27% antagonistic interaction by endophytic morphospecies in vitro against cacao pathogens, Moniliophthora roreri, P. palmivora and Crinipellis perniciosa, respectively, while in the ﬁeld the endophytic C. gloeosporioides produced a signiﬁcant decrease in pod loss (Mejia et al., 2008). Cryptosporiopsis quercina is an endophytic fungus of a medicinal plant, Tripterigium wilfordii. It was observed that C. quercina produced an antimycotic compound, cryptocandin, which was active against a number of human and plant pathogenic fungi, including C. albicans, S. sclerotiorum and Botrytis cinerea (Strobel et al., 1999a). A number of antifungal compounds have been identiﬁed by Yue et al. (2000) from the cultures of Epichloe and Neotyphodium species which showed activity against chestnut blight fungus, Cryphonectria parasitica. These compounds were indole derivatives, indole-3-acetic acid and indole-3-ethanol, a sesquiterpene and a diacetamide. Consequently, a tetramic acid, cryptocin, has also been isolated from the cultures of C. quercina, which exhibits strong antifungal activity against Pyricularia oryzae, the causal agent of blast of rice, as well as some other plant pathogenic fungi (Li et al., 2000). Colletotrichum gloeosporioides was isolated from A. mongolica, which produced antifungal metabolite colletotric acid, against the fungus Helminthosporium sativum (Zou et al., 2000). Another Colletotrichum sp., isolated from A. annua, produced bioactive metabolites that were fungistatic to several plant-pathogenic fungi (Lu et al., 2000). Pestalotiopsis microspora is a commonly isolated and well-identiﬁed fungus from every rainforest plant and, as a single endophytic species, it contributes a high percentage to the total mass of fungal endophytes in any host. Pestalotiopsis is observed to produce many antimicrobial secondary metabolites. One such secondary metabolite is ambuic acid, an antifungal agent which has been described from several isolates of P. microspora (Li et al., 2001). P. jesteri, isolated from the Sepik River area of Papua New Guinea, produced jesterone and hydroxyjesterone which exhibited antifungal activity against a variety of plant-pathogenic fungi (Li and Strobel, 2001). Two new metabolites, ethyl 2,4-dihydroxy-5,6-dimethylbenzoate and phomopsilactone, have been isolated from P. cassiae, an endophytic fungus in Cassia spectabilis, with strong antifungal activity against the phytopatogenic fungi, C. cladosporioides and C. sphaerospermum (Silva et al., 2005). An aquatic plant, Rhyncholacis penicillata, is known worldwide to harbour a potent antifungal microbe, Serratia marcescens, which produces an antioomycetous compound named oocydin A (Strobel et al., 1999b). Oocydin A provides the plants with a strong protection against several water moulds. Antibacterial activity of endophytes The antimicrobial activity of endophytic fungi has been observed in a range of bacteria Role of Fungal Endophytes representing pathogens to plants and humans. The broths of 16 endophytic fungi isolated from the medicinal herb, Cynodon dactylon (Poaceae), were identiﬁed as having potent anti-Helicobacter pylori activity. The most active endophyte, identiﬁed as Aspergillus sp. (strain number: CY725), produced four active fractions and was identiﬁed as: (i) helvolic acid; (ii) monomethylsulochrin; (iii) ergosterol; and (iv) 3β-hydroxy-5α, 8α-epidioxy-ergosta-6, 22-diene with corresponding MICs of 8.0, 10.0, 20.0 and 30.0 µg/ ml against H. pylori, respectively (Li et al., 2005). Bioactivity of endophytic fungi of Coffea arabica and C. robusta was screened against Salmonella choleraesuis, Staphylococcus aureus, Pseudomonas aeruginosa and four different Escherichia spp. Out of these endophytic fungi, T. harzianum, Guignardia sp. and Phomopsis sp. have inhibited four to ﬁve bacterial species successfully (Sette et al., 2006). Out of 377 isolates of endophytic fungi from Garcinia plants, 18.6% isolates displayed antimicrobial activity against at least one pathogenic microorganism, such as S. aureus, a clinical isolate of methicillin-resistant S. aureus, C. albicans and C. neoformans (Phongpaichit et al., 2006). Epicoccum purpurascens and Truncatella hartigii were found to have signiﬁcant action against human pathogenic bacteria. E. purpurascens expressed a good antibacterial effect on S. aureus and P. aeruginosa and a very good antibacterial effect on E. coli, while T. hartigii exhibited a signiﬁcant antibacterial effect on Enterococcus faecalis (Janes et al., 2007). Fusarium was the most frequently isolated endophyte from the Chinese traditional medicinal plant, Dioscorea zingiberensis, and F. redolens showed the most potent antibacterial activities against B. subtilis, S. haemolyticus, E. coli and X. vesicatoria (Xu et al., 2008). Two antibacterial cerebrosides, one new and another known, were isolated from Fusarium sp., an endophytic fungus found in Quercus variabilis. The new cerebroside was named fusaruside with structure (2S,2′R, 3R,3′E,4E,8E,10E)-1-O-b-d-glucopyranosyl2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy9-methyl-4,8,10-sphingatrienine. Both of them were active against B. subtilis, E. coli 187 and P. ﬂuorescens (Shu et al., 2004). Periconicins A and B were isolated from endophytic fungus Periconia sp. of Taxus cuspidata and exhibited antibacterial activity against many pathogenic bacteria. The minimum inhibitory concentration (MIC) of periconicin A was even less (3.12 µg/ml) than that of gentamicin (12.5 µg/ml) against Klebsiella pneumoniae (Kim et al., 2004). The endophytic fungus, Xylaria sp., isolated from Ginkgo biloba, showed strong antibacterial activity in vitro against S. aureus (MIC 16 µg/ml), E. coli (MIC 10 µg/ml), S. typhae (MIC 20 µg/ml) and S. typhimurium (Liu et al., 2008). Recently, some bioactive nitronaphthalenes have been isolated from endophytic fungus, Coniothyrium sp. (Krohn et al., 2008). Javanicin, an antibacterial naphthaquinone, has been isolated from neem endophyte, Chloridium sp., which was signiﬁcantly active against Pseudomonas spp. (Kharwar et al., 2009). Antiviral activity of endophytes Viruses are an important causal agent of various diseases in plants and animals. Endophytes can induce plant resistance against viral diseases, but there is a contradiction and Guy (1992) found no correlation between virus infection and the incidence of endophyte in perennial ryegrass (L. perenne), whereas other correlative studies have revealed that some endophyte-infected tall fescue (Festuca arundinaceum) seem to be more resistant to barley yellow dwarf virus (BYDV) than the others (Mahmood et al., 1993; Guy and Davis, 2002). Lehtonen et al. (2006), when releasing the viruliferous aphid vectors to endophyte-infected and endophyte-free L. pretense plants in a common garden, found the number of aphids and the percentage of BYDV infections were lower in endophyte-infected plants compared to endophyte-free plants. Human cytomegalovirus (hCMV) is a ubiquitous opportunistic pathogen. Two novel human cytomegalovirus protease inhibitors, cytonic acids A and B, have been isolated from the solid-state fermentation of the endophytic fungus, Cytonaema sp. (Guo et al., 2000). 188 S.K. Gond et al. Nematicidal activity of endophytes Insecticidal activity of endophytes Endophytic fungi are known to produce some compounds which are toxic to nematodes. The ﬁrst report on antagonistic activity of endophytic fungi against plant parasitic nematodes was observed in tall fescue (F. arundinacea) infected by Pratylenchus scribneri. The nematode population was found to be comparatively less in the soil surrounding endophyte-infected plants. Since the root of tall fescue (F. arundinacea) was infected by Acremonium coenophialium, it was considered that the presence of A. coenophialium deterred the nematode population. The colonization of fungal endophyte, F. oxysporum, in the roots of tomato plant reduced 60% infection of Meloidogyne incognita successfully. Endophyte-free perennial ryegrass plants are shown to have a larger number of M. incognita population in roots than endophytecontaining plants (Ball et al., 1997). Pregaliellalactone and structurally related lactones were isolated with nematicidal activity from non-graminaceous endophytes and related saprophytic ascomycetes (Kopcke et al., 2002a,b). Another endophytic microbe, Burkholderia ambifaria, isolated from corn root, produced some toxic metabolites which inhibited egg hatching and mobility of second-stage juveniles of M. incognita (Li et al., 2002). Diedhiou et al. (2003) demonstrated the successful nematicidal activity of an arbuscular mycorrhiza, Glomus coronatum, and an endophytic fungus, F. oxysporum, against the M. incognita in tomato plant. Several endophytic fungi isolated from above-ground plant organs produced 3-hydroxypropionic acid (HPA) by bioactivity-guided fractionation of extracts and showed selective nematicidal activity against the plant-parasitic nematode, M. incognita, with LD50 values of 12.5–15 µg/ml (Schwarz et al., 2004). Radopholus similis is an important parasitic nematode on banana and other plants. It is suggested that the dual inoculations of endophytic fungal isolates reduce a large number of the R. similis population (Felde et al., 2006). Fungi are known to produce a large number of insecticidal metabolites such as destruxins, ibotenic acid, pantherine, tricholomic acid, etc. Endophytic fungi are also known to deter insect pests (Clay, 1989; Carroll, 1991, 1995; Azevedo et al., 2000). Several toxins are produced by endophytic fungi and these substances confer host protection against different herbivores. The endophytic fungus, P. oblonga, was responsible for reducing the spread of Dutch elm disease causal agent, Ceratocystis ulmi, by controlling its vector beetle (P. brevilineum) (Webber, 1981). In 1985, Claydon and his co-workers conﬁrmed that endophytic fungi belonging to the Xylariaceae family synthesized secondary metabolites in host Fagus sp. and that these substances affected the beetle larvae. Susceptible and resistant cultivars of perennial rye grass (L. perenne L.) against sod webworms (Crambus spp.) were analysed for the presence of an endophytic fungus. All resistant cultivars were found to have a high infection of endophytic fungi. Several highly infected ryegrass species with endophytic fungi consequently have shown less attack frequency of Argentine stem weevils (Listronotus bonariensis) (Gaynor and Hunt, 1983). Barker et al. (1984) and Prestidge et al. (1984) also observed that the same grass infected with endophytic Acremonium sp. was more resistant to stem weevils in New Zealand. In the white spruce, Picea glauca, the death rate of the Homoptera, Adelges abietis, was considerably higher when galls were infected with the endophytic fungus, C. sphaerosperum (Lasota et al., 1983). In L. perenne and a few members of genus Cyperus, insectpest Spodoptera frugiperda was affected adversely by endophytic fungus like Balansia cyperi (Clay et al., 1985a,b). Ahmad et al. (1985) showed that endophytic Acremonium sp. deterred the grasshopper, Acheta domesticus. Patterson et al. (1992) observed the production of alkaloids by endophytic Acremonium in plants Lolium and Festuca that reduced the attack of the Japanese beetle, Popilla japonica. Muscodor Role of Fungal Endophytes vitigenus, an endophytic fungus of Paullinia paullinioides, from the Peruvian Amazon, is known to produce naphthalene, which effectively repels the adult stage of the wheat stem sawﬂy, Cephus cinctus (Daisy et al., 2002). Endophyte-mediated resistance was reported in strong creeping and chewings fescue species against red thread (Bonos et al., 2005). Beauveria bassiana is a highly effective entomopathogen of a wide range of insects. Grass varieties infected by Neotyphodium endophyte have affected the feeding performance and preference of newly hatched nymphs of the hairy chinch bug, Blissus leucopterus hirtus, a common turfgrass pest in north-eastern USA (Steeve et al., 2007). Akello et al. (2007) incorpotated B. bassiana as an artiﬁcial endophyte in banana plants to combat the banana weevil, Cosmopolites sordidus. The endophytic fungi, B. bassiana and Clonostachys rosea, isolated from coffee plant, showed strong antagonistic activity against coffee berry borers (Vega et al., 2008). Two new insecticidal compounds, 5-hydroxy-2-(1′-oxo-5′-methyl-4′-hexenyl) benzofuran and 5-hydroxy-2-(1′-hydroxy5′-methyl-4′-hexenyl) benzofuran were isolated via bioassay-directed fractionation of culture extracts of an unidentiﬁed endophytic fungus obtained from wintergreen, Gaultheria procumbens (Findlay et al., 1997). These compounds exhibited toxicity to spruce budworm (Choristoneura fumiferana Clem.) cells. Peramine and lolines, potent insecticides, are produced in endophyte-infected perennial ryegrass and protect them from the Argentine stem weevil, Listronotus bonariensis (Rowan and Latch, 1994; Tanaka et al., 2005). Nodulisporic acids, novel indole diterpenes, have potent insecticidal properties against the larvae of the blowﬂy by activating insect glutamate-gated chloride channels. Nodulisporium, an endophytic species from the plant, Bontia daphnoides, produces such nodulisporic compounds (Demain, 2000). A strain of endophytic Penicillium sp., isolated from the fresh roots of Derris elliptica, produces some insecticidal compound analogues to rotenone against the adult turnip aphid, Lipaphis erysimi (Hu et al., 2005). 189 Plant Protection in Abiotic Stresses Endophytes are also involved in the protection of plants in various abiotic stresses like drought, temperature, pH, heavy metals, etc. (Rodriguez et al., 2004). Water stress tolerance was observed in epacrids and their endophytic partners in south-west Australia (Hutton et al., 1996). In drought conditions, water content of some endophyte-associated, ﬁeld-grown tall fescues may be maintained at higher levels than those of endophyte-free plants (Elbersen and West, 1996; Buck et al., 1997). This phenomenon may be explained by enhanced accumulation of solutes in tissues of endophyte-infected plants as compared to non-infected plants, or by reduced leaf conductance and a slowdown of the transpiration stream, or due to thicker cuticle formation (Malinowski and Belesky, 2000). The endophytic mutants and wild-type C. magna confer drought tolerance that allows symbiotic tomato and pepper plants to survive desiccation for 24 and 48 h longer than non-symbiotic plants, respectively (Redman et al., 2001). Endophytic colonization was observed to increase the minimum leaf conductance in Theobroma cacao, a measure of leaf water loss after maximal stomatal closure under drought stress (Arnold and Engelbrecht, 2007). However, no evidence for endophyte-mediated drought tolerance was observed in Acremonium-infected tall fescue (White et al., 1992). It is suggested that endophyte-mediated drought resistance may be due to alterations in drought avoidance. Malinowski and Belesky (1999) observed that the pH of a limed, acidic soil increased faster as a result of the root activity of endophyte-infected tall fescue compared with non-infected plants under phosphatedeﬁcient conditions. Liu et al. (1996) observed that aluminium tolerance in endophyteinfected ﬁne fescues (Festuca spp.) was greater as compared to non-infected plants. In an experiment, endophyte-infected clone grew signiﬁcantly better in high aluminium soils relative to the endophyte-free clone (Zaurov et al., 2001). L. perenne, symbiotic with N. lolii, showed higher values of total dry weight and tiller number compared 190 S.K. Gond et al. to non-symbiotic plants in Zn stress (Monnet et al., 2001). In low NaCl salt stress condition, the endophyte Piriformospora indica-infected barley plants showed higher biomass than non-infected plants (Waller et al., 2005). The mechanism of endophyte-conferred salt tolerance has not been investigated so far. In the USA, a plant species, Dichanthelium lanuginosum, has been found growing in the geothermal soils of Yellowstone National Park (YNP) and Lassen Volcanic National Parks (LVNP) at temperatures as high as 57°C (Stout and Al-Niemi, 2002). Redman et al. (2002) observed that those plants colonized by an endophytic fungus, Curvularia protuberata, were able to tolerate the higher temperature and, thus, we might conclude that endophytes supported the plant to withstand heat or drought stresses. However, an in-depth investigation by Marquez and his colleagues (2007) showed that this was not only because of plant–fungus symbiosis but it also included a virus as a third partner, which parasitized on C. protuberata. Thus, it is a complex tripartite symbiosis and the heat tolerance ability of the fungus is, in fact, related to the virus. That mycovirus is called a Curvularia thermal tolerance virus (CThTV). Two mechanisms are involved in the endophyte-conferred biotic and abiotic stress tolerance: (i) rapid activation of host stress response systems in exposure to stress (Redman et al., 1999); and (ii) synthesis of anti-stress biochemicals in the host, either by endophytes or through endophyte induction (Bacon and Hill, 1996). Endophyteproduced anti-stress biochemicals are mostly alkaloids. In addition to anti-stress biochemicals, plant and fungal mutualism has been maintained over an evolutionary time by the ability of fungi to control the activation of host stress response systems and, in core, act as ‘biological triggers’ (Rodriguez et al., 2004). When a plant interacts with environmental biotic and abiotic stresses, it produces several damaging reactive oxygen species (ROS). Therefore, it is hypothesized that endophytes inside plants scavenge these ROS rapidly and protect their host (Rodriguez and Redman, 2005; Tanaka et al., 2006). Indian Contributions to Fungal Endophyte Research The past history of endophytic research in India, especially with fungi, is not so encouraging. It seems that workers who started this research in India are still actively involved in advancing their research manifesto with this ‘under-studied’ group of microbial population and have not advanced to the ﬁelds and forests of the countryside looking for novel microbe/plant associations. Prof Suryanarayanan and his group (Chennai) have initiated biodiversity and distribution patterns of fungal endophytes with some medicinal plants in India and have published several papers along this line. He has also isolated some bioactive compounds and melanin from endophytic fungi (Suryanarayanan et al., 2004). Several research groups have started paying more attention to various aspects of endophytic fungi. No more than a dozen research groups at various locations in India are vigorously involved in either biodiversity or natural product discovery from this untapped and alternative resource (Table 15.1). It has become obvious to many workers throughout the world that endophytic microbes have enormous potential to solve many of mankind’s problems. Thus, with the discovery of new compounds, we can protect our agriculture and medicine industries, as well as plant health. After more than 20 years of effort, the total number of publications from Indian researchers, including some fairly recent ones (Shankar et al., 2003; Seena and Sridhar, 2004; Amna et al., 2006; Tejesvi et al., 2007; Gangadevi and Muthumarry, 2008), is relatively small. Due to the great variation in plant biodiversity and seasonal changes in India, we may have a better opportunity to collect/ isolate various types of promising endophytic fungi, especially from rainforests and mangrove swamps, which may be able to produce an enormous variety of potential bioactive natural compounds. An increasing population of AIDS and immunocompromised patients in India compels us to bear them in mind when searching for safe drugs. Table 15.1. List of Indian workers involved in endophytic fungal research. Name of group leader Place of work Work specialization E-mail addresses 1. Dr T.S. Suryanarayanan 2. Dr K.R. Sridhar Dr H.S. Prakash 4. 5. Dr D.J. Bhat Dr J. Muthumarry 6. 7. Dr Arun Arya Dr R. Uma Shaankar 8. Dr Absar Ahmad National Chemical Laboratory, Pune 9. 10. Dr S.K. Singh Dr S.C. Puri/ R.K. Khajuria Dr R.N. Kharwar Agharkar Research Institute, Pune Regional Research Laboratory, Kanal Road, Jammu Tawi Department of Botany, B.H.U., Varanasi-221005 Endophytic fungal diversity and natural product discovery Endophytic fungal diversity and bioactive molecules Endophytic fungal diversity and bioactive molecules Endophytic fungal diversity Endophytic fungal diversity and bioactive fungal compounds Endophytic fungal diversity Endophytic fungal diversity and bioactive molecules Synthesis of silver and gold particles from endophytes Endophyte diversity Natural product development tssury@md3.vsnl.net.in ts_sury2002@yahoo.com sirikr@yahoo.com 3. Department of Botany, Vivekanand College, Chennai Department of Biosciences, Mangalore University, Mangalagangotri, Mangalore Department of Botany, University of Mysore, Manasagangotri Department of Botany, Goa University, Panji, Goa Department of Botany, University of Madras, Chennai Department of Botany, MSU, Baroda University of Agriculture, Karnataka singhsksingh@rediffmail.com khajuriark@yahoo.com Endophytic fungal diversity and natural product discovery rnkharwar@yahoo.com kharwar1@rediffmail.com 11. hasriprakash@gmail.com bhatdj@rediffmail.com mm_j@rediffmail.com aryaarunarya@rediffmail.com umashaanker@gmail.com aahmad@dalton.ncl.res.in Role of Fungal Endophytes Sr. No. 191 192 S.K. Gond et al. India really needs a variety of novel antimicrobial compounds of biological origin, so that we can solve the problems of ecofriendly farmers and the weaker sections of society in which the above-mentioned diseases are prevalent. The fungi, as a group, hold enormous potential as sources of antimicrobials. Observations prove that this group of organisms resides inside healthy plant tissues as endophytes without causing any detectable symptoms. Therefore, we feel strongly that India needs to gear up and exact its research to exploit the maximum potential of the promising endophytes for natural product discovery, which could at least facilitate some of the existing problems of its huge population. Conclusions In the study of mycodiversity, we often forget the endospheric fungi as researchers focus their attention on the phyllospheric and rhizospheric fungi. The endosphere is a special niche where endophytic microorganisms reside and, in response, produce a variety of metabolites, which are mostly toxic to plant and human pathogens. In this aspect, plant pathogens interact with the plant itself, as well as the plant’s endophytes. The role of endospheric or so-called endophytic fungi in plant protection is quite clear in the above-mentioned examples. Besides protecting plants from biotic and abiotic stresses, endophytes also improve the health and yield of plants by producing some growth-regulating phytohormones. Although endophytes are still poorly investigated microorganisms, they have shown that they are going to play a prominent part in the discovery of many bioactive natural compounds. Bioactive natural products of endophytic origin can change the scenario of existing agropesticides because of their easy and sustainable production. Many scientists throughout the world are engaged in the search for bioactive compounds from endophytes. There is a gap in the knowledge on the genetic and biochemical communications between the plant and endophytic symbionts. We have to minimize this gap for better utilization of endophytic microorganisms. Acknowledgements The authors are thankful to the Head of the Department of Botany, BHU, Varanasi, for providing the necessary facilities. They also extend their thanks to the CSIR, New Delhi, for ﬁnancial support. References Ahmad, S., Govindarajan, S., Funk, C.R. and Johnson-Cicalese, J.M. (1985) Fatality of house crickets on perennial ryegrass infected with a fungal endophyte. Entomologia Experimentalis et Applicata 39, 183–190. Akello, J., Dubois, T., Coyne, D. and Hillnhú´tter, C. (2007) Beauveria bassiana as an artiﬁcial endophyte in tissue-cultured banana plants: a novel way to combat the banana weevil Cosmopolites sordidus (http://www.banana2008.com/cms/details/Paper1.doc, accessed 26 July 2008). Amna, T., Khajuria, R.K., Puri, S.C., Verma, V. and Qazi, G.N. (2006) Determination and quantiﬁcation of Camptothecin in an endophytic fungus by liquid chromatography-positive mode electrospray ionization tandem mass spectrometry. Current Science 91, 208–211. 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Patil1 and Anjali Patil2 1Department of Botany, Shivaji University, Kolhapur, India; 2Department of Botany, Rajaram College, Kolhapur, India Abstract The rust fungi (Uredinales) consist of 7000 species belonging to 163 genera in 14 families and comprise about 10% of all described species in the Kingdom Fungi. All the rust fungi are ecologically obligate parasites on ferns, gymnosperms and angiosperms. There are six vital processes in plants and, correspondingly, six ways in which rusts affect their hosts adversely. Rusts as pathogens damage foliage, the main organ of photosynthesis, destroy seedlings, impair growth and interfere in the metabolism of the hosts. Management of any disease begins with correct identiﬁcation of the pathogen; hence, some important concepts in rust systematics are discussed, along with detailed information about rust diseases of some economically important crops. Of course, discussion on plant diseases would not be complete without recent management strategies. The discussion includes the following; 1. Rust systematics, including characteristic features of rust fungi, their occurrence and geographical distribution, vegetative and reproductive propagules, pleomorphism, autoecious and heteroecious nature and host range, etc. 2. Rust diseases of crops, including ﬁeld crops – medicinal, ornamental, cereals, pulses, millets, oilseeds, fruit and plantation crops, etc. – nature of disease, epiphytotics, disease development index; X = XoeRT, assessment of crop losses. 3. Management strategies citing food crisis, need for another green revolution, crop losses, famines, social impact of rust diseases, e.g. change in coffee-drinking habit due to coffee rust, management methods – Sharvelle’s strategy (1961): (i) protective, (ii) preventive; and (iii) corrective (physiological disorders), cultural, chemical, biological, breeding, biotechnology – transgenic plants are described in detail. Introduction: Rust Systematics The rust fungi (Uredinales) consist of 7000 species belonging to 163 genera in 14 families and comprise c.10% of all described species in the Kingdom Fungi (Kirk et al., 2001; Ono, 2002). The Uredinales are believed to be monophyletic taxa and recent molecular–phylogenetic analysis (Swann and Taylor, 2001) supports this perspective. Very recently, a rust, Uredo vetus Henne, has been reported for the ﬁrst time on Selaginella sp. The parasitism of rust fungi to the host plant is highly speciﬁc; however, this specialization varies with species, for example, two well-known rusts of soybean, namely Phakopsora pachyrhizi Syd. and Syd. and P. meibomiae Arthur (American rust) (Ono et al., 1992), occur on a large number of species of family Leguminosae. The rust fungi  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 201 202 M.S. Patil and A. Patil are unique in having complex life cycle patterns with elaborate spore forms. Accordingly, many types of life cycles are known (Laundon, 1973) with modiﬁed spore types with immense functional diversities; for example, aecioid teleutospores in endoform rusts like Endophyllum, Monosporidium and Kulkerniella or uredinoid teleutospores in Hemileia vastatrix Berk. & Br., which Rajendren (1967) described as ‘Kamat Phenomenon’. The spermogonial–aecial host/s and uredinial–telial host/s are closely associated eco-geographically. Heteroecious life cycle is widespread in the uredinales. If all spore forms are produced in unidirectional order, the life cycle is said to be macrocyclic (Puccinia graminis, P. helianthi, the heteroecious and autoecious rusts of wheat and sunﬂower, respectively). There is a tendency to omission of spore form in life cycles and thus many different patterns exist, for example, demi-cyclic, in which the uredial stage is absent. In rust fungi, a widespread assumption is that parasitism and host specialization are acquired at an early stage of rust fungus evolution. Nine types with 11 variations are found in nuclear cycles associated in metabasidium development of microcyclic rust fungi (where only a telial stage with or without a spermogonial stage is formed on a plant throughout the season). Thus, rust species that produce only teleutospores that germinate without dormancy to initiate new generations repeatedly in a single growing season would be highly adaptive, e.g. P. pampeana Speg. on chillies (Capsicum spp.), P. alyxiae Arthur on Alyxia spp., P. xanthii Schw., M. machili (Hennings) T. Sato, E. acacia Hodges and Gardner. Microcyclic species exhibit two or more patterns of nuclear cycles and different metabasidium development, indicating that microcyclic lineages might have evolved independently and repeatedly from macrocyclic parental species. many economically important pathogens of vascular plants. Dietel (1900) divided the order Uredinales into four families based on sessile or pedicillate teleutospores as follows: Families of Rust Fungi Based on Teleutospores 1. Puccinia Pers. ex Pers. (c.3000–4000 spp.). 2. Uredo Pers. ex Pers. (c.3000 spp.). 3. Uromyces (Link) Unger (c.600–700 spp.). 4. Ravenelia Berkeley (c.200 spp.). Rust fungi comprise one of the largest and best described groups of fungi and include Pedicillate teleutospores -------Pucciniaceae Sessile teleutospores In a single layer --------Melampsoraceae In 1–2 layers forming waxy crust ----------------------------------Coleosporiaceae In chains ---------------------Cronartiaceae Families Pucciniastraceae (Arthur) Gaumann Coleosporiaceae Dietel Cronartiaceae Dietel Melampsoraceae Schroeter Phakopsoraceae (Arthur) Cummins & Hiratsuka Mikronegeriaceae Cummins & Hiratsuka Chaconiaceae Cummins & Hiratsuka Uropyxidaceae (Arthur) Cummins & Hiratsuka Pileolariaceae (Arthur) Cummins & Hiratsuka Raveneliaceae (Arthur) Leppik Phragmidiaceae Corda Sphaerophragmidiaceae Cummins & Hiratsuka Pucciniaceae Chevalier Puccinosiraceae (Dietel) Cummins & Hiratsuka Unassigned genera Total Genera 06 02 01 02 11 01 10 10 03 14 10 06 15 09 99 08 107 However, this dependence on teleutospore morphology has brought many unrelated genera into the same family. Some of the larger genera are: The Rust Fungi 5. 6. 7. 8. Melampsora Castagne (c.100 spp.). Hemileia Berk. & Br. (c.50 spp.). Coleosporium Lev. (c.80 spp.). Phragmidium Link (c.60 spp.). In some cases, the original function has been changed irrespective of its basic nature or structure; for example, the species of Endophyllum, Monosporidium or Kulkerniella produce aeciospores morphologically in aecial cups, but they function like teleutospores producing promycelium bearing basidiospores and are thus called aecioid teleutospores. In H. vastatrix, urediniospores occasionally function like teleutospores and are called uredinoid teleutospores. Of course, in the ﬁrst example, no teleutospores are produced, while in the second example, teleutospores normally are produced in the life cycle. In the course of the evolution of rust fungi, there is a tendency to eliminate spores, narrowing the host range and surviving in spite of unfavourable environmental conditions or non-availability of the required host. There are numerous examples in which a rust survives or continues its life cycle by producing one type of spore, e.g. Aecidium, Uredo, Peridermium, Caeoma, etc. 203 repeating like conidia, germinate very easily within 24 h at high temperatures and relatively higher humidity, but their viability is lost at very high temperatures. At lower temperatures, spores remain viable for a long time. They germinate mostly by germ tubes, except in coffee rust, where they behave like uredino teleutospores. Urediniospores are most signiﬁcant in disease development and spread on epiphytotic scale (van der Plank, 1963, 1968). Hence, this spore state is also considered as the conidial state during sporulation and the release of spores form spore clouds in the air and serves as secondary and tertiary inoculum within the crop in a favourable season. Rust diseases, due to their repeating nature, are known as compound interest diseases. Log e(x/1 – x) where x is a proportion of infected susceptible tissue, if the pathogen is systemic. Urediniospores are light, airborne and travel long distances at various heights; for example, wheat rust urediniospores travel from Mexico to Canada; in Indian wheat rust, the rust originates in South India from the Nilgiri and Pulney Hills and travel via Central India from the plateau of Mahabaleshwar and Panchgani to North India. Spore morphology In groundnut rust, the uredinospores are one-celled, spherical to oval or angular, stalked, mostly brown coloured, faint or dark, thick-walled with spiny, verrucose, or a modiﬁcation of these two, rarely smooth, bearing visible areas in the walls through which germination takes place. Tulasne and Tulasne (1847) observed for the ﬁrst time, pores/oscules varying from 2 to 20 in number. The germ pores may be distributed equatorially, zonal or scattered. Cummins (1936) also recognized their phylogenetic signiﬁcance in the rust taxonomy. There appears to be some correlation between the arrangement of pores and the shape of urediniospores; globoid spores usually have scattered pores, while ellipsoid, oblong or asymmetrical spores usually have zonnate pores. Urediniospores are bi-nucleate, Rust physiology – urediniospore germination Spore liberation is active and the terminal velocity of fungal spores in the air is 0.05– 2.5 cm/sec. In calm weather, only 0.05% spores travel more than 100 m from their source of origin. Spores of black stem rust fall from a height of 1.6 km to the ground at a speed of 12 mm/sec and travel from place to place at 11–32 km/h (Gregory, 1973). Urediniospores of different rust species have a different period of viability as they are affected by environmental factors such as RH, light intensity, as well as their own structural characteristics, namely wall thickness, etc. In northern India, urediniospores are killed by high temperatures in the ﬁeld and cannot serve as a source of inoculum the following year. 204 M.S. Patil and A. Patil Viability of urediniospores in rust fungi Rust urediniospores show different periods of viability at 20–40% RH and 23°C, e.g. P. graminis tritici 36 days, P. recondita 63 days, P. coronata 87 days, P. menthae 173 days, P. helianthi 185 days and U. pisi 75 days. Viability of urediniospores decreases at higher or lower humidity. Spores germinate generally at 90–100% RH, while the temperature requirement varies greatly in different species. The period required for sporulation (urediniospores) in black stem rust of wheat is found to be 5 days at 24°C. If the temperature is lowered to 0°C, then sporulation occurs after 85 days. Spore longevity depends on light, temperature, relative humidity, species of rust and type of spore. Basidiospores and pycniospores are delicate and have least viability. But if the spores are kept at a low temperature, viability lasts for 18 days. In the case of sunﬂower rust, relative humidity is more important than temperature. At 80% RH, only 5% aeciospores remain viable after 56 days. Teleutospores spores are produced at the end of a rust fungus’s life cycle, i.e. spores terminating the life cycle of rusts. They are produced in telia in or on the host, are innate or erumpent, covered or exposed in telial sori, in the leaves or in the stem. There is a tendency in rusts to eliminate spore states showing progressive reduction either due to non-availability of host or climatic conditions, for example, rusts in temperate regions on the family Liliaceae. Rust systematics, a dynamic science, is far from perfect; hence, there is still a lot of work to be done on their taxonomy and pathology. Study of their host’s behaviour during development, varieties, races, physiological forms and pathotypes is beyond the scope of taxonomists. Rust Diseases of Some Economically Important Crops Plant pathology originated in Europe and migrated to North America, where it ﬂourished and spread to different parts of the world. Among all known living organisms, three groups are dominant, namely insects, ﬂowering plants and fungi. Among all these plant pathogens, fungi are the most dominant and successful plant pathogens and are estimated to produce more than 25,000 diseases. Among these, 8000 diseases of cultivated and plantation crops are extremely damaging in the ﬁeld every year. Kuhn, (1858) wrote a book entitled Diseases of Cultivated Plants. Rusts are complex; hence, it is difﬁcult to understand how they damage standing crops in the ﬁeld qualitatively and quantitatively, creating problems of food crisis and insecurity; a global problem today. Epidemiological Studies Epidemiology is the science of epidemics or diseases in plant population. Types of epiphytotics are: 1. Based on the rate of disease development: (i) Tardiv (slow epiphytotics); (ii) Explosive (rapid epiphytotics). 2. Area covered and time of development: (i) Pandemic – developing on a continental scale; (ii) Sporadic – seasonal and irregular incidence. There are also secondary epiphytotics known. Epiphytotics is also deﬁned as ‘a host– pathogen system, out of genetic balance in favour of the pathogen’. Such epiphytotics of crop plant diseases are known, in the history of plant pathology, to be followed by food famines: 1. Wheat rust epidemics occurred in 1916 in America and Canada; 1935 and 1937 in America; 1951 in Europe; and 1827, 1907, 1947, 1949–1950, 1957, 1971–1972 in India. 2. Coffee rust epidemics occurred in 1867 and 1875 in Sri Lanka; 1891 in the Philippines; 1891–1892 in Java; 1911–1913 in Central Africa; 1871–1878 in South Africa; and 1970–1971 in Brazil. The coffee rust famines inﬂuenced the coffeedrinking habit, which then changed to tea. The Rust Fungi Rusts are compound interest diseases and an increase of infection at a compound interest rate exponentially/logarithmically increases the rate of compound interest of disease by primary and secondary infection. The compound interest equation can be given as: X = XoeRT where X = the amount of disease at time T, Xo = initial amount of disease at O time, R = infection rate, which is variable, and e = 2.718 for cereal rusts. The rate (R) of increase % per unit of time is a fundamental concept in epidemiology, e.g. 12.5%/day in P. recondita and 57%/day in Phytophthora infestans. Development of epiphytotics is basically a transport problem to get enough inoculum to the right place at the right time. Plant–pathogen– environment is a triple interaction and may be complicated by vectors and humans; according to van der Plank (1963, 1968), the pathogen must be virulent. To express virulence quantitatively, the disease reaction type is expressed in numerical values as: R (resistant) = 01, MR (moderately resistant) = 02, S (susceptible) = 03 Aggressiveness corresponds to disease severity on a 0–9 score scale: 0 = absent, 9 = more than 75% leaf area in 12 days after inoculation therefore VI (virulent index) = [1 + (virulence × aggressiveness) × latent period] VI = [1 + VAL – 1] where VI = virulent index, A = aggressiveness and L = latent period. 205 The estimated annual crop losses worldwide (Agrios, 2005) are: US$ Attainable crop production (2002 prices) Actual crop production Production without crop protection Losses prevented by crop protection Actual annual losses to world crop production Losses caused by disease 1–5 trillion 995 billion 445 billion 415 billion 550 billion 220 billion Rusts damage plants and plant products, causing economic losses. Crop protection measures result in increased prices of primary products to consumers and pollution of the environment. Rusts are also pathogenic to animals and humans. Diseases are responsible for minor aesthetic losses – in domestic gardens, avenues and forests. There are six vital processes in plants and, correspondingly, six ways in which rusts affect their hosts adversely. Rusts as pathogens damage foliage, the main organ of photosynthesis, destroy seedlings, impair growth and interfere in the metabolism of the hosts. Rusts keep hosts alive and active for their own growth, development and spread. Hence, pathogens and hosts have coevolved. At the same time, rusts are a useful means of controlling weeds as their infection results in thinning of plants in the ﬁeld. Artiﬁcial infection of rust fungi in fodder grasses brings about an increase in protein content. Crop Losses Green Revolution and Grain Production in India Conservative estimates of total annual losses in crop production by diseases, insects and weeds worldwide are 220 billion US$ corresponding to 31–42% of all losses, of which diseases are 14.1%, insects 10.2% and weeds 12.2%, while 6–12% losses are postharvest losses. In India, a green revolution began in 1960. In 1965, hybrid varieties were introduced, followed by an increased consumption of fertilizers (N, K, P). From the 53% of total area under cereal cultivation, hybrid cultivars have been introduced in 16% of the area. These innovations in agricultural practices 206 M.S. Patil and A. Patil revolutionized grain production in India from 1900 to 1971. From 1900/01 to 1910, grain production was 67.6 Mt. It then remained stable until 1948, in the preindependence era. During 1948–1949, there was a wheat famine. During the ﬁrst three of the Five Year Plans (1950–1965), the rate of grain production increased by 2.5%. In the fourth Five Year Plan, grain production was approximately 100 Mt, i.e. grain production increased by 5%. The food crisis provided not only a warning but also an opportunity for new thinking. Still, there is some hope as the International Grains Council has forecast a 7% increase in global wheat production. India is the world’s second largest producer of wheat and rice and the expected wheat harvest this year is 76.8 Mt and rice production is 95.7 Mt. Among the major cereal crops, wheat is the most suitable due to its superior quality of grain, coupled with its wide adaptability for cultivation under varied conditions; humans and wheat will survive in any environment. Even today, the wheat rust management mission is still incomplete and awaiting novel solutions to increase the yield of quality grains. Lord John Boyd Orr, the ﬁrst Director of the FAO, said in 1948, ‘a lifetime of poor nutrition and actual hunger is the fate of at least 2/3rd of the world’s population’. This is still true, even today. Diseases of Crop Plants and Associated Pathogens 4. Rust of jowar (Sorghum bicolor (L.) Moench): (i) P. purpurea Cke.; (ii) P. levis Arthur; (iii) P. nakanishiki Dietel. 5. Rust of pearl millet or bajara (Pennisetum glaucum (L.) R. Br.): (i) P. substriata Ell. and Barth var. indica Ramachar and Cummins, India; (ii) P. substriata Ell. and Barth. var. decrospora Eboh, Nigeria; (iii) P. substriata Ell. and Barth. var. penicillaris Ramachar and Cummins. 6. Rust of maize (Zea mays L.): (i) P. sorghi Schw. (common corn rust); (ii) P. polysora Underw. (southern corn rust); (iii) Physopella zeae (tropical corn rust). Oilseed crops 1. Soybean rust (Glycine max (L.) Merr.): Malupa sojae (P. Henn.) Ono, Y. et al. or Malupa state of P. pachyrhizi H. and P. Sydow. 2. Groundnut rust (Arachis hypogaea L.): P. arachidis Speg. 3. Sunﬂower rust (Helianthus annuus L.): P. helianthi Schwein. 4. Safﬂower rust (Carthamus tinctorius L.): (i) P. carthami Corda; (ii) P. caleitrapae var. centaureae (DC.) Cummins. 5. Linseed/ﬂax rust (Linum usitatissimum L.): M. lini (Ehrb.) Lev. Grain crops Plantation crops 1. Wheat rusts (Triticum spp.) (i) Black stem rust: P. graminis Pers. tritici Eriks. and Hennen; (ii) Brown rust: P. recondita Rob. ex Desm.; (iii) Yellow or stripe rust: P. striiformis West. 2. Leaf rust of rye (Secale cerealis L.): P. graminis Pers. secalis. 3. Leaf or crown rust of oat (Avena sativa L.): P. coronata Corda and P. graminis Pers. avenae Fraser & Ledingham. 1. Coffee leaf rust (Coffea arabica L. and other spp.): (i) H. vastatrix Berk. and Br.; (ii) H. coffeicola (reported only from Cameroon, West Africa). 2. Mulberry rusts (Morus alba L. and other spp.): (i) A. mori Barclay; (ii) Cerotelium ﬁci (Butler) Arthur. 3. Dalbergia rust (Dalbergia spp.): Sphaerophragmium dalbergiae Dietel = U. dalbergiae The Rust Fungi P. Henn. (1895) = U. sisso Syd. & Butl. (1906). 4. Teak rust (Tectona grandis L.): Olivea tectonae (Ramkr., T.S. and K.) Mulder = Chaconia tectonae Ramkr., T.S. and K. 2. 3. 207 Rust of Vitex spp.: (i) O. ﬁmbriata (Mains) Cumm. & Hirat.; (ii) O. scitula H. Sydow; (iii) O. viticis Ono, Y. and Hennen. Rust of Vinca major L.: P. vincae Berk. Pulses and vegetables Ornamental plants 1. Green gram rust (Cicer arietinum L.): Uromyces ciceris arietini (Gron.) Jack. 2. Rust of Phaseolus sp: U. appendiculatus var. appendiculatus (Pers.) Unger. 3. Cowpea rust (Vigna sp.): U. vignae Barclay. 4. Bean rusts (Pisum, Vicia, Lens, Lathyrus spp.): (i) U. viciae-fabae (Pers.), Schroeter, autoecious rust in Europe and America; (ii) U. pisi (Pers.) Wint., heteroecious rust in Europe, rarely in India. 5. Chilli rust (Capsicum annuum L.): P. pampaeana Speg. (Mexico, Peru, Brazil, Columbia and Gautemala). 1. Rose rusts (Rosa spp.): Phragmidium spp. (10 spp.). 2. Gladiolus rust (Gladiolus spp.): P. gladioli (Duby) Cast. 3. Tulip rust (Tulipa spp.): P. prostii Moug. (on wild species). 4. Canna rust (Canna spp.): P. thalie Dietel. 5. Chrysanthemum rust (Chrysanthemum spp.): P. chrysanthemi Roze. 6. Saxifraga rust (Saxifraga spp.): P. saxifragae Schlecht. Forage crops (Fabaceae) Rust of wheat (Triticum spp.) 1. Bersim/clover rust (Trifolium spp.). 2. Lucerne (Medicago spp.). 3. Alfalfa rust (M. sativus L.): U. striatus Schroeter. 4. Clover rusts (Trifolium spp.): (i) U. trifolii-repentis Liro; (ii) U. fallens (Arthur) Barth. In India, wheat is cultivated on 16m ha, but the average yield is very low, that is, 810–1000 kg/ha as compared to the wheat yield in other countries, namely Argentina 1210 kg/ha, America 1610 kg/ha, Belgium 3700 kg/ha and Denmark 4000 kg/ha. The ﬁve major wheat-growing areas in India are in the north-western zone, the north-eastern zone and the central, peninsular and northern hilly zones. Gene exchange of the new allopolyploids inevitably would have resulted through hyphal fusion, nuclear exchange and genetic recombination in urediniospore population. This results in widening the host range of the hybrid rusts because of gene diversity and increases the value of survival and nutritional status of the rust. Continuous introduction of hybrid cultivars in the ﬁeld through plant breeding serve as new hosts to rust. Three species of wheat and their cultivars are used mainly for cultivation of durum wheat (T. durum Desf.) or macaroni wheat, which covers about an 85% area. Bread wheat (T. aestivum L.) covers a 14% Fibre crops 1. Cotton rusts (Gossypium spp.): (i) P. gossypi (Arthur) Hiratsuka = U. gossy = C. desmium; (ii) A. gossypi, an aecial state of P. cacabata Arthur & Holway. 2. Linseed/ﬂax rust (Linum usitatissimum L.): M. lini (Ehrb.) Lev. Medicinal plants 1. Adhatoda zeylanica Nees. rust: Chrysocelis butteri (Dietel and Sydow) Laundon. The key to species and varieties of Puccinia and Uromyces producing rust diseases can be found in the Appendix at the end of this chapter. 208 M.S. Patil and A. Patil area and emmer wheat only about a 1% land area. Rusts of wheat have many physiological races and wheat cultivars are recommended by plant breeders in speciﬁc regions for cultivation to avoid rust disease development. Epidemiological studies of three rusts have shown that collateral hosts have a restricted role, while alternate hosts virtually have no role at all. The survival of these rusts in India is primarily through urediniospores that survive on self-growing plants or volunteer plants. The airborne urediniospores favoured by wind and rain due to tropical cyclones in the months of October and November get dispersed and deposited over Central India from the Nilgiri Hills in South India. Brown and black stem rusts become established there and then subsequently spread to the eastern and northern states of India over the Indo-Gangetic Plain. Brown rust appears ﬁrst in the Himalayan foothills, eastern Uttar Pradesh and north Bihar in the month of January. The western lines associated with disturbances and rain spread the pathogen to the north-western states of India, along with yellow rust. The Nilgiri and Pulney Hills are the primary focal point providing the source of inoculum, that is urediniospores migrating upward with air currents towards the north via Central India, periodically trapped and studied by Mehta (1929, 1952). The Mahabaleshwar and Panchgani plateaus also serve as a focal point for the secondary source of inoculum (Joshi et al., 1986). Rust of coffee Coffee rust is a disease of the coffee plantation crop, namely Coffea arabica L., C. libarica and C. canephora, cultivated for berries to produce coffee, a well known non-alcoholic drink like tea, in Ethiopia, Yemen, Sri Lanka, South and Central Africa, Cameroon, Bahamas, Brazil and India. The world production of coffee is 3.16 Mt/year. In India, coffee is cultivated mainly on the hill slopes of Karnataka, Tamil Nadu and Kerala. Coffee production is estimated to be 964,000 t/year on a worldwide basis and its production in India is 230,000 t. The pathogen of the coffee plant is H. vastatrix Berk. and Br., which develops very serious disease on foliage, leading to defoliation. Rust of groundnut (Arachis hypogaea L.) Groundnut is the world’s second largest source of edible oil and ranks 13th in production among world food crops. India is the largest producer of groundnut. Groundnut is cultivated in 26m ha of land worldwide and produces 34.5 Mt/year. Groundnut is cultivated in India on 7.6m ha and produces 7.8 Mt/year. The major states in India cultivating groundnut are Gujarat, Maharashtra, Tamil Nadu and Andhra Pradesh. This crop is attacked by 55 pathogens. Among all the diseases, three diseases, namely groundnut rust and early and late leaf spot diseases, are more serious. They generally develop simultaneously and pod yield decreases by up to 10–70% (Ghewande and Savalya, 1999). Rust is caused by P. arachidis Speg.; this perpetuates by uredinia, the only spore state throughout the world except teleutospores, which were recorded in Paraguay only once. It is not known how groundnut rust perpetuates and it occurs regularly every year in India without having a telial state, alternate or collateral host. It is said that groundnut rust develops ﬁrst in South India and then migrates to North India. This disease, along with early and late leaf spot disease, renders the crop uneconomical in the rainy season, which is the major period of groundnut cultivation in India. Rust of jowar (Sorghum spp.) The genus Sorghum Moench has 23 species (Simon, 1993). The crop is damaged by four different fungal diseases: seed and seedling disease, foliage disease, head disease and root and stalk disease. Sorghum rust is a foliage disease which infects almost all species of Sorghum. High temperatures (75–80°F) and humid weather is favourable for disease development. The species of Puccinia that infect Sorghum (Cummins, 1971) are P. purpurea, P. levis and P. nakanishiki. The most The Rust Fungi prevalent rust of jowar throughout the world is P. purpurea Cooke, which is heteroecious and its aecial host is O. corniculata L. But the aecial stage plays a negligible role in rust disease. The rust infection and host reaction results in the formation of bright purple-coloured spots on the leaves. There are 32 races in cultivated Sorghum distributed in Southeast Asia (11) and Africa (21). Rust of maize (Zea mays L.) Maize rust or leaf rust of maize, P. sorghi Schw., is an American rust. However, maize is susceptible to two more rusts, i.e. P. polysora, southern corn rust, and P. zeae, tropical corn rust. It is a heteroecious rust and its aecia are produced on species of Oxalis, namely O. stricta, according to Arthur (1929). Aecia are more common in this rust than in Sorghum rust. However, Mishra (1962) has claimed that the alternate host of maize rust is O. corniculata, on which aecia were collected from Nepal. The rust infects all types of maize with a varying degree of severity. Rust of bajra/pearl millet (Pennisetum spp.) The genus Pennisetum Rich. has c.80 species and is distributed throughout the tropics. Pearl millet (P. glaucum (L.) R. Br.) is a staple food crop of the semi-arid tropical parts of the world, mainly Asia and Africa. There are about 14 rusts reported on bajra (Cummins, 1971). However, only two are well-known, namely P. substriata Ell. and Barth. var. indica Ramachar and Cummins in India and P. substriata Ell. & Barth. var. decrospora Eboh., recently reported from Nigeria. The ﬁrst rust is predominant in India and is heteroecious. The alternate aecidial host is the species of Solanum. Rust infection produces pustules on both sides of the leaf with necrotic spots, due to which premature drying of leaves may result. Occasionally, pustules also develop on leaf sheaths and stem. 209 Rust of ﬂax/linseed (Linum usitatissimum L.) This crop is cultivated mainly for oil and ﬁbre. It is affected by rust in most of the linseed-growing areas of the world such as Asia, America and Europe. The rust appears in India in February. This rust, M. lini (Ehrb.) Lev., also infects wild species of Linum. It is an autoecious rust and infects all the green parts of the plant. Telia develop late on stems and form crusts covered by epidermis. Aecia of this rust are caeomoid. Flor (1956) studied this rust and differentiated 179 races from America alone. Eighteen races are reported from India. L. mysorense L., a wild host, has been reported to harbour the rust from India. Rust of pea (Pisum sativum L.) Pulse crops are affected by two rusts, namely U. pisi (Pers.) Wint., a heteroecious rust reported from Europe only and rarely in India, while U. viciae-fabae (Pers.) Schroeter, an autoecious rust, is found in Europe, America and Asia. It was found that aeciospores played a major role in the dissemination of lentil rust during the active growing season. It was also suggested that secondary aecia are produced at low temperatures (17–22°C), while higher temperatures induced uredinia. Teleutospores are dormant spores, survive for 2 years and remain viable at low temperatures (3–18°C). Rust of gram (Cicer arietinum L.) Gram rust is caused by U. ciceris-arietini (Gron.) Jack. It is a heteroecious rust, but no alternate host or pycnia and aecia have been collected. The same rust has been collected on wild species of Trigonella polycerata, a weed of Fabaceae growing at higher altitudes. It is claimed to be the source of urediniospores, the primary inoculum. Rust of bean Beans belong to different genera of the family Fabaceae, namely Phaseolus, Vicia, Lathyrus, 210 M.S. Patil and A. Patil Pisum, Dolichos and Vigna. Their commercial cultivars are a source of vegetables, pulses and forage crops. They are cultivated extensively all over the world as kharif and rabi crops. These crops are infected in the ﬁeld by many rusts. The pathogens are U. appendiculatus (Pers.) Unger var. appendiculatus, U. vignae Barclay, U. viciae-fabae (Pers.) Schroeter and U. pisi (Pers.) Wint. The ﬁrst three pathogens are autoecious and the fourth is heteroecious. Many races of these pathogens are known. Heavy infection of leaves results in defoliation and poor productivity. Rust of rose (Rosa spp.) All rusts of roses belong to the genus Phragmidium and ten species infect roses worldwide. However, seven species are very common. The most common species is P. disciﬂorum, which perpetuates on hybrids of R. canina and R. gallica, but is less likely to attack climbing or rambling roses like R. multiﬂora. The rusts are autoecious and all spore types, except pycniospores, are equally harmful to the foliage. As a result of infection in some seasons, severe defoliation ensues and plants are greatly weakened. Keeping a garden clean is the most effective method of keeping roses healthy. Rust of soybean (Glycine max (L.) Mill.) Soybean as an oilseed crop is cultivated all over the world as kharif. About 25 diseases are known on soybean crop. Among these, there are 19 predominant fungal diseases. In fungal pathogens, rust is the most serious in India. The rust entered India in 1970 from the New World to Japan via Nepal in northern India and spread to the south-western parts of India up until 1995. The rust was ﬁrst reported from Taiwan. The fungus inciting soybean in Asia was ﬁrst described as U. sojae P. Henn. from Japan in 1903. It was subsequently described and renamed by Sydow, as U. sojae H. and P. Sydow. However, this was erroneous due to the host not being soybean but Mucuna spp. (Butler and Bisby, 1931). Moreover, U. sojae P. Henn. is not considered as an anamorph of U. mucunaei Rabenh. The anamorph and teliomorph connection was ﬁrst proved by Sawada (1931) and named as P. sojae Sawada. In the two rusts of soybean, namely P. pachyrhizi and P. meibomaiae (Arthur) Arthur, the former species shows wide geographical distribution in Asia, Australia and Africa, while the latter is restricted to America. In India, soybean rust does not produce telia and perpetuates only by uredinia, possibly due to environmental factors. Rust of ﬁg (Ficus carica L.) Rust of cotton (Gossypium spp.) The genus Gossypium is known by 4–5 species. Use of cotton ﬁbre in India is found in ‘Rig Veda’. Cotton cultivation is mainly for ﬁbre and oil from seeds. Cotton is cultivated as a cash crop in 80 countries of the world. The cotton crop is infected heavily by a large number of pathogens, including rusts. Among all the rusts, P. gossypi (Arthur) Hiratsuka is the most troublesome to cotton, not only in cultivated varieties but also in perennial cotton. The rust disease seriously damages the cotton crop and is responsible for c.20–70% ﬁnancial loss to cotton growers annually. Rust of ﬁg and other species of Ficus is produced by C. ﬁci (Butler) Arthur. It is a common rust found throughout tropical and subtropical parts of the world. Telia have been observed only in India on F. glomerata Roxb., an evergreen shade tree. In the commercial ﬁg, F. carica L., this rust appears late in the season (monsoon) and does not affect the quality of the fruits, but defoliation exposes them to sunburn. It also reduces host vigour. It is possible to have a cell-free culture using a complex culture medium to culture rust urediniospores. The method was ﬁrst used successfully in black stem rust, P. graminis tritici, for sporulation. Nowadays, compounds like kinetin and benzimidazole, The Rust Fungi which exert a cytokinin effect, are used to culture detached leaves in solution in test tubes for up to a month to determine the races of rusts. 211 ‘Deities – rust gods, Robigan and Robigus’. Thomas Knight, the English plant physiologist, gave experimental proof of the ability of aeciospore to infect cereals and after that same year, voluntary eradication started in Denmark. Rust Disease Management Strategies There is a need for effective disease forecasting and warning systems, as well as a disease calendar for each crop. Crop protection/ management varies widely with different crops, due to different factors such as: ● ● ● ● varietal susceptibility of crops soil types agronomic practices and cropping patterns. The study of diseases, disease development, disease outbreak, pathogens, varieties, races, biotypes, ecotypes, pathotypes, specialization, host plants, their hybrids, cultivars, ﬂuctuating factors like soil, water, fertilizers, pesticides, host–pathogen complex, etc. is very vast and difﬁcult. The outbreak of disease on an epiphytotic scale resulting from the interaction of pathogen, host and environment can be represented through disease progress curves (DPC). The problem becomes more serious due to a pathogen having high pathogenicity, which includes: ● ● virulence and aggressiveness (vigorous races). Today, virulence, based on evidence, can often be considered to be oligogenic, in which a few genes are involved, as suggested by van der Plank (1968), while aggressiveness is generally polygenically inherited. Virulence is conditioned due to gene diversity and aggressiveness by variation in the doses of enzymes. Hence, disease reaction is a chemical process which entails changes in the host and parasite cell metabolism. Black stem rust is found to be more severe on wheat than barley and yellow rust on barley than wheat. To avoid heavy losses, early sowing was recommended by Pliny. In Greek and Roman civilizations, the appearance of plant diseases was attributed to Strategy of management of plant diseases (Sharvelle, Strategy of Plant Disease Control, 1961) Most control measures either reduce the initial inoculum or rate of spread of plant pathogens (van der Plank, 1963). It is important to reduce and delay the initial infection as much as possible, by disease forecasting in advance so that farmers can protect the crop plants to avoid monetary losses. Sharvelle (1961) classiﬁed the strategies for plant disease control into two categories: (i) immunization; and (ii) prophylaxis (to eradicate the pathogen). Strategy of plant disease control Immunization 1. 2. Genetical resistance. Induced resistance. Prophylaxis 1. 2. 3. Protection: (i) Chemical prophylaxis; (ii) Environmental manipulation. Eradication: (i) Crop rotation; (ii) Sanitation; (iii) Alternate host elimination; (iv) Chemical eradication. Legislation: (i) Quarantine; (ii) Regulatory measures. Developing New Strategies for Disease Management: Role of Oxidative Burst The molecular biology of interactions between disease-resistance genes, defence genes and 212 M.S. Patil and A. Patil their role in genetic engineered diseaseresistance elicitor (signal) molecules has been detected in fungal, bacterial and viral pathogens. These molecules serve as signals to elicit defence mechanism of the host. Host resistance genes may function as receptors of these signals. Only a few disease-resistance genes have been cloned from plants. Analysis of these genes shows the presence of leucine-rich repeats (LRRS), leucine zippers and nuclear localization signals (NLS). LRRS are involved in protein–protein interactions of the signal transduction pathway. Several defence genes are widely found in both resistant and susceptible plants and are involved in the production of antimicrobial compounds, namely phenols, phytoalexins and pathogen-related (PR) proteins; PR-1, 2,3,5,6 and 8, co-enzyme reductase (HMGR), transgenic plant expressing phenylalanine ammonial-yases (PAL) showed enhanced disease resistance (Vidyasekaran, 1997). Fungicides in the Control of Rust Diseases The different fungicide dosages recommended for rust disease control are shown in Table 16.1. Other Methods of Plant Disease Management Biological methods Biological control using different microbes is the most popular and ecologically safe method, but is not used practically due to many constraints. The following are some potential and promising parasites of rusts which can be used in biological control in the future. 1. Aphanoderma album (Preuss.) W.Gams This Hyphomycetes is characterized to produce a metabolite which switches off sporulation of urediniospores to teleutospores, thus terminating the life cycle of rust. 2. Cladosporium spp. (i) C. aecidiicola Theum.; (ii) C. exobasidii Jaap; (iii) C. uredinicola Speg. on P. recondita (UK). 3. Verticillium spp. (i) V. hemileiae Steyaert; (ii) V. lecanii (Zimm.) Viegas. V. hemileiae and V. lecanii, as a virtue of their growth on uredinia of coffee rust in a moist environment, produce a chitinase enzyme to weaken the wall of the spores, as a result of which the spores burst. Even the cultural ﬁltrates are effective (Ellis, 1971, 1976). 4. Sphaerellopsis ﬁlum (Biv. – Bern. ex. Fr.) B.C. Sutton = Darluca ﬁlum (Biv.) Cast. This hyperparasite was considered by Tarr (1972) as an ecologically balanced mycoparasite on rust fungi, especially the uredinia of the species of Puccinia and Uromyces of grasses. It is distributed in the tropical and subtropical moist regions of the world. However, it has not been used commercially. 5. Tuberculina costaricana H. Sydow. 6. Olpidium uredinis, an endoparasite in urediniospores. Plant breeding 1. Cultivation of hybrid cultivars recommended by plant breeders in different regions for different rusts. The hybrids have high resistance coupled with good quality and high productivity. Some rust-resistant cultivars are ‘Maris Ranger’, ‘Heines VII’, ‘Fenman’, ‘Hybrid 46’, ‘Minster’, ‘Opal’, CS 2D/2M, T. spelta 391, T. spelta G652 (ICARDA), CIM 25, ‘Dove’ (CIMMYT), HD 4502, ‘Arkan’, ‘Blueboy II’, ‘Centurk’, ‘Chris’ (USA), ‘Banks’ and ‘Egret’ (Australia). 2. Use of defence activators – spray of salicylic acid (SA), ferric chloride (FeCl3) and dipotassium hydrogen phosphate (K2HPO4). 3. Adult plant resistance (APR) – there has been an increasing interest in adult plant resistance, especially against leaf rust or brown rust pathogens of wheat in North India, because of its widespread occurrence in germplasm and its durability. The Rust Fungi 213 Table 16.1. The various fungicide dosages recommended for different diseases. I – Sulphur fungicides Sulphur 2–4 kg/ha Spray or dust 2–3 times Lime – sulphur Ziram 0.75 g/100 g 0.2% solution Sprays Spraying at the outbreak of disease and repeated at weekly intervals Ferbam 0.15% solution Sprays as required Thiram 0.3% solution 0.15–0.2% spray Seed treatment Sprays 0.2–0.3% solution 0.5% solution Dry seed dressing Two sprays at 10–15-day intervals Applied at 4-week intervals from petal fall 3 sprays at 14-day intervals Sprays at disease outbreak and repeat after 10–15-day intervals 5 sprays at 10-day intervals Applied at 4-week intervals from petal fall 3 sprays at 15-day intervals starting in last week of September before disease outbreak 5 weekly sprays in the growing season Spray at disease outbreak and repeat at c.10-day intervals as necessary Sprays at disease outbreak and repeat at 7–10-day intervals Sprays at 14-day intervals along with benomyl or carbandazim 4 sprays at 10-day intervals from disease outbreak. Add triton at rate of 2 ml/l suspension Seed treatment 4 sprays at 12-day intervals Sprays Dusting 3–6 applications at 10-day intervals Sprays 4–5, beginning when disease appears Zineb 0.2% solution 0.2% solution 0.15% solution 0.2% solution 0.2% solution 0.2% solution 0.2% solution 0.2% solution Maneb/ mancozeb 0.2% solution 0.25% solution 0.2% solution 0.3% solution 0.2% solution 400 ppm 2–3 kg/ha Nabam 0.15–0.25% solution Rust of beans – Uromyces appendiculatus var. appendiculatus, U. fabae Bean rust – U. fabae Bean – U. appendiculatus or U. phaseoli Mint – Puccinia menthae Sunﬂower – P. helianthi Rose – Phragmidium mucronatum Safﬂower – P. carthamii Apricot – Tranzschelia discolor Plum – T. discolor Safﬂower – P. carthamii Beet – U. betae Almond and apricot – T. discolor Barley – P. striiformis Chrysanthemum – P. chrysanthemi Garlic – P. allii Peach – T. discolor Phalsa – Dasturiella grewiae Soybean – Phakopsora pachyrhizi Wheat – P. recondita and P. graminis tritici Bean – U. appendiculatus var. appendiculatus Groundnut – P. arachidis Peas – U. fabae Safﬂower – P. carthamii Sorghum – P. purpurea Sunﬂower – P. helianthi Wheat – P. graminis tritici and P. recondita Wheat – P. graminis tritici and P. recondita continued 214 M.S. Patil and A. Patil Table 16.1. continued. II – Copper fungicides Bordeaux mixture 5:5:1/2:50 Copper oxychloride 1.8 kg/400 l Colloidal copper 1500 ml/400 l 0.35% solution (kocide 110) and 0.5% (cupravit) Sprays, 5–10 applications, add urea and ZnSO4 Sprays Sprays Sprays in March–April 300 l/ha Coffee – Hemileia vastatrix Beans – U. appendiculatus Beans – U. appendiculatus Coffee – H. vastatrix III – Mercury fungicides Agrosan GN 0.3% solution Seed treatment Safﬂower – P. carthamii IV – Heterocyclic nitrogenous compounds Captafol 0.3% solution 0.2% solution Seed treatment 3 sprays at 10-day intervals as soon as disease appears Safﬂower – P. carthamii Brown rust of wheat: P. recondita V – Systemic fungicides Benzimidazole 1.5 kg/ha Oxathiin-carboxin 1 g/kg 2.5 g/kg Oxycarboxin 2630 g/100 kg Dust or spray with carbendazim or carbendazim (0.07%) plus mancozeb (0.15%) Seed dressing with benomyl Seed treatment/foliar spray and soil treatment Seed treatment 1.12 kg/ha 1.68 kg/ha 3 kg/ha Granules in soil Spray Spray – single in Europe or 2–3 sprays common 15 kg/ha Soil treatment Soil treatment with benomyl spray Soil treatment with benomyl as spray Groundnut – P. arachidis Speg. and early and late leaf spot disease Safﬂower – P. carthamii Bean – U. appendiculatus Stripe rust of wheat – P. striiformis Leaf rust wheat – P. recondita Black stem rust – P. graminis tritici and P. recondita Safﬂower (seedling) stage – P. carthamii Fig rust – Cerotelium ﬁci Peanut – P. arachidis Sunﬂower – P. helianthi VI – Benzanilide derivatives Benzanilide 1.87 kg/ha Foliar spray Benodanyl/ Vitavax 500 mg/ml 2 sprays or soil drenching 0.75 l/ha 0.3% 250 ml/l 0.1125% 100 mg/l 10 g/kg 0.2% Spray/seed treatment 4 sprays at 15-day intervals 2 sprays or soil drenching Sprays at 6–9-day intervals 2 sprays or soil drenching Spray/seed treatment Spray/seed treatment Oxycarboxin Plantavax w.p. Stripe rust of wheat – P. striiformis and barley – P. hordei Effective against most of the rusts of cultivated plants Stripe rust of wheat – P. striiformis Leaf rust of wheat – P. recondita Sunﬂower – P. helianthi Sunﬂower – P. helianthi Sunﬂower – P. helianthi Stripe rust of wheat – P. striiformis Black stem rust – P. graminis tritici The Rust Fungi 215 Integrated disease management Acknowledgement 1. Cultural methods include early sowing. 2. Spraying of micronutrients such as Na2B4O7, CuSO4 increases resistance in plants. The authors express their gratitude to The Principal, Agriculture College, Kolhapur, India, for providing the facilities of their library. References Agrios, G.N. (2005) Plant Pathology, 5th edn. Academic Press, New York. Arthur, J.C. (1929) The Plant Rust (Uredinales). John Wiley and Sons, Inc, New York, 446 pp. Butler, E.J. and Bisby, G.R. (1931) The Fungi of India. Science Monograph I. Indian Council of Agricultural Research, India, 65 pp. Cummins, G.B. (1936) Phylogenetic signiﬁcance of the pores in the Uredinales. Mycologia 23, 1–102. Cummins, G.B. (1971) The Rust Fungi of Cereals, Grasses and Bamboos. Springer-Verlag, Berlin, 570 pp. Dietel, P. (1900) Uredinales. In: Engler, A. and Prantl, H. (eds) Naturlichen Pﬂangen Families, 1(1), pp. 28–88, 546–553. Ellis, M.B. (1971) Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, UK. Ellis, M.B. (1976) More Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, UK. Flor, H.H. (1956) The complementary genetic systems in ﬂax and ﬂax rust. Advances in Genetics 8, 29–55. Ghewande, M.P. and Savalya, S.D. (1999) Integrated management of groundnut diseases in S.D. India. (Abstract). In: Kambale, S.Y. (ed.) Proceedings of National Seminar on Recent Advances in Plant Pathology, Department of Botany, University of Pune, Pune, India, pp. 4–5. Gregory, P.H. (1973) The Microbiology of the Atmosphere, 2nd edn. Leonard Hill Books Aylesbury, UK, 377 pp. Joshi, L.M., Srivastava, K.D. and Singh, D.V. (1986) Monitoring of wheat rust in Indian subcontinent. Proceedings of Indian Academy of Sciences (Plant Science) 84, 387–406. Kirk, P.M., Cannon, P.F., David, J.C. and Stalper, J.A. (eds) (2001) Dictionary of Fungi, 9th edn. CAB International, Wallingford, UK. Kuhn, J.G. (1858) Diseases of Cultivated Plants. 312 pp. Mehta, K.C. (1929) Annual recurrence of rust on wheat in India. Presidential Address (Botany section) at Madras. Proceedings of Indian Science Congress, pp. 199–223. Mehta, K.C. (1952) Further studies on cereal rust in India-II. ICAR Science Monograph 18, India, 368 pp. Mishra, D.P. (1962) Natural occurrence of the aecial stage of Puccinia sorghi Schw. on Oxalis corniculata L. in Nepal. Indian Phytopathology 16, 8–9. Ono, Y. (2002) The diversity of nuclear cycles in microcyclic rust fungi (Uredinales) and its ecological and evolutionary implications. Mycoscience 43, 421–439. Ono, Y., Burticia, P. and Hennen, J.F. (1992) Delimitation of Phakopsora , Physopella and Cerotelium and their species on Leguminosae. Mycological Research 96, 825–850. Rajendran, R.B. (1967) Atypical and typical germination of uredinoid teliospores of Hemileia vastatrix. Mycologia 59(5), 918–921. Sawada, K. (1931) Materials of the Formosan fungi. Transactions of the Natural History Society of Taiwan 21, 227–235 (in Japanese). Sharvelle, E.G. (1961) The Nature and Uses of Modern Fungicides. Burgess Publishing Co, Minneapolis, Minnesota, 30 pp. Simon, B.K. (1993) A Key to Australian Grasses. Queensland Department of Primary Industries, Brisbane, 206 pp. Swann, E.C. and Taylor, J.W. (2001) Urediniomycetes. In: McLaughlin, D.J., McLaughlin, E.G. and Lemke, P.A. (eds) et al. The Mycota Volume VII: Systematics and Evolution, Part B. Springer, Berlin, pp. 37–56. Sydow, H.P. (1914) Beitrg zur kenntnis der parasitischen Pilz der insel Formosa. Ann. Mycologia 12, 105–112. Tarr, S.A.J. (1972) The Principles of Plant Pathology. The McMillan Press, London. Tulasne, L.-R. and Tulasne, C. (1847) Memoire sur les ustilaginees compares Uredinees- Annales des Sciences Naturelles Botanique, Série 3, (7), 12–27. van der Plank, J.E. (1963) Plant Disease Epidemics and Control. Academic Press, New York, 349 pp. van der Plank, J.E. (1968) Disease Resistance in Plants. Academic Press, New York. Vidhyasekaran, P. (1997) Fungal Pathogenesis in Plants and Crops. Marcel Dekker, New York. 216 M.S. Patil and A. Patil Appendix 1 Key to species and varieties of Puccinia and Uromyces producing rust diseases to cultivated crops which are very common but confusing to identify Wheat rusts I. I’. II. II’. Uredinial infection produces chlorotic streaks with halos on leaves -----------P. striformis Uredinial infection does not produce chlorotic streaks with halos on leaves ----------- II Urediniospores with 5–6 germ pores arranged equatorially -------------P. graminis tritici Urediniospores with 4–5 germ pores, scattered ----------------------------------- P. recondita Jowar rusts I. I’. II. II’. Uredinia aparaphysate ---------------------------------------------------------------------------- P. levis Uredinia paraphysate -------------------------------------------------------------------------------------II Urediniospores with 5–8 germ pores, scattered ---------------------------------P. nakanishiki Urediniospores with 3–5 germ pores, equatorial ---------------------------------- P. purpurea Bajra rusts I. Infection mostly hyphophyllous, teleutospores measure 21–49 µm long, 2-celled, pedicel coloured and short ---------- P. substriata Ell. & Barth. var. indica Ramachar & Cummins I’. Infection amphigenous, teleutospores large, up to 5-celled ---------------------P. substriata Ell. & Barth. var. decrospora Eboh. Maize rusts I. Teleutospores stalked and 2-celled -------------------------------------------------------------------II I’ Teleutospores sessile, in chain and innate -------------------------------------------------------- P. zeae II. Telia exposed/erumpent, urediniospores 26–31 µm long (aecia on Oxalis stricta L.)-------------------------------------------------------------------------------------------------------------------- P. sorghi II’. Telia covered, urediniospores 29–30 µm long (aecia not known) -------------- P. polysora Rusts of Phaseolus and Vigna spp. I. Urediniospores measure 24–29 × 17–19 µm, germ pores 4, equatorial (on Phaseolus spp.) ------------------------------------------- U. appendiculatus (Pers.) Unger var. appendiculatus I’. Urediniospores measure 29–32 × 20–22 µm, germ pores 2, equatorial (on Vigna spp.) -------------------------------------------------------------------------------------------------------------- U. vignae Rusts in forage crops I. Urediniospores have 4–7 germ pores, scattered (on red clover) ------------------U. fallens; T. pratens (only uredia and telia) I’. Urediniospores have 2–4 germ pores, equatorial (on white clover) --------------------------------------------------- U. trifolii-repentis T. repens and other spp. autoecious and macrocyclic Rusts on vegetable and pulse crops I. I’. Autoecious, occurs in America and Europe ------------------------------------ U. viciae-fabae Heteroecious, occurs only in Europe (Arthur, 1929) on P. sativum L. -------------- U. pisi 17 Etiology, Epidemiology and Management of Fungal Diseases of Sugarcane Ayman M.H. Esh Biotechnology and Tissue Culture Laboratories, Sugar Crops Research Institute, Agricultural Research Center, Giza, Egypt Abstract Sugarcane (Saccharum ofﬁcinarum L.) is one of the most important commercial crops in many countries of the world. It contributes nearly 70% of world sugar and provides the base materials essential for many other industries. Sugarcane crop is attacked by numerous foliar and root pathogens. Some of these diseases cause serious quantitative and qualitative losses which have negative effects on sugarcane production, as well as in the sugar industry. About 56 diseases of sugarcane have been reported so far from different parts of the world. Of these, 40 are caused by fungi, several of which can cause economic losses. The major sugarcane fungal diseases in different tropical and subtropical regions are: smut disease (Ustilago scitaminea); rust disease (Puccinia melanocephela); red rot (Glomerella tucumanensis [Colletotrichum falcatum]); eye spot disease (Bipolaris sacchari), pokkah boeng disease (Fusarium moniliforme); and pineapple disease (Ceratocystis paradoxa). This chapter includes the major fungal diseases of sugarcane and the various control practices used against them. Introduction Sugarcane (S. ofﬁcinarum L.) is a monocotyledonous plant from the family Poaceae of the subfamily Andropogoneae (Cox et al., 2000) and is considered as one of the oldest cultivated crops known to man. Sugar, along with honey, is the oldest natural sweetener (Peng, 1984; Naik, 2001). Sugarcane is grown in the tropical and subtropical regions of the world and is cultivated in nearly 60 countries as a commercial crop, with Brazil, India, China, Cuba, Thailand and Pakistan as the major sugarcane-growing countries (FAO, 2005). Due to its wide range of adaptability, it supplies more than 60% of world sugar demand and basic raw material in many industries, which makes it one of the most important cash crops that plays an enormous role in the economy. Various biotic and abiotic factors are responsible for yield reduction and economic losses. Among these factors, fungal diseases are the major cause. Over 100 fungi, 10 bacteria, 10 viruses and about 50 species of nematodes are pests of sugarcane in different parts of the world (Singh and Waraitch, 1981). Sugarcane, being a long duration crop (10–12 months), remains in the ﬁeld for several years (ratoons  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 217 218 A.M.H. Esh remain in the ﬁeld for up to more than 6 years). A serious drawback of this practice, however, is that pathogens may build up within the ﬁeld and be disseminated during propagation of new seed cane. The pathogen within the seed pieces is transmitted easily into young plants, which in turn serve as sources of inoculum for secondary infections in adjoining healthy plants. Several management strategies have been developed as a result of research and development work. The endless struggle between varieties and the complexity of disease have led correspondingly to the development of a variety of approaches for control. The role of fungicides in modernizing and changing the condition of agriculture is quite signiﬁcant (Mehta, 1971; McFarlane et al., 2006). Sugarcane Smut Disease Sugarcane smut, caused by the Basidiomycetes fungus Ustilago scitaminea Syd., is cosmopolitan in distribution and has been an important disease in nearly every sugarcane-producing country of the world. It can reduce crop yields by over 50% and make ratoon crops unproﬁtable to maintain. It is highly infectious and even developed countries have been unable to stay smut free with the use of appropriate quarantine measures (Antony, 2008). The disease was ﬁrst noted in South Africa in 1877, then in the early 1930s it caused severe problems in India and other countries in Asia. Years later, the disease started to establish and cause serious problems in different parts of the world: 1943, Argentina, (Cross, 1960); 1950, Brazil and Paraguay; 1957, Bolivia; 1960 and 1971, Hawaii (Byther et al., 1971); and 1974, Guyana (James, 1976). By 1981. the disease had been found in most of the Caribbean and North, South and Central America (Ferreira and Comstock, 1989). In 1998, the disease was reported for the ﬁrst time in the Ord River area of Western Australia (Riley et al., 1999). Australia is a major exception since the disease is present only in Western Australia. The sugar industries of eastern Australia, Fiji and Papua New Guinea are still free of the disease (Braithwaite et al., 2004b). Causal agents The fungus belongs taxonomically to Phylum: Basidiomycota; Class: Ustilaginomycetes, (Bisby et al., 2007). Classiﬁcation of U. scitaminea H. & P. Sydow, the causal agent of sugarcane smut disease, is based mainly on differences in spore morphology and the characteristics of germinating spores (LeeLovick, 1978). Smut races have been reported subsequently based both on observations and inoculation studies (Gillaspie et al., 1983). Usually, races are suggested when a cultivar succumbs to smut after being grown for several years without being infected (James, 1976). U. scitaminea races have been reported in Hawaii, Pakistan, the Philippines and Taiwan; the presence of the actual number of races and their prevalence are unknown (Ferreira and Comstock, 1989). Pathogenic races of sugarcane smut have been observed in several countries, including two races A and B from Hawaii (Comstock and Heinz, 1977) and three races (1, 2, 3) reported in Taiwan (Leu and Teng, 1972; Lee et al., 1999). However, Ferreira and Comstock (1989) considered the true prevalence of races to be controversial. Many claims are based on the reaction of the same cultivar in different countries, but the interpretation of these claims is confused by testto-test variation and the use of different inoculation methods. Two international collaborations have attempted to standardize race typing. Gillaspie et al. (1983) performed race typing under glasshouse conditions to standardize the environment and six races were identiﬁed. Grisham (2001) coordinated a race typing study in nine countries using local isolates tested against a standardized set of 11 differential cultivars. On the molecular level, many researchers have studied the genetic diversity among U. scitaminea isolates, either between local isolates or between isolates collected from different parts of the world (Braithwaite et al., 2004a,b; Xu et al., 2004; Singh et al., 2005). Genetic variation estimated from 12 AFLP primer combinations showed that, overall, there was little variation in the smut population across the world. However, isolates from Fungal Diseases of Sugarcane the Philippines, Taiwan and Thailand form a distinct cluster; it is therefore suggested that genetic variation is limited between the isolates and the phylogeny of U. scitaminea is poorly understood (Braithwaite et al., 2004b). Disease symptoms Smut-infected plants are distinguished by the emergence of a ‘smut whip’. The whips are the ﬂowering structures of the pathogen which produce teliospores. The ﬂowering structures transform into a whip-like sori that grows out between the leaf sheaths. At ﬁrst, it is covered by a thin silvery peridium (this is the host tissue), which peels back easily when desiccated to expose the sooty black-brown teliospores. Whips begin emerging from infected cane by 2–4 months of age, with peak whip growth occurring at the 6th or 7th month. Spindle leaves are erect before the whip emerges. Affected sugarcane plants may tiller profusely, with the shoots being more spindly and erect with small narrow leaves (i.e. the cane appears ‘grass-like’). Less common symptoms are leaf and stem galls and bud proliferation (Ferreira and Comstock, 1989; Agnihotri, 1990). Pathogenesis Ustilago scitaminea produces diploid spores called teliospores. When teliospores germinate, they undergo meiosis, which gives rise to a septate promycelium bearing four haploid sporidia (basidiospores). U. scitaminea, like most parasitic Heterobasidiomycetes, has a diallelic bipolar mating system (Alexander and Srinivasan, 1966; Leu, 1978; Moosawi-Jorf et al., 2006) in which only sporidia of opposite mating types conjugate. Of the four initial sporidia or basidiospores from each teliospore, two have a positive mating allele and two have a negative mating allele. U. scitaminea can thus both selfand outcross, but the frequency of natural selﬁng versus outcrossing is unknown. A 219 dikaryotic mycelium develops after fusion of compatible sporidia. This dikaryotic mycelium is infectious, penetrates behind bud scales and invades the meristematic zone of the bud. Entry into the meristem in the bud occurs between 6 and 36 h after the teliospores are deposited on the surface (Alexander and Ramakrishnan, 1980). Finally, the apical meristem of smut-infected cane produces a long whip-like structure bearing billions of teliospores (i.e. sorus). Sugarcane smut is spread by spores which have an aerial dispersal mode. The whip serves as a source of spores that release approximately one billion spores/whip/day into the air to infect the buds of the standing sugarcane. The infected buds remain dormant until the cane is cut for seed. The spores mixed with the soil of cropped or newly prepared ﬁelds also become a source of infection to the disease-free seed pieces. Under normal soil moisture, the spores only survive for a short time in the soil. On the other hand, several species of insects have been associated consistently with smut whips; this suggests insects could play a role in spore dispersal (Ferreira and Comstock, 1989; Agnihotri, 1990). Disease control The best control method is to use resistant cultivars. There is a strong genetic basis for resistance and resistant varieties have been readily available and used to control outbreaks of smut in several countries (Churchill et al., 2006). Disease-free planting material usually can be obtained by subjecting seed to hot water treatment. Hot water treatment, however, may not be practical on a large scale and its effectiveness may be subject to varietal differences (McFarlane et al., 2007). Several fungicides (triadimefon, ﬂudioxonil:mefenoxam:azoxystrobin, mancozeb, metalaxyl + carboxin + furathiocarb, pyroquilon, benomyl and chlorothalonil) have been used to control sugarcane smut when used as pre-planting fungicidal dips of planting setts (Wada et al., 1999; Wada, 2003). 220 A.M.H. Esh Sugarcane Rust Disease (Common and Orange Rust) Throughout the world, the important leaf rust disease causes severe losses in sugarcane ﬁelds (Magarey et al., 2008). In 2000, sugarcane rust was once considered a minor pathogen in the Australian sugar industry. In 2000, it devastated most plantations of the cultivar Q124 in Australia, causing yield losses of up to 40% (Apan et al., 2003; Braithwaite et al., 2004a; Magarey et al., 2008). In the USA, the yield loss caused by a rust epidemic due to cultivar CP 72-1210 in 1987 was 20% (Raid and Comstock, 2000). Sugarcane rust is caused by two species belonging to the genus Puccinia, P. melanocephala and P. kuehnii, the former causes common rust disease, while the later causes orange rust. Common rust caused by P. melanocephala H. & P. Syd. was ﬁrst reported on sugarcane in 1949 in the Deccan area in India (Patel et al., 1950). The disease has been also reported from: Japan (Ohtsu, 1975 [cited by Muta, 1987]); the Philippines (Serra et al., 1983); Australia (Egan and Ryan, 1979); Taiwan (Hsieh et al., 1977); Dominican Republic (Presley et al., 1978); Jamaica (Burgess, 1979); Puerto Rico (Liu, 1979); Cuba (Sandoval et al., 1983); Carribean and Central America (Purdy et al., 1983); Hawaii (Comstock et al., 1982); and Angola, Kenya, Madagascar, Tanzania, Uganda, Zambia, Zimbabwe, South Africa, Mozambique and Malawi (Egan, 1980; Sivanesan and Waller, 1986). Orange rust caused by P. kuehnii Butl. was ﬁrst reported on sugarcane in Java in 1890 (Ryan and Egan, 1989). The disease has been reported from Japan (Ito, 1909), Australia, Indonesia, the Philippines, Taiwan, Paciﬁc Islands, Sri Lanka, Malaysia, Thailand, New Caledonia, China (Egan, 1980; Sivanesan and Waller, 1986) and India (Mukerji and Bhasin, 1986). Disease symptoms The initial symptoms of common rust are small, elongated yellowish spots, which are visible on both leaf surfaces. The spots increase in size up to 1.5 mm in diameter and usually turn brown to orange-brown or redbrown. The lesions occur irregularly and typically range from 2 to 10 mm in length, but occasionally reach 30 mm. The spots are raised and are surrounded by a pale yellow halo (Raid and Comstock, 2000). The raised pustules are formed predominantly on the undersurface of the leaves and the urediospores formed therein are orange to orange-brown. On a highly susceptible variety, considerable numbers of pustules may occur on a leaf, coalescing to form large, irregular, necrotic areas. High rust severities may even result in premature death of young leaves. Severe rust has caused reductions in both stalk mass and stalk numbers (Rao et al., 1999; Raid and Comstock, 2000). Causal agent Puccinia melanocephala Syd. and P. Syd. (common rust) and P. kuehnii Butler (orange rust) are reported to cause rust diseases on sugarcane (Butler, 1914; Cummins and Hiratsuka, 1983; Shine et al., 2005; Ido et al., 2006; Comstock et al., 2008; Ovalle et al., 2008). The two obligate parasitic fungi belong to Phylum: Basidiomycota; Class: Urediniomycetes, Order: Uredinales. The causal agents of common and orange rust cannot be clearly distinguished based on colour of lesions and uredinia and the size of urediniospores. However, they are distinguishable based on the presence or absence of abundant capitate paraphyses in uredinia, echinulation, colour and wall thickness of urediniospores, colour of the telia and colour and wall thickness of teliospores. P. melanocephala has abundant capitate paraphyses in uredinia and urediniospores with dense echinulation, darker brown and uniformly thick walls. They also have dark brown to blackish telia with brown to dark brown teliospores with apically thickened walls. P. kuehnii has morphologically indistinct paraphyses in uredinia and urediniospores with moderate echinulation, lighter brown and Fungal Diseases of Sugarcane sometimes apically or uniformly thickened walls (Virtudazo et al., 2001). Pathogenesis The life cycle of sugarcane rust is simple, with the urediniospore being the only known infectious spore. These are produced in, and are released from, pustules that develop on the underside of sugarcane leaves. The development of substomatal vesicles, infectious hyphae, haustoria and subsequent infection processes are similar to other Puccinia spp. Urediniospore production occurs 8–18 days after the initial urediniospore lands on a leaf, depending on varietal susceptibility and environmental conditions (CABI CPC, 2006). Spread of rust disease occurs primarily by wind and water-splash movement of urediniospores. The movement of diseased vegetative parts of sugarcane, contaminated equipment and workers from one location to another may also provide a means of spread. The expression of the disease is inﬂuenced by the interaction of genetic, environmental (primarily air temperature and leaf wetness) and physiological (age of infected plants) factors. The infection may occur within the temperature range of 5–34°C; however, the optimal temperatures for spore germination are between 15° and 30°C. Heavy rains tend to remove spores from the atmosphere, rendering them infective if they land on the soil (Egan, 1964; Comstock and Ferreira, 1986). On the other hand, it has been found that rains favour the development of orange rust but inhibit the development of common rust (Croft et al., 2000). Sugarcane plants appear to be most susceptible at 3–6 months old (Ryan and Egan, 1989). Disease control The best control of sugarcane rust is use of resistant sugarcane varieties. The development of resistant cultivars has decreased the economic losses caused by this disease (Ryan and Egan, 1989). Nevertheless, existing 221 resistant cultivars are threatened by the establishment of new races of the pathogen. For example, cultivar CP 78-1247 was considered to be resistant or moderately resistant until 1988, and then it exhibited extremely high rust susceptibility throughout south Florida (Raid, 1989). However, resistance has not been stable or durable on certain varieties, presumably because of rust variants. For this reason, it is highly recommended that growers should diversify their varietal holdings (Raid and Comstock, 2000). Chemicals like propiconazole/mancozeb, cyproconazole, triadimefon and triadimenol have been used for the control of sugarcane rust. Several soil factors inﬂuence rust infection levels on sugarcane signiﬁcantly. Studies have shown that rust levels are higher on sugarcane grown on low pH soils, high soil moisture and high levels of phosphorus and potassium nutrients present in the soil (Johnson et al., 2007). Red Rot Disease of Sugarcane Red rot is one of the oldest known diseases of sugarcane. It occurs in most cane-growing countries. The disease was ﬁrst described from Java by Went (1896) and then the disease was reported from Australia, India, Hawaii and the USA. It is clear that the disease was widely distributed before the knowledge of its impact on sugarcane crop (Singh and Singh, 1989). Symptoms The pathogen, Colletotrichum falcatum Went, can attack any part of the sugarcane plant – stalk, leaf, buds or roots – but it is usually considered a stalk and a seed-piece disease. C. falcatum completes its life cycle on the sugarcane leaf and usually the damage to the leaf does not pose a serious threat to cane or cause much harm to the plant (Singh and Singh, 1989; Raid, 2006). The most damaging phase of this disease occurs when the pathogen attacks the stalk. Depending on the age of the stalk, 222 A.M.H. Esh time of infection and susceptibility of the cane genotype, it produces different types of symptoms. The typical stalk symptoms, that is, presence of white spots in otherwise rotten (dull red) internodal tissues and nodal rotting, appear when the crop is at the fag end of the grand growth phase in subtropical areas. These white patches are speciﬁc to the disease and are of signiﬁcance in distinguishing red rot from other stalk rots. At a later stage, some discoloration of rind often becomes apparent when internal tissues have been badly damaged and are fully rotten (Singh and Singh, 1989; Raid, 2006; Duttamajumder, 2008). In susceptible varieties, the red colour, sometimes along with some grey colour, may be seen throughout the length of the stalk. The infection is conﬁned largely to the internodes in resistant varieties. On the leaves, the pathogen may produce elongated red lesions on the midribs, reddish patches on the leaf sheaths and, infrequently, small dark spots on the leaf blades. Eventually, the lesions may develop a straw colour in the centre. In seed pieces, the entire seed piece may become rotten and the internal tissues turn various shades of red, brown or grey (Singh and Singh, 1989). Causal agent The fungus causing red rot of sugarcane is commonly known by its imperfect state, i.e. C. falcatum Went (Glomerella tucumanensis). The perfect state of the fungus belongs to Phylum: Ascomycota; Class: Ascomycetes; Family: Glomerellaceae; Genus: Glomerella (Bisby et al., 2007). Conidia are falcate (but not markedly so), fusoid, apices obtuse, 15.5 (25–26.5)– 48 µm × 4 (5–6)–8 µm and contents are granular and sometime contain oil globules. At least two races have been identiﬁed. The variations in the asexual state of the fungus (Colletotrichum state) may originate through: (i) heterokaryosis; (ii) by recombination through parasexual mechanism; and (iii) by the universal mechanism of mutation, selection and adaptation in response to the changes in the host environment. Heterokaryosis is the mechanism through which the fungus collects and consolidates two or more genetically different nuclei in the hypha and derives the beneﬁt of the introduced genetic material. These newly gathered nuclei also multiply in tandem with the native nuclei (Duttamajumder, 2008). Pathogenesis The pathogen mainly infects the stalks through the nodes. Once the infection is established in the stalk, the fungal mycelium grows intracellularly and is sparse in the reddened areas. The dead cells are packed in white patches with profuse hyphae. The size and number of these white areas are correlated with the susceptibility of the variety. The lesions become dark red, narrow and sharp margins, with a few white spots in resistant varieties, while in susceptible varieties the lesions become wide, light red and ill-deﬁned margins with prominent white spots (Singh and Singh, 1989). One of the major sources of inoculum is midrib lesions. Also, diseased stalks and crop debris and infected plant material are important sources of inoculum and cause secondary infections. Wind, rain, heavy dews and irrigation water play a role in the dispersal of the inoculum. The pathogen spores washed into the soil may produce infection in planted seed pieces. Climatic factors affect both the spread and severity of red rot. In newly-planted cane, the disease is favoured by excessive soil moisture, drought conditions and low temperatures. Disease control The use of resistant varieties is the most effective method of prevention and control of sugarcane red rot disease (Singh and Singh, 1989; Raid, 2006; Singh et al., 2008b). Management of the disease by the use of diseasefree seed canes for planting is impractical due to the difﬁculty in diagnosing dormant infections of the fungus in seed canes under Fungal Diseases of Sugarcane ﬁeld conditions (Viswanathan and Samiyappan, 2002). It is difﬁcult to manage red rot through chemotherapy because the impervious nature of rinds and ﬁbrous nodes at cut ends does not allow sufﬁcient absorption in setts (Agnihotri, 1990). However, better crop stands have been achieved from enhanced germination obtained by treating seed pieces with a fungicide before planting (Raid, 2006). Thermotherapy (moist hot air or hot water) is thought useful for inactivation of red rot pathogen, but it is difﬁcult to remove deep-seated infections. It is limited in checking secondary infections (Singh, 1973; Singh and Singh, 1989; Raid, 2006). In India, extensive studies about the possibilities of using biological control to control sugarcane red rod disease have been carried out (Mohanraj et al., 1999). Seventy-ﬁve per cent of canes may be protected against secondary infection of red rot by dipping the setts for 15 min in 2.5% culture ﬁltrate of Trichoderma harzianum (Th 38) and also by applying Trichoderma multiplied culture in press mud 20 kg/ha beneath the setts in furrows. Besides the biological control of red rot, the growth in improved resulting is to enhanced yield by 15.4 t/ha (Singh et al., 2008a,b). Sugarcane Eye Spot Disease Eye spot has been reported in many sugarcane-growing areas of the world. The disease was ﬁrst described by van Breda de Haan in Java (1892, cited in Comstock and Lentini, 2005). The disease is prevalent and is found in 66 sugarcane-growing countries (Agnihotri, 1990). Generally, the disease has a minor economic impact on sugarcane yield in most areas because of the use of resistant varieties (Comstock and Lentini, 2005). In India, in 1976, the disease was in epidemic form and affected 1600 ha sugarcane crop in Mandya district of Karnataka only (Kumaraswami and Urs, 1978). The disease can reduce sugarcane yield by 15–20% (Sharma et al., 2004). 223 Disease symptoms Typical mature eye spot symptoms are characterized by a reddish-brown elliptical lesion (0.5–4.0 mm long, 0.5–2.0 mm wide) with yellowish-brown margins. Reddish-brown to yellowish-brown streaks, sometimes called ‘runners’, extend upward from individual lesions toward the leaf tip. These streaks are 3–6 mm wide and 30–90 cm long. The entire leaf eventually may become necrotic (Comstock and Steiner, 1989; Comstock and Lentini, 2005). Causal agent Eye spot disease is caused by Bipolaris sacchari (Butler) Shoemaker. The fungus belongs taxonomically to Phylum: Ascomycota; Class: Ascomycetes; Order: Pleosporales. B. sacchari is the teleomorphic stage of Helminthosporium sacchari Butler. The name H. sacchari is still used occasionally. Pathogenesis Sugarcane eye spot fungus B. sacchari (H. sacchari) produces a host-speciﬁc toxin (HST). HSTs are a group of structurally complex and chemically diverse metabolites produced by plant pathogenic strains of certain fungal species and function as essential determinants of pathogenicity or virulence. HSTs are referred to as ‘host selective’ because they are typically active only toward plants that serve as hosts for the pathogens that produce them and disease never occurs in the absence of toxin production (Wolpert et al., 2002). The HST is a mixture of three isomers (A, B and C) (Lesney et al., 1982; Livingston and Scheffer, 1984) and the HST produced by the sugarcane red rot pathogen is responsible for the disease symptoms (Steiner and Byther, 1971). The fungus causes eye-shaped lesions on the leaves, followed by the development of reddish brown streaks or ‘runners’ extending from the lesions toward the tip of 224 A.M.H. Esh the leaf. The toxic compound from the fungus causes the runner (Steiner and Byther, 1971). Eye spot spores, which are produced abundantly on leaf lesions, are dispersed by wind and rain. High humidity and dew formation are the favoured conditions for spore germination. The disease is not transmitted by seed pieces and mechanical transmission by equipment and by humans is unimportant. a ladder-like appearance. These lesions sometimes break through the surface of the rind, causing curvature and distortion of the stalk. Exaggerated versions of these depressions may look like neatly made ‘knife-cuts’ in the stalk. In the most advanced stage of pokkah boeng, the entire top (growing point) of the plant dies (referred to as ‘top rot’). The ladder-like lesions are due to rupturing of the diseased cells that cannot keep up with the growth of the healthy tissue (Martin et al., 1989; Raid, 2009a). Disease control Causal agents The only practical and efﬁcient method of control of eye spot disease is with resistant clones. Chemical control using foliar fungicides is not practical (Comstock and Lentini, 2005). Pokkah Boeng Disease Pokkah boeng, which is a potentially destructive disease of sugarcane, is caused by Gibberella moniliformis (Sheldon) Wineland. There have been many reported outbreaks of the disease which have been severe, like the Java outbreak in 1896, but they have caused little economic loss (Martin et al., 1989). Disease symptoms In the early stages of infection, the symptoms of the disease are chlorotic areas at the base of young leaves, distortion (wrinkling and twisting) and shortening of the infected leaves and ﬁnally stalk death in severe cases. The infected leaves can be distinguished by their narrow base. Irregular reddish stripes and specks develop in the chlorotic parts which appear in mature leaves. The infection is present in the stalk and dark reddish streaks may be found extending through several internodes. Also, in the internodes, the infection may form long lesions with cross-depressions that give them The disease is caused by the fungi, Fusarium moniliforme (G. fujikuroi) and F. moniliforme var. subglutinans (G. subglutinans). The perithecia of G. fujikuroi occur only on dead plant material, while the perithecia of G. subglutinans are rarely formed in nature; thus, the perithecia of these two pathogens are rarely associated with infected sugarcane plants (Martin et al., 1989). The pathogens have a wide host range, i.e. rice, corn, sorghum and many other grasses. These fungi also cause other diseases, such as seedling blight, scorch, stalk rot, root rot and stunting in different crops (Martin et al., 1989; Raid, 2009a). Pathogenesis The pathogens of pokkah boeng disease are transmitted by the movement of spores from one locality to another by air currents (Martin et al., 1961; Raid, 2009a,b). Pokkah boeng disease of sugarcane may also spread from seeds contaminated with the fungus (Martin et al., 1961). It appears to be favoured by dry climatic conditions being followed by a wet season. Cane that is 3–7 months old and growing vigorously appears to be most susceptible (Martin et al., 1989). The pathogen spores enter the spindle along the margin of a partially unfolded leaf, then germinate and grow into the inner tissue of the spindle leaves. The conidia germinate and the mycelium can pass through the soft cuticle of young leaves to the inner Fungal Diseases of Sugarcane tissues because the epidermal tissues are still fragile and not protected by the plant system (Dillewijn, 1950). The mycelium spreads to vascular bundles of the immature stem and blocks the vessels, which eventually leads to growth distortions and rupture, and this development shows the ladder-like lesions (Holliday, 1980; Martin et al., 1989). Disease control The only efﬁcient control for pokkah boeng disease is the use of resistant varieties. Sugarcane resistance to pokkah boeng has been shown to be highly heritable (Martin et al., 1989). Pineapple Disease of Sugarcane Pineapple disease is an economically important sugarcane disease that is widely distributed in almost all the regions where sugarcane is grown (Wismer and Bailey, 1989). In India, the disease has been noticed on different sugarcane varieties (Singh et al., 1990). The disease is caused by the fungus Ceratocystis paradoxa, which induces seedpiece decay following planting. The affected setts emit a smell resembling that of the mature pineapple fruit (Went, 1896). 225 acetate content in the infected tissue may rise up to 1%, which is sufﬁcient to inhibit the germination of buds (Kuo et al., 1969). As a result, gappy stands are evident and young crops have a patchy and uneven appearance. In the early stages of rotting, the disease may be diagnosed by a strong odour of overripe pineapple. Although pineapple disease is not considered important in standing cane, infection may occur if the stalks are physically damaged or stressed (Wismer and Bailey, 1989). Causal agents Pineapple disease is caused C. paradoxa. The fungus belongs to Phylum: Ascomycota; Class: Ascomycetes. The fungus produces two types of imperfect spores, conidiospores 6–24 um × 2–5.5 µm (thin-walled cylindrical conidia) and chlamydospores 10–25 um × 7.5–20 µm (thick-walled and oval). These spores are produced intensively on the internal tissues of the infected seed pieces. They are released into the soil on seed piece decay. The spores may survive for several years in the soil, serving as a source of inoculum for the next crop (Wismer and Bailey, 1989). The perfect stage of the fungus has been reported and it occurs naturally on cacao (Dade, 1928) and sugarcane (Kuo et al., 1969). The pathogen also causes diseases of pineapple, banana, cacao, coconut and oil palm (Wismer and Bailey, 1989). Disease symptoms The disease affects sugarcane setts in the ﬁrst weeks after planting. The fungus spreads rapidly through the parenchyma and colonizes all the internal tissue of the seed piece, which turns red and eventually black. The black coloration results from the production of fungal spores within the seed piece. Nodes act as partial barriers to the spread of rotting, but with susceptible varieties, entire seed pieces may become colonized by the fungus (Wismer and Bailey, 1989). The pineapple odour resulting in the decayed seed pieces is due to ethyl acetate, formed by the metabolic activity of the pathogen. The ethyl Disease control Control is a priority, especially where the soil inoculum level is high through the amelioration of conditions that favour the germination of buds and the emergence of young shoots (e.g. good quality cuttings, adequate irrigation, right planting time and depth). The ends of cuttings are dipped in a fungicidal solution, either as a cold dip or the fungicide may be incorporated into the water tank at the time of the hot water treatment (Antoine, 1956). Other fungicides used to control pineapple disease include 226 A.M.H. Esh benomyl, propiconazole carbendazim, etc. (Autrey, 1974). Systemic fungicides are found more efﬁcient than non-systemic fungicides (Vijaya et al., 2007). Since pineapple disease is a soilborne disease, crop rotation or a fallow period between cane crops may prove to be of some beneﬁt in reducing its impact. Seed-piece infection by pineapple disease frequently proceeds from the exposed cut ends to the centre of the seed piece. Therefore, the use of seed pieces containing at least three nodes increases the likelihood that buds closer to the centre will germinate (Wismer and Bailey, 1989). For disease control, sett treatment with chemical fungicides before planting is an effective method and is widely followed. 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(2004) Genetic diversity of Ustilago scitaminea in mainland China. Sugar Tech 6, 267–271. 18 New and Emerging Fungal Pathogens Associated with Leaf Blight Symptoms on Wheat (Triticum aestivum) in Argentina Analía Edith Perelló CIDEFI (Centro de Investigaciones de Fitopatología) – CONICET (Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas), Facultad de Ciencias Agrarias y Forestales de la Universidad Nacional de La Plata, La Plata, Provincia de Buenos Aires, Argentina Abstract Regional surveys are being conducted at the CIDEFI to investigate the presence of wheat (Triticum aestivum L.) pathogens on leaves and seeds across the Argentinian cropping area. During the past 5 years in the wheat cropping area of Buenos Aires Province, Entre Ríos and Santa Fe Provinces, Argentina, several unusual diseases have been found on wheat leaves. From the symptomatic tissues, the fungi were isolated and identiﬁed. To test pathogenicity and fulﬁl Koch’s postulates, inoculations of different wheat cultivars under greenhouse conditions were carried out; disease symptoms and the causal agents are described. Introduction Wheat ranks as a primary source of food and livelihood for hundreds of millions of people globally, especially in developing countries. Several serious foliar diseases caused by necrothrophic pathogens occur in this crop in Argentina. Among them, Septoria tritici leaf blotch and tan spot caused by Drechslera tritici-repentis are the most important. Both can cause serious yield and quality losses under the right conditions. Among biotrophic diseases, leaf rust is a very dangerous one. Samples were obtained from different wheat cultivars from 2001 in 13 different locations of the main wheat-growing area of Argentina (northern, central and eastern regions of the Buenos Aires Province, Entre Ríos and Santa Fé). Most symptoms were observed on the upper leaves at growth stage 69 (anthesis complete) and at growth stages 80 (early dough) to 85 (soft dough) according to the scale of Zadok et al. (1974). Samples of 20–40 plants of each disease area were used for late laboratory identiﬁcation of leaf-spotting fungi. The frequency of the pathogens was different among localities and cultivars over the past 10 years. Pyrenophora tritici-repentis was predominant, followed by Mycosphaerella graminicola. In a total of 193 and 240 infected leaf samples collected in 2006– 2007, D. tritici-repentis was observed in 63 and 77% of leaf samples, respectively, suggesting an increasing trend in incidence over the years. This may be explained in  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 231 232 A.E. Perelló part due to the adoption of reduced-tillage cropping practices. Surprisingly, a signiﬁcant increase in the frequency of Alternaria spp. isolates was observed, with 41 and 53% of the samples harbouring tan spot in the same period. The isolates were identiﬁed as belonging mostly to the A. infectoria species group. Moreover, mapping of pathogen distribution during the past years in different agroclimatic zones of Buenos Aires Province shows that A. infectoria is a widespread pathogen that is gaining prominence as an emerging wheat pathogen in latter years. Also, it was found, together with A. alternata, on almost 70% of wheat seed samples. Other diseases were sporadic, isolated or minor and included foliar blight or spots caused by the fungus A. triticina, A. infectoria species group complex, Bipolaris sorokiniana, Cladosporium herbarum, Phoma sorguina, Ascochyta hordei, Pyricularia grisea, Cephalosporium gramineum and others. Some disease symptoms are characteristic and obvious. Other diseases, however, may be difﬁcult to diagnose without microscopic or laboratory analysis. Since different diseases require different control strategies, their accurate diagnosis is essential. Leaf Blight of Wheat Caused by Alternaria triticina in Argentina Alternaria species are perhaps the most common fungi encountered by mycologists working in plant pathology. As plant pathogens, over 4000 Alternaria/host associations are recorded in the USDA Fungal Host Index and the genus ranks 10th among nearly 2000 fungal genera listed based on the total number of host records. In Argentina, this genus has been studied on wheat plants since the last decade (Perelló, 2007; Perelló et al., 1992, 1996, 2002, 2003, 2005a,b, 2008; Perelló and Sisterna, 2005, 2006). Several Alternaria species and numerous uncharacterized Alternaria taxa have been found associated with leaf blight symptoms. Alternaria species are some of the most prodigious producers of toxic secondary metabolites, producing over 70 compounds of varying toxicity (Kumar and Rao, 1979). Some of these metabolites are powerful mycotoxins not yet characterized in Argentina. During routine investigations across the wheat (T. aestivum)-growing area of the Buenos Aires Province, diseased leaf samples were collected from different wheat cultivars. Discoloured oval lesions appear on lower leaves. The disease progresses upwards, lesions enlarge and coalesce to irregular, dark blotches, often with chlorotic margins. Severely infected seeds are discoloured and shrivelled. Necrotic tissue fragments were surfacesterilized and plated on potato dextrose agar (PDA), from where Alternaria specimens were isolated. Morphobiometrical and cultural features of the fungus were examined on potato carrot agar (PCA). Conidia were irregularly oval, ellipsoid conical, gradually tapering into a beak, 15–92 × 8–35 µm, with 1–10 transverse septa and 0–5 longitudinal septa, light brown to dark olive buff, becoming darker with age. All isolates obtained were identiﬁed as A. triticina following the morphological descriptions by Anahosur (1978) and conﬁrmed by comparison with reference strains of CABI Bioscience (IMI 289962 and IMI 178784) kindly sent by Dr D. Mercado (Université Catholique de Louvain, Unité de Phytopathologie, Belgique). One of the isolates has been lodged in the culture collection of La Plata Spegazzini (LPSC) (accession number 798). Pathogenicity tests were conducted in the greenhouse. Susceptible wheat cultivars were inoculated at tillering and heading stages with a conidial suspension (2 × 105 conidia/ml). After 10 days, typical leaf blight symptoms developed and A. triticina was recovered from the lesions. No symptoms appeared on the control plants. Alternaria triticina causes signiﬁcant yield losses in wheat on the Indian subcontinent, from where it originates and has spread throughout the world (Agarwal et al., 1993). Although A. triticina has been detected previously in Argentina on wheat leaves and seeds (Perelló et al., 1992), it has probably existed as a minor pathogen for many years without being noticed. The recent increase in the severity of leaf blight may be New and Emerging Fungal Pathogens due to new cultural practices such as conservation tillage, nitrogen fertilization, irrigation, use of new germplasm, as well as favourable weather conditions. As A. triticina is a quarantine pathogen in many countries, it is important to investigate the incidence and importance of this disease in Argentinean wheat areas. This is the ﬁrst published record of A. triticina on wheat in Argentina and on any host in this country. Alternaria leaf blight Alternaria triticina Prasada & Prabhu causes signiﬁcant yield losses in wheat in the Indian subcontinent, from where it originated and has spread throughout the world. The disease was ﬁrst reported in 1924 but remained incompletely characterized for several years. Early studies associated Alternaria spp. with the disease, but the causal organism, A. triticina, was not identiﬁed until 1962 (Prasada and Prabhu, 1962). From 1960 to 1964, leaf blight damaged all commercial cultivars on the Indian subcontinent (Prabhu and Prakash, 1973; Bhowmik, 1974; Sokhi, 1974). It developed on plants approaching maturity, caused premature death of the uppermost leaves and heads and reduced yield signiﬁcantly. Today, durum wheats, their derivatives and introduced Mexican wheats are considered most susceptible (Frisullo, 1982). In addition to wheat, the disease affects triticale in India and other graminaceous hosts in the Middle East and Nigeria (Chaudhuri et al., 1976). Symptoms The pathogen may infect all foliar parts. Alternaria leaf blight is characterized by small, chlorotic, oval- or elliptical-shaped lesions scattered on lower leaves. As the plant matures, the disease progresses upwards and lesions darken to brown-grey, enlarge and coalesce to irregular, dark blotches, irregular in shape and may have a yellow margin. The chlorotic borders of the lesions may become diffuse and turn light to dark brown 233 in colour. The lesions develop progressively from lower to upper leaves and blighting may extend to heads and leaf sheaths. Symptoms appear on leaves, seeds and spikelets. Severely infected seeds are discoloured and shrivelled (Rault et al., 1983). Under humid conditions, the lesions support visible clusters of dark, powdery conidia. Lesions are difﬁcult to distinguish from those caused by Helminthosporium spp. Alternaria leaf blight is likely to develop near irrigation ditches, in low areas, or wherever humidity and soil moisture are high. It develops rapidly once wheat plants are 6–8 weeks old, and especially as the crop approaches maturity. Bread and durum wheat, barley and triticale are the primary hosts. Causal organism Isolations from leaf lesions routinely yield Alternaria spp., many of which are saprophytic and mask the pathogen. Alternaria triticina is distinguished by its wheat-speciﬁc virulence and cultural characters. The mycelium and conidia of A. triticina are initially hyaline and later olive buff. Conidiophores are septate, usually unbranched but occasionally branched, erect, single or fasciculate, emerging through stomata, geniculate, straight, length variable, between septa 17–28 µm, 3–6 µm wide. A chromogenic variant in A. triticina was studied (Jain and Prabhu, 1976). Conidia of A. triticina are acrogenous, borne singly or in short chains (two to four spores). They vary from 8 to 35 µm in width and 15 to 92 µm in length, are dark, ellipsoid to conical, tapering to a beak. A. triticina grows on a variety of simple media (Rao and Subrahmanyam, 1974). Colonies on PDA are discrete or effuse, dark blackish brown to black, margin smooth and entire. Growth is optimal between 20° and 24°C, with limits near 5° and 35°C. Physiological specialization of the pathogen exists and six races have been characterized and reported using 15 differentials. Nonspeciﬁc phytotoxins produced by the pathogen apparently play a role in wheat pathogenesis. 234 A.E. Perelló Alternaria infectoria Complex Associated with Black Point and Leaf Blight Symptoms in Argentina Among the ﬁeld fungi found in cereals, Alternaria is the dominant genus and, within this habitat, taxa of the A. infectoria species group predominate by far. Information available on the A. infectoria species group is limited as the taxa it comprises have often been misidentiﬁed as other small-spored Alternaria species, due to the use of insufﬁcient methods for identiﬁcation. Members of the A. infectoria species group are morphologically distinguishable from other small-spored species of Alternaria by their long secondary conidiophores and formation of white or grey colonies on dichloran rose bengal yeast extract sucrose media (DRYES) (Andersen et al., 2002). Furthermore, this species group is the only one among Alternaria where the teleomorph, Lewia, has been identiﬁed in Argentina. To date, the A. infectoria species group comprises the known species A. arbusti, A. conjuncta, A. infectoria, A. oregonensi, A. triticimaculans, A. metachromatica, A. viburni, A. intercepta and A. novae-zelandiae, as well as an unknown number of distinct taxa yet to be described. Members of the A. infectoria species group produce a range of unique secondary metabolites that are useful for metabolic proﬁling and chemotaxonomy of Alternaria. Until now, four different proﬁles have been identiﬁed, suggesting that there is a potential risk of Alternaria mycotoxins in wheat in Argentina (Pich et al., 2007). Black point and leaf blight caused by A. infectoria species group complex is a new disease of wheat in Argentina. Four hundred and ten isolates collected from 17 different geographical zones of the Argentinian cropping area were tested for their morphological variation. The isolates differed in their morphobiometrical and cultural characteristics on PCA. On this basis, they were categorized into four morphotypes. Further, 20 isolates were characterized according to their pathogenic and biochemical variability. Pathogenicity tests were conducted under greenhouse conditions on wheat plants of the cultivars Buck Arriero, Buck Charrúa, Buck Granar, Buck Poncho, Buck Yatasto, Klein Cacique, K. Estrella, ProINTA Cinco Cerros, ProINTA Elite and Pro INTA. Signiﬁcant differences between cultivars, isolates and the interaction isolate × cultivar were shown according the ANOVA results. Analysis of severity means (Tukey’s test) showed cultivar Buck Charrúa as the one with the best behaviour against all the isolates tested, and cvs. Pro INTA Cinco Cerros and Pro INTA Elite as the most susceptible. Symptoms observed were: chlorosis and/or apical or general necrosis (blight), or elongated necrotic spots surrounded by a chlorotic halo. Additionally, 20 samples of wheat seeds from different localities in the Buenos Aires Province were analysed by blotter test (ISTA) (Neergaard, 1979). After 7 days incubation (20 ± 2°C and cycles of 12 h light plus NUV light), the microorganisms developed were identiﬁed and the A. infectoria complex in particular was characterized according to its morphobiometrical features on PCA. A prevalence (samples infected/samples analysed) of 55% and infection values of 37% of A. infectoria species group members was registered. Twenty isolates were tested in a comparative analysis of ﬁve isoenzyme patterns (phosphatase, peroxidase, A-esterase, B-esterase, glutaminotransaminase) and total proteins. Mycelium for electrophoresis in polyacrylamide gel was obtained from monosporical cultures of the fungus on PCA over 10 days. The results obtained revealed differences between the strains in the isozyme banding patterns. Each isolate had a characteristic electromorph, showing different main bands of enzyme activity and some minor bands varying in intensity for all the patterns assayed. Isozyme data corroborated the morphological and pathogenic variability observed previously on A. infectoria isolates collected from wheat. These results support the usage of isoenzymatic patterns for the characterization of isolates of A. infectoria complex associated with black point and leaf blight symptoms on wheat, as a valuable additional tool to aid the traditional taxonomy, base on morphocultural characters only. New and Emerging Fungal Pathogens Detection of Lewia infectoria and its Alternaria Anamorph from Wheat in Argentina Occurrence of L. infectoria (Fuckel) Barr & Simmons (teleomorph of A. infectoria) developed in culture is described, illustrated and reported for the ﬁrst time. Monosporic isolates, obtained from infected wheat plants, produced conidia within a week and ascomata with fully mature ascospores within 7 months when stored on slants of PCA at 4 degrees in darkness. The anamorph exhibited the sporulation pattern of A. infectoria species group and was identiﬁed on the basis of axenic colony morphology and by the prominence of their secondary conidiophore structure. Critical examination of the teleomorph proved it to be L. infectoria. The importance in interpreting the teleomorph– anamorph pair is discussed. A. infectoria species group, causing leaf blight and black point of wheat, was not signiﬁcant for many years, but currently this constraint has become a new problem (Perelló and Sisterna, 2006). During 2005, wheat samples were collected and typical symptoms of tan–dark brown leaf spot were observed on several cultivars from the cropping area of Buenos Aires Province. Different isolates of a fungus with characteristics of Alternaria were obtained from this material. In laboratory conditions on PCA, cultures of this fungus produced conidia within a week. Then, these isolates were stored to maintain a fungal collection. Within 7 months, ascomata with fully mature ascospores of a previous undescribed genus were observed in connection with this anamorphic state. Based on morphological characters (Simmons, 2002), the teleomorph proved to be a Lewia species, described here as L. infectoria with its Alternaria anamorph. Wheat leaves exhibiting necrotic symptoms were collected during September and October in 2005 from different cultivars of farmers’ ﬁelds and research stations of eight localities of Buenos Aires. Lesions on leaves were viewed through a stereoscope at × 12 and speciﬁc morphological characteristics of pathogens were recorded. Fungi were cultured on PDA. After screening of the cultures, 235 180 belonged to the Alternaria genus. Single spore cultures were obtained on PCA. Based on morphological characters like the conidial sporulation pattern and the prominence of their secondary conidiophore structure, most of the strains were identiﬁed as members of the A. infectoria species group. The cultures were stored on slants of PCA at 4°C in darkness. Numerous conidia appeared on the surface of the agar within a week and groups of fruiting bodies within 7 months. To determine their stage of development, the fungal fruiting bodies were placed on a microscope slide, stained with 0.25% Trypan blue in lactid acid:glycerol:water (1:1:1) and examined with a light microscope (× 400). After 7 months, groups of fertile ascomata (pseudothecia) with septate hyaline mature ascospores developed in isolates obtained from the cultivar Klein Estrella, from a particular ﬁeld in the locality of Balcarce (Buenos Aires Province). There were detectable differences between isolates with regard to pseudothecial density and speed of ascospore maturity. In some cases, the production of immature asci was observed. The morphobiometrical and cultural features of these ascomata on PCA allowed the identiﬁcation of the teleomorph of A. infectoria, L. infectoria (Fuckel) Barr & Simmons. Its description is as follows: ascomata ellipsoid, 400–500 × 150 µm, with a short, papillate beak, dark, thin-walled at maturity. Asci 105–125 × 13–16 µm, subcylindrical, straight or somewhat curved. Ascospores 8, 18–22 × 7–8 µm at full development, broadly elliptic, muriform, becoming 5-septate (3 primary septa), only end cells not longitudinally septated, constricted, yellow-brown. Although most Alternaria species do not have teleomorphic afﬁnities, a number of anamorphically deﬁned taxa within the Pleosporaceae have recognized teleomorphs and most are not commonly encountered (Simmons, 1986, 2002). These teleomorphs are representative of nearly all major lineages within the Pleosporaceae. An evaluation of the teleomorphic characters of well-known Pleospora spp. with anamorphs of Alternaria, namely P. infectoria and P. scrophularieae, revealed that Pleospora spp. with Stemphylium anamorphs were morphologically 236 A.E. Perelló distinct from Pleospora spp. with Alternaria anamorphs, particularly in the size of the ascomata and ascospores. This resulted in the designation of the genus Lewia for Pleospora-like fungi with Alternaria teleomorphs (Simmons, 1986). Evidence of production of Alternaria-related teleomorphs in axenic culture was previously reported by Bilgrami (1974) in L. infectoria, Simmons (1986) in L. photistica, Kwasna and Kosiak (2003) in L. avenicola and Kwasna et al. (2006) in L. hordeicola. Other described Lewia species, like L. chlamidosporiformans, L. ethzedia, L. intercepta, L. sauropi, L. viburni and L. eureka, usually produce ascomata on tissues of infected plants (Simmons, 1986, 2002; Vieira and Barreto, 2005). During the present study, ascomata of L. infectoria were produced in vitro, in axenic culture on PCA slants, in connection with the anamorph. However, the fungus does not often produce both anamorph and teleomorph on the same slant. Crossing between isolates is evidently not necessary for production of the teleomorph since single-ascospore axenic cultures continued to produce ascomata on PCA. These results were similar to observations made by Kwasna and Kosiak (2003) for L. avenicola. In addition, its ﬁnding in Argentina has provided an important framework for hypothesis testing in advanced studies on Alternaria/Lewia epidemiology and pathogenicity variability on wheat plants. Lewia infectoria forms pseudothecia on wheat straw in the ﬁeld under determined weather conditions. This could play an important role as a source of inoculum in Alternaria/Lewia disease able to infect wheat and wild grasses as a result of the dispersal of airborne ascospores. The discovery of the sexual stage in nature may have a large inﬂuence on localized development of the diseases in different regions of the country. Occurrence of Ascochyta hordei Hara var. europaea Punith. on Wheat Leaves in Argentina Ascochyta leaf spot is often overlooked in association with other leaf spot diseases. It is reported on wheat as a pathogen of minor economic importance but there are some reports pointing out that high humidity conditions could favour the occurrence of outbreaks of the disease (Scharen and Krupinsky, 1971). During September–October 2002, leaf spot symptoms on wheat cultivar Baguette 10, growing in farmers’ ﬁelds in Tandil, eastern area of Buenos Aires Province, were commonly observed. The leaves showed symptoms similar to those described for other necrotrophic foliar pathogens (D. triticirepentis) and Stagonospora nodorum, suggesting that any of these might have been involved. Ascochyta was commonly isolated from affected tissues of the samples collected. A. tritici Hori & Enj. is generally accepted as the cause of Ascochyta leaf spot, but A. graminicola Sacc. is cited in some literature (Zillinsky, 1984). Punithalingam stated the status of A. tritici was uncertain but it might be a synonym of A. hordei (Farr et al., 1989). Sprague and Johnson (1950) also stated that A. tritici was close to A. hordei Hara, differing mainly in the symptoms on barley. The identity of the culture of Ascochyta isolate A1102 of this study was determined as A. hordei Hara var. europaea by experts of the Centraalbureau voor Schimmelcultures (CBS), The Netherlands, and deposited in the CBS culture collection under the number 112525. Ascochyta was not previously reported on wheat and other grasses in Argentina. In this sense, the ﬁrst occurrence of this fungus as a member of the leaf spotting complex on wheat plants in the Argentinian cropping area is signiﬁcant. Diseased leaves were collected, stored in paper bags and transported to the laboratory. The pathogen was isolated from typical necrotic symptoms. Morphobiometrical and cultural studies of the fungus were studied. Inoculation experiments to conﬁrm pathogenicity were performed in the greenhouse at 15–25°C and 80% relative humidity on 16 wheat cultivars: Buck Arriero, Buck Yatasto, Buck Poncho, Buck Charrúa, Buck Halcón, ProInta Granar, ProInta Cinco Cerros, Desimoni Caudillo, ProInta Imperial, ProInta Puntal, ProInta Guazú, ProInta New and Emerging Fungal Pathogens Colibrí, ProInta Elite, Klein Estrella, Klein Cacique and Klein Dragón. Plants were grown in plastic pots 12 cm in diameter (4 seeds/plot in all samples) with a standard potting mix. Plants were inoculated when they had reached the third expanded leaf stage. Inoculum was prepared from 10-dayold cultures of A. hordei var. europaea (isolate A1102) growing on PDA and was obtained by ﬂooding each sporulating plate with sterile distilled water and gently scraping the fungal colony with a ﬂame-sterilized scalpel to dislodge conidia. The conidial suspension was ﬁltered once through a single layer of cheesecloth and spore concentration was determined with a haemocytometer. The inoculum consisted of 12 × 106 conidia/ml. Twenty seedlings of each cultivar were used for the inoculation. Leaves were sprayed to runoff with a manually operated sprayer. The inoculated plants and controls were kept in a moist chamber for 48 h. The ﬁrst symptoms appeared 92 h after inoculation. Between 40 and 60% were necrotic 18 days after inoculation. In natural ﬁeld infections, it was observed that plants were affected rather severely, especially the basal leaves. Lesions at ﬁrst are distinct, chlorotic, ellipsoidal or round and 1–5 mm across. Later, they become diffused and grey-brown internally. Pycnidia sometimes form and appear as black dots within necrotic lesions. They are submerged in host tissues, except for a papillate projection. In culture on PDA, pycnidia measured 142.5–225 × 93.75–206.25 µm. Conidia (pycnidiospores) are straight, hyaline and oblong, 3.75–5.60 × 15–18.70 µm, typically with one median septum. All wheat plants inoculated with A. hordei var. europaea in the greenhouse developed symptoms identical to those observed on naturally infected plants in the ﬁeld. The amount of damage to seedlings was measured as per cent necrotic leaf area from the ﬁrst leaf of 15 plants per each inoculated cultivar in comparison with controls. Cultivars Buck Arriero and Buck Poncho showed the most conspicuous symptoms 9 days after inoculation, with a disease severity rating of between 8–35% of necrotic leaf area. The rest of the cultivars showed no symptoms or 237 had low disease severity. Buck Arriero, Buck Charrúa, Buck Halcón, Buck Poncho, Buck Yatasto and Klein Dragón showed the most severe symptoms of the disease (between 12–45% of the necrotic leaf area) 20 days after inoculation. The rest of the cultivars showed little evidence of infection with only a 10% necrotic foliar area, except ProInta Imperial and ProInta Puntal, which showed no evidence of infection, indicating that the disease pressure on Buck cultivars was much higher than on the rest of the wheat cultivars examined. These observations are consistent with other reports (Wiese, 1977), which indicates that the fungus is apparently of little economic consequence as a foliar pathogen of wheat. Inoculation studies proved that A. hordei var. europaea was the cause of this outbreak on wheat in Argentina. New cultural practices (reduced tillage, nitrogen fertilization, irrigation), the use of new germplasm and favourable environmental conditions could have contributed to creating ideal conditions for the increase and spread of inoculum, not only of A. hordei var. europaea but of the foliar complex of necrotrophic pathogens in general. Other wheat cultivars may also be susceptible to isolates of the pathogen. Phoma sorghina (Sacc.) Boerema, Dorenbosch & van Kesteren in Wheat Leaves in Argentina Phoma sorghina (Sacc.) Boerema, Dorenbosch & van Kesteren is plurivorous, ubiquitous and common in the tropics and subtropics, causing diseases of cereals and other Gramineae and forage crops (Punithalingham, 1985; Manavolta and Bedendo, 1999; Kumar and Kumar, 2000). Although P. sorghina has been found causing leaf spots in different hosts such as Agave americana, Gossypium hirsutum, Lycium halimifolium, Lycopersicum esculentum, Oryza sativa, Populus nigra, Sorghum spp. and Zea mays, the disease it causes to aerial parts of plants is of minor importance (White and Morgan-Jones, 1983). It causes considerable loss of seedlings of Macroptilum, Stylosanthes and Sorghum 238 A.E. Perelló through pre- and post-emergence death. The fungus has been found on or associated with sorghum grains in the humid Argentinian Pampa (Gonzalez et al., 1997), but there are no previous reports of its presence on wheat plants in Argentina and therefore the conﬁrmation of this fungus as a foliar pathogen of wheat in Buenos Aires Province is signiﬁcant. The ﬁrst occurrence of leaf spot diseases of wheat by P. sorghina was in the Buenos Aires Province of Argentina in 2002. The fungus was detected on samples from two localities, Olavarría and Los Hornos, on experimental ﬁeld plots with wheat cultivars, Buck Poncho and Buck Diamante. Diseased leaves were collected, stored in paper bags and transported to the laboratory. The pathogen was isolated from typical necrotic lesions on PDA Petri dishes. Fifteen plants of each of the cvs. Buck Arriero, Buck Poncho, Klein Cacique, Klein Estrella, ProInta Cinco Cerros, ProInta Elite, ProInta Granar, ProInta Guazú, ProInta Imperial and ProInta Puntal were grown in plastic pots 12 cm in diameter and containing a potting mix of clay 21.2%, lime 56%, sand 22.8%, soil organic matter (SOM) % = 3.35; C % = 1. Plants were inoculated at the third expanded leaf stage and heading stage. Inoculum was prepared from 10-day-old cultures of P. sorghina growing on PDA by ﬂooding each sporulating plate with sterile distilled water and gently scraping the fungal colony with a sterile scalpel to dislodge conidia. The resulting suspension was ﬁltered once through a single layer of cheesecloth and the spore concentration was determined with a haemocytometer. The spore concentration in inoculum was adjusted to 1 × 106 conidia/ml. Control plants were sprayed with sterile distilled water. Leaves were sprayed to runoff with a manually operated sprayer. The inoculated plants and controls were kept in a moist chamber for 48 h and observed daily. The ﬁrst symptoms appeared 72 h after inoculation under greenhouse conditions and 15–50% of plants showed necrotic lesions 10 days after inoculation. The wheat cvs. showed different degrees of susceptibility to the pathogen. The cvs. Buck Arriero, Buck Poncho and Klein Cacique became severely infected with up to 40% of their leaf surface spotted. The cvs. ProInta Cinco Cerros, ProInta Elite, ProInta Granar and ProInta Imperial were slightly spotted, with 1–5% of their foliage covered by spots. The cvs. Klein Estrella, ProInta Guazú and ProInta Puntal were free of spots. Elongated necrotic yellowish to light-brown lesions could be observed on the upper surfaces of affected leaves of wheat cvs. Leaf spots later coalesced to form large irregular spots with yellow margins. Under high humidity, pycnidia developed within spots on leaves after 21 days. A gelatinous spore mass was extruded in cirri from pycnidia. No symptoms or spots were seen on the control plants. All wheat plants inoculated with P. sorghina in the greenhouse developed symptoms identical to those observed on naturally infected plants in the ﬁeld. In culture, the fungus developed dark, greyish colonies with dense aerial mycelium with abundant, solitary or sometimes aggregated pycnidia with characteristic beaks. Conidia were globose to ovoid or shortly cylindrical, usually straight, hyaline, unicellular 4–7 × 2 µm. Abundant chlamydospores and dictyochlamydospores were observed. Newly formed chlamydospores quickly became covered with a black coating that obscured their brown colour. The identiﬁcation was conﬁrmed by the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. One representative isolate of P. sorghina has been lodged in the CBS culture collection with the accession number 112525. Cephalosporium gramineum Nisikado & Ikata on Wheat Leaves in Argentina Cephalosporium stripe is a disease of cereals that is sporadic in its distribution and occurrence but can cause severe yield losses when it occurs. The disease is found most consistently in areas where frost heaving, resulting from ﬂuctuating winter temperatures, heavier soils and higher soil moisture damages roots (Bruehl et al., 1976). Cephalosporium stripe is caused by Hymenula cerealis (synonym C. gramineum). This fungus New and Emerging Fungal Pathogens is slow growing in culture and probably in nature, too. It produces tiny conidia on sporodochia in the saprophytic stage on wheat straw, but as a parasite it invades the vascular system, where it interferes with water movement. It is the only true vascular parasite known to attack wheat. Hosts C. gramineum attacks most winter cereals, but especially wheat. It invades several grasses (Bromus, Dactylis, Poa) and probably was indigenous to the region in native grasses. Until now in Argentina, it has been detected on Bromus and wheat plants only. Disease symptoms Cephalosporium stripe is ﬁrst observed in the spring as distinct yellow stripes on leaf blades, sheaths and stems. The stripes may contain thin brown streaks (necrotic vascular tissues) surrounded by yellow. Frequently, a yellow stripe on the leaf blade continues as a single brown line down the leaf sheath. Nodes are darker than normal on diseased plants and, when cut lengthwise, the inner nodal tissue is brown in colour. Plants are stunted and the heads are white and sterile. If any seed is set, it is usually shrivelled. Diseased plants have a scorched appearance when hot weather accentuates moisture stress. The fungus survives for as long as 4–5 years in undecomposed infested straw. In 2004, on a non-tilled wheat assay sown at the Julio Hirschhorn Experimental Station, belonging to the Facultad de Ciencias Agrarias y forestales de la Universidad Nacional de La Plata, Argentina, chlorotic stripes, which became necrotic, were observed on leaves of wheat cultivar Buck Biguá. Samples were collected and remitted to the laboratory at the CIDEFI. Morphocultural and morphobiometrical characteristics allowed identiﬁcation of the fungus as C. graminearum. The fungus was cultured on 239 2% PDA Petri dishes. Wheat plants of the same cultivar were inoculated with a pathogen conidial suspension by a manual sprayer under greenhouse conditions. Similar symptoms developed from 7 to 21 days after the inoculation. Occurrence of Cladosporium herbarum on Wheat Leaves in Argentina Cladosporium on wheat was reported to be a common and mild parasite affecting dead or half-dead plant tissues in association with some other fungi. It often appears on the ear heads, causing a greenish black mouldy growth on the affected parts (Wiese, 1987). It was not found to cause severe symptoms on leaves and stems of wheat, but there were some reports pointing out that moist and shady conditions could favour the occurrence of outbreaks of the disease on leaves (Arya and Panwar, 1955). During the past 5 years, leaf spot symptoms on wheat cultivars Buck Pingo, Buck Biguá, Buck Brasil and Buck Poncho growing in the north-east of the Buenos Aires Province, were commonly observed. Infected leaf samples were collected during September– November in an extensive survey conducted in 2002. Samples were collected from different cultivars in farmers’ ﬁelds and one experimental research station across the wheat region of Buenos Aires and Entre Ríos Provinces, in ﬁve of the eight sites surveyed (Los Hornos, Nogoyá, Olavarría, Tandil and Victoria). In most of the plants, leaves showed symptoms sufﬁciently similar to those described for the complex of necrotrophic foliar pathogens, i.e. D. triticirepentis (Died.) Shoem., S. tritici Rob. in Desm., A. triticimaculans Simmons & Perelló and B. sorokiniana (Sacc.) Shoem., suggesting that any of these might have been involved. C. herbarum was commonly isolated from affected tissues of the samples collected and it has emerged as a widespread and serious foliar disease. The fungus was previously reported in Argentina (Marchionatto, 1948) as C. herbarum Link. 240 A.E. Perelló var. cerealinum Sacc. on leaves and spikes of wheat and other grasses without describing the symptoms in detail. Since then, there have been no other reports of this disease on wheat leaves in Argentina. Diseased leaves were collected, stored in paper bags and transported to the laboratory. The pathogen was isolated from typical necrotic symptoms. On PDA Petri dishes, morphobiometrical and cultural studies of the fungus were conducted on single spore colonies grown in Petri dishes containing PDA, cultured at 20 ± 2°C under cool-white ﬂuorescent light supplemented with near UV with a 12 h photoperiod. Inoculation experiments to conﬁrm pathogenicity were performed in the greenhouse at 15–25°C and 80% relative humidity. Fifteen plants of each of the cultivars Buck Biguá, Buck Brasil, Buck Pingo and Buck Poncho were grown in plastic pots (12 cm diameter) with a standard potting mix. Plants were inoculated when they had reached the third expanded leaf stage and heading stage. Inoculum was prepared from 10-day-old cultures of C. herbarum (isolates Ch101 and Ch500) growing on PDA and was obtained by ﬂooding each sporulating plate with sterile distilled water and gently scraping the fungal colony with a ﬂame-sterilized scalpel to dislodge conidia. The conidial suspension was ﬁltered once through a single layer of cheesecloth and spore concentration was determined with a haemocytometer. The inoculum consisted of 2 × 105 conidia/ml. Control plants were sprayed with sterile distilled water only. Leaves were sprayed to runoff with a manually operated sprayer. The inoculated plants and controls were kept in a moist chamber for 48 h. The plants were observed periodically. The ﬁrst symptoms appeared between 72 and 92 h after inoculation. All cultivars showed susceptibility to both of the isolates tested. Between 12 and 75% were necrotic 10 days after inoculation. Reisolation from leaves with lesions was performed and the isolates were compared morphologically with those used for inoculation to fulﬁl Koch’s postulates. In natural ﬁeld infections, amphigenous, irregular yellowish brown spots were observed that especially affected the basal leaves rather severely. They often conﬂuenced and elongated, developing progressively from lower to upper leaves. The margin of top leaves became brittle when dried and the tissue tore. When lesions spread over the leaf surface, they caused the death of the entire leaf. A velvety olivaceous grey mould of spores and mycelia developed on the surface of the infected tissue, forming dense tufts. Microscopic examination revealed the presence of conidiophores more or less erect, septate, sparsely branched; the spores are often in chains of 2 or 3, subcylindric, pale olive, 1-(2-3) septate, 10–15 × 4–7 µm. The teleomorph, M. tulasnei (Jancz.) Rothers was not seen. All wheat plants inoculated with C. herbarum in the greenhouse developed symptoms identical to those observed on naturally infected plants in the ﬁeld. No differences in degree of infection were noted among the cultivars. Nevertheless, adult plants showed more severe symptoms than younger ones. No symptoms were observed in the control non-inoculated plants. Isolation from symptomatic tissue has consistently yielded cultures of C. herbarum. The fungus sporulated on the diseased tissue in the Petri dishes. Comparison of morphological characteristics of C. herbarum isolates revealed no differences between ﬁeld- and glasshouse-produced spores, according to the shape and size of conidia. The isolates of C. herbarum have been lodged in the culture collection of the CIDEFI (Centro de Investigaciones de Fitopatología), Facultad de Ciencias Agrarias y Forestales de la Universidad Nacional de La Plata, Buenos Aires, Argentina, with the accession numbers 111-01, 209-02, 210-02, 212-02 and 215-02. Inoculation studies proved that C. herbarum was the cause of this outbreak on wheat in Argentina. In the past few years, the increased incidence of the disease may be related to new cultural practices (reduced tillage, nitrogen fertilization, irrigation), the use of new germplasm and favourable weather conditions. This contributed to a major spread, not only of C. herbarum but also of the foliar complex of necrotrophic pathogens in general. New and Emerging Fungal Pathogens The fact that other wheat cultivars apart from those checked may also be susceptible to the pathogen shows the importance of conducting thorough research to determine the reactions of those cultivars currently used in the Argentinian cropping area. Pyricularia grisea on Wheat Leaves in Argentina During 2006/2007, P. grisea (Cooke) Sacc. was detected for ﬁrst time in the north-east region of Argentina, a non-traditional, marginal culture area. Plants of wheat cv. Klein Chajá presented spots or blight symptoms on all aerial parts. Isolates and pathogenicity tests conﬁrmed the presence of P. grisea associated to blight symptoms on leaves, sheets and spikles. Pyricularia grisea also affects rice and other gramineous spontaneous species in the region. Simultaneously, during 2007, the fungus was isolated from wheat plants cvs. Cronox, Baguette 11, ACA 304 and BioINTA from Bragado, Baradero, Rojas, Alberti and 9 de Julio localities from the typical wheat area in Argentina. Conclusion Wheat (T. aestivum L.) cultivars currently grown in Buenos Aires Province, Argentina, are susceptible to different leaf spotting fungi. Surveys conducted over several years in Argentina have determined that the main fungi involved in this disease complex are M. graminicola (Fuckel) Schroet. in Cohn (anamorph S. tritici Roberge in Desmaz.) (leaf spot), Cochliobolus sativus (Ito & Kuribayashi) Drechs. ex Dastur (anamorph B. sorokiniana (Sacc.) Shoemaker (spot blotch) and P. tritici-repentis (Died.) Drechs. (anamorph D. tritici-repentis (Died.) Shoemaker) (tan spot). A. triticimaculans Simmons & Perelló was ﬁrst described on wheat in Argentina in 1996 and commonly observed since then, like others members of the infectoria complex. During surveys of wheat commercial ﬁelds from 2001 to 2002 to now 241 on the northern, central and southern prairies of the Buenos Aires Province, a dramatic difference was observed between fungal diseases. A high incidence of D. tritici-repentis was commonly observed in all locations analysed. Tan spot, caused by the fungus P. triticirepentis (Died.) Drechs. (anamorph D. triticirepentis) (Died.) Shoem., is a major disease of wheat (T. aestivum L.) worldwide (Wiese, 1977; Hosford, 1975). The disease has a fast growth in the Southern Cone region of South America including Argentina, where it was found for the ﬁrst time affecting wheat crops in the north-central region of the Buenos Aires Province in the early 1980s (Annone, 1985). Subsequently, tan spot has gained predominance among other wheat diseases in most wheat-growing areas in the country (Kohli et al., 1992; Annone, 1997; Carmona et al., 1999; Perelló et al., 2003). Tan spot was often observed throughout the growing season and was the most common leaf disease observed each year, in 72.6% of all wheat ﬁelds in 2001 and 90.4% in 2002. Additionally, strains of Alternaria spp. from wheat plants with symptoms of leaf blight sufﬁciently similar to those described for tan spot were collected from 11 localities. All isolations corresponded to the A. infectoria species group (Simmons, 1994; Simmons, personal communication, 2001; Perelló et al., 2002). Leaf blight of wheat caused by Alternaria spp. isolates was not signiﬁcant in complex of wheat foliar diseases in Argentina for many years but, currently, this pathogen has become a new problem in the Buenos Aires Province. Symptoms are often difﬁcult to distinguish in the ﬁeld from those caused by D. tritici-repentis (Died.) Shoem. Other pathogens for wheat, like C. herbarum, P. sorghina, C. gramineum, Pirycularia oryzae and A. tritici were registered for the ﬁrst time in Argentina (Perelló, 2007). The pattern of diseases produced by these phytopathogens in some areas of cultivation is changing drastically due, among other causes, to new market trends that induce changes in agricultural practices and the introduction of new crops. Monitoring these changes is important in order to 242 A.E. Perelló take appropriate and timely action to prevent disease dispersal. Information on the most common leaf spotting fungi would help to identify appropriate benchmarks for selecting for disease resistance in different environments. It would also help breeders to set priorities in the incorporation of disease resistance to the leaf spotting complex into adapted wheat cultivars. Moreover, widespread occurrence of these fungal diseases in the major wheatgrowing region of Argentina described warns regional breeders and pathologists to increase efforts to manage the spotting wheat leaf complex in order to avoid future epidemics. Acknowledgement The author is grateful for the ﬁnancial support of Project 11/A142 ‘Patógenos fúngicos del trigo y su posibilidad de biocontrol con microorganismos antagonistas en el marco de una agricultura sustentable’ of the Programa de Incentivos a la Docencia e Investigación de la Universidad Nacional de la Plata. References Agarwal, V.K., Chahal, S.S. and Mathur, S.B. (1993) Alternaria leaf blight. In: Mathur, S.B. and Cunfer, B.M. (eds) Seed-borne Diseases and Seed Health Testing of Wheat. Danish Government Institute of Seed Pathology for Developing Countries, Hellerup, Denmark, pp. 9–13. Annone, J.G. (1985) Presencia de la mancha tostada del trigo (Helminthosporium tritici – repentis). Carpeta de Production Vegetal Trigo. Tomo VII Informe No. 88, EEA, INTA Pergamino, Buenos Aires, pp. 1–9. Annone, J.G. (1997) Tan spot of wheat in Argentina: importance and disease management strategies. In: Duveiller, E., Dubin, H.J., Reeves, J. and McNab, A. 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Basu1,2 1Agriculture 2Department and Agri-Food Canada Research Centre, Lethbridge, Canada; of Biological Sciences, University of Lethbridge, Lethbridge, Canada Abstract Fenugreek (Trigonella foenum-graecum L.) is an annual legume crop cultivated in India, the Mediterranean region, China, parts of Africa, Europe and Australia and, in recent years, in North America. Although traditionally used as a spice crop, fenugreek has important medicinal and nutraceutical properties and is also grown as a forage crop in some countries. This multi-use crop has the potential to expand into new areas, as well as increase in the area where it is traditionally grown. Therefore, its reaction to biotic and abiotic factors that can limit its production deserves special attention. Although this review contains a discussion on all fenugreek diseases and insect pests, the main focus is on the causal organisms, symptoms and corresponding control measures for all of the major and minor fungal diseases affecting its productivity. It is interesting to note that only a few diseases have been reported to affect this crop adversely. The two major fungal diseases that affect fenugreek are powdery mildew caused by Erysiphe polygoni and Cercospora leaf spot caused by Cercospora traversiana. However, disease problems may change as this crop is grown more widely and with larger acreages outside of its natural area of adaptation. Ongoing vigilance in disease monitoring and development of new resistant varieties is needed to ensure productivity and usefulness of this crop in the future. Introduction Fenugreek (T. foenum-graecum L.) is an annual crop belonging to the legume family Fabaceae. Although widely cultivated in India, China, northern and eastern Africa, parts of Mediterranean Europe, Argentina and Australia (Acharya et al., 2006a), it was only recently introduced to North America (Acharya et al., 2006b). Fenugreek is a dicotyledonous, self-pollinated plant with trifoliate leaves, branched stems and white ﬂowers, which typically produces golden yellow seeds (Basu et al., 2008). It has two morphological forms of ﬂowering shoots, the common one bearing axillary ﬂowers and an indeterminate growth habit, whereas plants with blind shoots possess both axillary and terminal ﬂowers with a more determinate growth habit (Busbice et al., 1972; Fehr, 1993; Acharya et al., 2008; Basu et al., 2008). Although both closed and open type ﬂowers are reported, the majority of fenugreek ﬂowers belong to the closed  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 245 246 S.N. Acharya et al. type (Petropoulous, 2002; Acharya et al., 2008). Fenugreek is capable of ﬁxing atmospheric nitrogen in the soil. The plants require a minimal amount of nitrogen for growth, reducing the need for nitrogen fertilizers to supplement crop growth and facilitating use of the plant in crop rotations. Fenugreek is considered a dryland crop; water requirements are low, making cultivation of fenugreek an increasingly attractive alternative to producers in regions with limited water supply. Use of fenugreek in arid and semiarid environments can reduce the cost of irrigation, reduce the potential for eutrophication of surface water and limit contamination of groundwater sources (Basu et al., 2004; Acharya et al., 2008). According to Acharya et al. (2004), dryland adaptation of fenugreek was a major consideration for introducing it as a forage crop for growth in the temperate climates of western Canada. Well-drained, loamy soils are most favourable for the crop (Rosengarten, 1969; Acharya et al., 2008), while heavy and wet soils are known to restrict fenugreek growth (Petropoulos, 1973; Acharya et al., 2008) and increase susceptibility of the plant to disease. Although fenugreek is known primarily as a spice crop used especially in India and Mediterranean regions for cooking, the species name foenum-graecum refers to ‘Greek hay’, highlighting its use as a forage crop in early years (Acharya et al., 2006b). Fenugreek also has been referred to as a medicinal herb, both in Indian Ayurvedic and traditional Chinese medicines (Tiran, 2003). Its leaves and seeds have been used extensively to prepare extracts and powders for medicinal use like wound healing and promotion of lactation in weaning mothers (Basch et al., 2003; Tiran, 2003; Acharya et al., 2007a). The medicinal value of fenugreek comes mainly from three chemical constituents; i.e. steroidal sapogenins, galactomannans and isoleucine (Acharya et al., 2006a, 2007a, 2008). Steroidal sapogenins are often used as a raw precursor for the production of steroidal drugs and hormones such as testosterone, glucocorticoids and progesterone (Fazli and Hardman, 1968) and are also effective in the treatment of hypercholesterolaemia (McAnuff et al., 2002). Fenugreek galactomannans appear to aid in the control of type 2 diabetes in both animals (Raju et al., 2001; Tayyaba et al., 2001; Puri et al., 2002; Vats et al., 2002, 2003) and humans (Sharma et al., 1996; Puri et al., 2002). The amino acid isoleucine is a precursor of 4-hydroxyisoleucine, which is known to regulate the secretion of insulin in animals (Broca et al., 2000) and also making it potentially useful in the control of diabetes. Since fenugreek can be used for multiple purposes (e.g. as a spice, forage crop, eco-friendly dryland crop and for medicinal and nutraceutical applications), there is interest in cultivation of the plant in new biogeographical areas of the world (Acharya et al., 2008; Basu et al., 2008). Acharya et al. (2006b) have described fenugreek as a traditional ‘Old World’ crop with signiﬁcant potential for use in the ‘New World’. ‘Tristar’ is the ﬁrst North American variety of fenugreek released by a research group in Canada (Acharya et al., 2007c). As a result of its increased economic and industrial importance, those involved in fenugreek production need to become more aware of the diseases that can affect both yield and quality of the plant adversely. In many fenugreekgrowing areas, infectious and non-infectious diseases are becoming an important production constraint because of their ability to cause variation in crop yield and quality (Basu et al., 2006a). When fenugreek is introduced to a new biogeographical area, new diseases may emerge that can cause a reduction in productivity, and even crop losses (McCormick and Hollaway, 1999; Fogg et al., 2000). This review looks at major and minor diseases of fenugreek reported worldwide, as well as emerging fungal diseases that are increasing in importance, to use of the plant commercially. Diseases of Fenugreek Fenugreek production is affected by both biotic and abiotic agents. Abiotic diseases or disorders are non-infectious and are often Diseases of Fenugreek caused by a deﬁciency in nutrients, extremes in temperature, moisture, soil acidity or alkalinity, an excess of certain micronutrients within the soil and toxic impurities in the atmosphere (Petropoulos, 2002; Acharya et al., 2008). For example, Sinskaya (1961) reported yellowing of some fenugreek plants under ﬁeld conditions due to mineral deﬁciencies in boron, magnesium, manganese or potassium. Physiological diseases resulting from abiotic agents can lead to premature death of the plant and loss of forage and seed yield. In western Canada, exposure of fenugreek crops to very dry and hot conditions has resulted in stunted growth and yellowing, with occasional loss of leaves from the plant. Diseases caused by living or biotic agents (pathogens) are often infectious (Acharya et al., 2008). The most important diseases of fenugreek are caused by plant pathogenic fungi. Bacterial diseases are next in degree of importance, followed by viral diseases (AAFRD, 1998; Fogg et al., 2000; Prakash and Sharma, 2000; Petropoulos, 2002; Weiss, 2002; Jongebloed, 2004). While information on speciﬁc pests and diseases damaging fenugreek is limited, in general, insects and pathogenic organisms that attack other common legume crops grown in the vicinity of fenugreek, such as alfalfa, can also attack fenugreek (Basu et al., 2006b). Viral diseases Bean yellow mosaic virus, alfalfa mosaic virus, cowpea mosaic virus, soybean mosaic virus, pea mosaic virus, potato virus A and Y and clover vein mosaic virus are all common viral infections of fenugreek (Petropoulos, 2002). Bhasker and Summanwar (1982) reported mosaic wilt on fenugreek. Flexuous rod-shaped viruses like bean yellow mosaic potyvirus (Singh, 1969) and pea streak carlavirus (Hagedorn and Walker, 1949) have also been reported on fenugreek. These viral diseases have been associated with moderate losses of fenugreek seed and forage yield (Table 19.1). 247 Bacterial diseases McCormick and Hollaway (1999) found that infection of fenugreek with Pseudomonas syringae resulted in bacterial blight. First reported in Victoria, Australia, infection with these bacteria caused small isolated patches, to entire crop loss in the ﬁeld. Fogg et al. (2000) reported the same disease on fenugreek in New Jersey, USA. It has also been suggested that the bacterium Xanthomonas alfalfa can infect fenugreek (Petropoulos, 2002), leading to loss in productivity (Table 19.1). Nematode diseases Various nematodes, typically not identiﬁed as a problem for other crops, can damage fenugreek roots (Weiss, 2002; Jongebloed, 2004). The soilborne nematode Meloidogyne incognita has been shown to cause root rot and the death of immature fenugreek plants in Australia (Jongebloed, 2004) (Table 19.1). However, it is interesting to note that fenugreek has also been reported to have some anti-nematicidal properties. Zia et al. (2003) reported that decomposed seeds of fenugreek caused a marked reduction in population densities of the soil nematode M. javanica, which causes root-knot development in mungbean. Decomposed seed and aqueous extracts of fenugreek were also able to enhance plant height and shoot fresh weight in mungbean. Insect pests In Australia, insects such as thrips, podborers and Heliothis can cause serious damage to forage yield in fenugreek (Lucy, 2004). Basu et al. (2006b) reported that in southern Alberta (Canada), a low level of insect pests such as Lygus bugs and, to a lesser extent, alfalfa plant bugs and aphids had been observed in fenugreek ﬁelds. In addition, the researchers reported that western ﬂower thrips (especially severe under greenhouse conditions), alfalfa looper, alfalfa weevil 248 S.N. Acharya et al. Table 19.1. The major non-fungal diseases of fenugreek reported worldwide. Disease groups Viral diseases Bacterial diseases Nematode diseases Insectmediated diseases Tolerant varieties/ genotypes Causal organisms Country reported Bean yellow mosaic virus England Potato virus A NA* Fluorescent, Ethiopian NA Cowpea mosaic virus NA NA Potato virus Y Tobacco etch virus Pea streak virus NA NA NA NA NA NA Pea mosaic virus Soybean mosaic virus NA NA NA NA Alfalfa mosaic virus NA NA Tomato black ring virus NA NA Clover vein mosaic virus NA NA Pseudomonas syringae pv. syringae Xanthomonas alfalfa Meloidogyne incognita Australia NA Australia NA NA Brunt, 1972; Petropoulos, 1973, 2002 Schmelzer, 1967; Anonymous, 1968 Vidamo and Conti, 1965; Anonymous, 1968 Schmelzer, 1967 Petropoulos, 2002 Hagedorn and Walker, 1949; Anonymous, 1968 Petropoulos, 2002 Quantz, 1968; Schmelzer and Wolf, 1971 Quantz, 1968; Schmelzer and Wolf, 1971; Latham and Jones, 2001 Quantz, 1968; Schmelzer and Wolf, 1971 Quantz, 1968; Schmelzer and Wolf, 1971 McCormick and Hollaway, 1999; Fogg et al., 2000 Petropoulos, 2002 Jongebloed, 2004 Lygus keltoni, L. elisus, L. borealis and L. lineolaris Adelphocoris lineolatus Acyrthosiphon pisum Frankliniella occidentalis Sitona sp. Hypera postica Autographa californica Aphis craccivora Canada Tristar Basu et al., 2006a Canada Canada Canada Canada Canada Canada India, West Asian countries India, West Asian countries Australia, India, the Mediterranean region India Sudan India India India India Tristar Tristar Tristar Tristar Tristar Tristar NA Basu et al., 2006a Basu et al., 2006a Basu et al., 2006a Basu et al., 2006a Basu et al., 2006a Basu et al., 2006a Weiss, 2002 NA Weiss, 2002 NA Weiss, 2002; Lucy 2004 NA NA NA NA NA NA Weiss, 2002 Weiss, 2002 Weiss, 2002 Weiss, 2002 Weiss, 2002 Weiss, 2002 Myzodes persicae Scirtothrips dorsalis Tetranychus cucurbitae Pachymerus pallidus Diacrisia oblique D. orichalcea Prodenia litura Maruca testulalis Note: *NA = not available. References Diseases of Fenugreek and Sitona sp., were attracted to standing fenugreek crops under ﬁeld conditions in western Canada. Aphis craccivora and Myzodes persicae have caused damage to fenugreek crops from west Asia to India, while various Thysanoptera (thrips), including Scirtothrips dorsalis, have been found on almost all fenugreek crops grown from the Mediterranean to India (Petropoulos, 2002; Weiss, 2002). There have also been reports of mite (Tetranychus cucurbitae) attacks on fenugreek in India (Weiss, 2002). Pachymerus pallidus, a seed beetle, which attacks a wide range of crops, is a major pest of fenugreek in the Sudan (Weiss, 2002). A number of polyphagous caterpillars belonging to the order Lepidoptera, including Diacrisia oblique, D. orichalcea and Prodenia litura, and especially the mung moth (Maruca testulalis), have been reported to affect fenugreek in India (Weiss, 2002) (Table 19.1). Common Fungal Diseases of Fenugreek The two most common fungal diseases infecting fenugreek are Cercospora leaf spot and powdery mildew (AAFRD, 1998). Powdery mildew on fenugreek, caused by E. polygoni, can seriously reduce crop yield (Prakash and Sharma, 2000; Jongebloed, 2004) and has the potential to affect biomass and seed yield in crops grown under moist agroclimatic conditions in North America. In Australia, yield of fenugreek was seriously affected by blight caused by C. traversiana and wilt caused by Fusarium oxysporum and Rhizoctonia solani (Jongebloed, 2004). The pathogen C. traversiana is spread by contaminated seed and is now found in many countries, ranging from India to Europe, eastern Africa including Ethiopia and in several countries in South America; it is slowly becoming a major fenugreek disease concern (Weiss, 2002). Other well-known fungal diseases observed to be associated with fenugreek are collar rot, leaf spot and pod spot diseases (Petropoulos, 2002) (Table 19.2). In India, 27 species of fungi have been isolated from fenugreek seeds (Prabha and 249 Bohra, 1999). Time of sowing can inﬂuence the damage caused by an infection. For example, in Haryana, India, seed sown in mid October as compared with the end of November exhibited a 30% reduction in damage caused by E. polygoni and Leveillula taurica (Sharma, 1999). Downy mildew caused by Peronospora trifoliorum and spring black stem and leaf spot caused by Phoma pinodella have recently become more common (Lakra, 2002, 2003; Bretag and Cunnington, 2005). Several leaf diseases causing varying degrees of damage generally or in speciﬁc seasons, including rust due to Uromyces anthyllidis, have been reported in India (Weiss, 2002). Root and collar rots caused by Rhizoctonia spp., typically R. solani and Alternaria spp., often A. alternata, can damage individual crops (Weiss, 2002). Antifungal activity for fenugreek has also been reported in the primary literature (El-Gizawy et al., 2000). Lupin and fenugreek seed extracts signiﬁcantly suppressed Pythium damping-off of cucumber and tomato seedlings, as well as radish damping-off caused by R. solani. Moreover, application of seed extracts had a signiﬁcant positive effect on seedling growth of the vegetables tested (El-Gizawy et al., 2000). A detailed description of major and minor fungal diseases of fenugreek reported all across the globe and their prescribed control measures are outlined individually in the following sections. Cercospora leaf spot Cercospora leaf spot is a seedborne fungal disease, considered to be one of the most serious threats to fenugreek. This disease is capable of causing considerable economic loss (Leppik, 1959, 1960; Khare et al., 1981; Zimmer, 1984; Ryley, 1989). The Cercospora leaf spot of fenugreek has been reported all across the world and is most common in Australia, several eastern European countries, South America, North America, in the Near East and India (Voros and Nagy, 1972; Cook, 1978; Khare et al., 1981; Ryley, 1989). 250 S.N. Acharya et al. Table 19.2. The major fungal diseases of fenugreek reported worldwide. Name of the disease Pathogenic fungal species Country reported Cercospora leaf spot Cercospora traversiana India, NA Australia, Canada Collar rot Rhizoctonia solani India Leaf spot Ascochyta sp. UK Powdery mildew Oidiopsis sp. Downy mildew Peronospora trigonellae Israel, Ethiopia, England India Powdery mildew Leaf spot Leveillula taurica Pseudoperiza medicaginis Phoma pinodella Israel Tolerant varieties/ genotypes References Leppik, 1959, 1960; Khare et al., 1981; Zimmer, 1984; Ryley, 1989 TG-18, UM-20, Hiremath et al., 1976; Pusa Early Hiremath and Bunching Prasad, 1985; Raian et al., 1991; Petropoulos, 2002; Datta and Chatterjee, 2004 Fluorescent, Walker, 1952; Ethiopian Petropoulos, 1973 Fluorescent Palti, 1959; Rouk and Mangesha, 1963; Petropoulos, 1973 HM-346, HM-350, Lakra, 2002, 2003; HM-444 HAU, 2008 NA* Palti, 1959 NA Glaeser, 1961 Australia NA Erysiphe polygoni Israel, Ethiopia, India, Canada HM-350, HM-444, Fluorescent Rust Pod spot Uromyces trigonellae Heterosporium sp. Israel UK Charcoal rot Macrophomina phaseolina Sclerotinia trifoliorum Fusarium oxysporum Pakistan NA Kenyan, Moroccan NA Spring black stem and leaf spot Powdery mildew Root rot Fusarium wilt UK India, Sudan, Malta NA NA Bretag and Cunnington, 2005 Petropoulos, 1973; Zimmer, 1984; Prakash and Saharan, 2000; Basu et al., 2006a; HAU, 2008 Ubrizsy, 1965 Petropoulos, 1973 Haque and Ghaffar, 1992 Petri, 1934 Borg, 1936; Komaraiah and Reddy, 1986; Hashmi and Thrane, 1990; Bansal and Gupta, 2000 Note: *NA = not available. The causal organism for this disease is C. traversiana, a member of the Ascomycetes (Agrios, 1997). Several researchers have suggested that C. traversiana is the only species of the Cercospora infecting fenugreek (Cook, 1978; Ryley, 1989). Conidiophores of C. traversiana are dark, paler towards the tip, unbranched, rarely geniculate and rarely septate. These conidiophores develop in fascicles of 3–5 conidiophores per fascicle, with a length of up to 420 µm and width ranging from 3 to 5 µm (Ryley, 1989). The conidia are Diseases of Fenugreek hyaline, acicular, straight or slightly curved, apex rounded, base truncate and multicellular. The main source of overwintering inocula is plant debris, where sclerotia or stromata can form. Conidia germinate best at a high relative humidity and at a high temperature. They are dispersed mainly by rain-splash and to some extent by wind (Agrios, 1997). Cercospora leaf spot initially presents itself as circular, sunken lesions that appear bleached in colour, with narrow (1–2 mm) chlorotic halos on the surface of the leaves. These lesions expand rapidly as the infection progresses, producing necrotic areas. Each area of infection is sharply deﬁned, with most lesions surrounded by a characteristic yellowish halo. Lesion size is increased signiﬁcantly on mature leaves, where sporulation becomes evident, giving the lesions a whitish, velvet-like appearance (Zimmer, 1984). Severely infected plants are reported to have only a few leaves situated towards Lesions surrounded by yellowish halo and lesion size increases considerably: advanced symptoms 251 the apex of the plant (Agrios, 1997). Stem and pods also can become infected. Disease symptoms on pods include discoloured infected areas, as well as severely infected areas that can become shrunken and twisted (Zimmer, 1984). The life cycle of the pathogen is shown in Fig. 19.1. Control measures As the pathogen is often seedborne, seed treatment before planting has been an effective control measure in some cases (Leppik, 1960; Khare et al., 1981). However, selection of healthy seeds as planting material may also provide an effective control (Cook, 1978). Rotation with crops outside of the host range for the fungal pathogen C. traversiana may also be useful. It appears that prevention of seed contamination by treating plants when the pathogen is ﬁrst detected will likely be the best approach to limiting spread Mycelia giving rise to conidiophores and conidia Lesions spread as infection advances Conidia disseminated by rain-splash and wind Appearance of circular, sunken bleached lesions on leaves: initial symptom Infected seed giving rise to an infected plant Life cycle of Cercospora leaf spot on fenugreek host plant Conidia infecting healthy plants and healthy leaf tissue Fungus overwintering in plant debris may produce sclerotia or stromata Fungus overwintering in non-treated seeds Fig. 19.1. The life cycle of Cercospora traversiana on fenugreek host plant. 252 S.N. Acharya et al. of this pathogen. Spraying the plants with fungicides such as benomyl, chlorothanolin, Bordeaux mixture, mancozeb and maneb has been suggested as an effective chemical control measure (Agrios, 1997). Collar rot Collar rot is another important fungal disease of fenugreek and has been reported in all parts of India (Hiremath et al., 1976; Hiremath and Prasad, 1985; Raian et al., 1991). The causal organism for this disease is a member of the Basidiomycetes. R. solani reduces yield of fenugreek causing foot-rot and damping-off where freshly emerged seedlings fall over and die (Petropoulos, 2002). The vegetative mycelium of R. solani is colourless when young but turns brown on maturity. The mycelium consists of hyphae partitioned into distinct individual cells by a septum consisting of a doughnut-shaped pore (Ogoshi, 1987; Alexopoulos et al., 1996). R. solani survives as sclerotia in the soil and on plant tissue, and as mycelia by colonizing soil organic matter as a saprophyte. Sclerotia and/ or mycelia present in the soil and/or on plant tissue germinate to produce fungal hyphae that can attack the subsequent year’s crop (Alexopoulos et al., 1996). The pathogen primarily attacks below-ground plant parts such as the root system, but is also capable of infecting other parts such as green foliage, seeds and hypocotyls. The most common symptom of the disease is damping-off (Petropoulos, 2002). Most of the severely infected seedlings may die at pre- or post-soil emergence stages. The infected seedlings may develop reddish-brown cankers on roots and stems at or near ground level (Anderson, 1982; Adam, 1988; Agrios, 1997). Control measures Cultivation of resistant varieties has been suggested as the best control measure for the disease (Prasad and Hiremath, 1985). According to Haque and Ghaffar (1992), seed dressing and soil drenching with Rhizobium meliloti, Trichoderma banatum, T. harzianum and T. pseudokonongii can control the infection effectively. The Gram positive bacterium Bacillus subtilis can also be used effectively as a biological control agent for R. solani (Tschen and Kou, 1985; Tschen, 1987). Prasad and Herimath (1985) demonstrated that carbendazim could be used as a seed and dry soil mix fungicide and that captan also could be used to drench the soil and kill the fungus. Leaf spot Leaf spot is another seedborne disease of fenugreek that is caused by fungal pathogens of the Ascochyta sp. belonging to the Ascomycetes (Walker, 1952; Petropoulos, 2002). The fungus attacks the leaves, stems and pods of fenugreek, reducing both yield and quality severely. It can survive in the soil, on infected seed and on crop residues. The pathogen is disseminated by both wind and rain-splash (Agrios, 1997; Petropoulos, 2002). Irregular brown to black spots with distinct margins are detected on infected leaves. As the disease progresses, the leaves on the plant may die and fall off. Infected seeds have round, dark brown lesions. Seedlings from infected seeds start rotting from the point of seed attachment and rotting advances towards the stem and taproot; subsequently, the young seedlings die (Petropoulos, 2002). Cool, moist weather is favourable for rapid dissemination and growth of the fungus (Anonymous, 1970; Agrios, 1997). Control measures Cultivation of tolerant genotypes is a good idea to avoid rapid infestation of the fungus (Agrios, 1997). To protect fenugreek plants from primary infection, seeds can be treated effectively with benlate, while to prevent secondary infection, use of a frequent foliar spray containing benlate is also recommended (Petropoulos, 2002). Fusarium wilt Fusarium wilt of fenugreek is caused by the fungus F. oxysporum, an Ascomycete that Diseases of Fenugreek has been reported by several investigators across the world (El-Bazza et al., 1990; Borg, 1936; Hashmi, 1988; Bansal and Gupta, 2000; Petropoulous, 2002). The pathogen F. oxysporum is both seed and soilborne (Komaraiah and Reddy, 1986; Hashmi and Thrane, 1990; Bansal and Gupta, 2000; Pierre and Francis, 2000). The pathogen can remain in infested soils for up to 10 years. Dissemination of the pathogen occurs through seed, soil and infested plant parts (Pierre and Francis, 2000). Fusarium wilt ﬁrst appears as a slight clearing in veins found on the outer portion of younger leaves, followed by downward drooping of the mature leaves. At the seedling stage, plants infected by F. oxysporum may wilt and die soon after the symptoms appear. In mature plants, vein clearing and downward drooping of the leaf are often followed by stunting, yellowing of the lower leaves and subsequent wilting of leaves and young stems. Marginal necrosis of the infected leaves, rapid defoliation and ﬁnally death of the entire plant typically follow (Agrios, 1997). Browning of the vascular tissue is strong evidence of Fusarium wilt infestation. Furthermore, symptoms become more apparent on mature plants during the period between blossoming and fruit maturation (Jones et al., 1982; Smith et al., 1988). 253 infections. Fungal spores produced within leaf spots during the growing season are spread by splashing rain (Petropoulos, 1973). Symptoms of the disease become visible at the third stage of pod development and can be seen as dark brown to black spots on the pods that extend to produce a dark olive, velvet-like cover. Initially, localized spots of infection elongate transversely to the pod axis but with time spread over the pod surface and transform into more rounded to oblong lesions. These spots are also visible on the stems, but are rarely found on the plant leaves. Petropoulos (2002) suggests that the fungus does not enter into the seeds as the mycelium of the fungus is not buried deeply in the epidermis of the pod and that contamination of fenugreek seeds by this fungus takes place speciﬁcally during the threshing process. Control measures Hot water treatment of the seeds before planting is efﬁcient to remove the fungus from the seeds (Pirone et al., 1960). Resistant cultivars tolerant to that fungus have been suggested as the best way to restrict rapid dissemination of the disease on a standing crop effectively (Petropoulos, 2002). Control measures Some effective means of controlling F. oxysporum include disinfection of the soil and planting of the seeds with thiram or captan, crop rotation with non-hosts of the fungus, or use of resistant cultivars (Singh, 2001). Pod spot Petropoulos (1973) ﬁrst investigated and described this disease in fenugreek and identiﬁed Heterosporium sp., an Ascomycete, as the causal agent. Heterosporium medicaginis is the only species of Heterosporium that has been reported to be pathogenic to legumes (Karimov, 1956). The fungus overwinters on dead leaves. Spores spread from old plant debris to initiate new plant Spring black stem and leaf spot This disease of fenugreek has been reported in Australia by Bretag and Cunnington (2005). These investigators also identiﬁed P. pinodella, an Ascomycete previously known as A. pinodella (Jones, 1927), as the causal agent of the disease. This observation is supported by another investigation conducted by Boerema et al. (2004). Phoma sp. has been isolated from the seeds of fenugreek in Egypt, India, Nepal, Pakistan, Sri Lanka, Sudan and Syria (Hashmi, 1988), suggesting that the organism is not new to fenugreek crops. The pycnidia of the fungus are more or less globose, glabrous, ovoid to ellipsoid and usually aseptate (Punithalingam and Gibson, 1976; Bretag and Cunnington, 2005). Onfroy et al. (1999) reported that the length 254 S.N. Acharya et al. of conidia could range from 7.3–9.6 µm. The pathogen persists as pycnidia and mycelia in plant debris. It is dispersed mainly by splashing rain and to some extent by wind. Numerous small, irregular-shaped, dark brown to black leaf lesions surrounded by small chlorotic areas often appear as disease symptoms on the leaves, petioles and stems of the growing plant. Elongated black lesions may also develop on the taproot (Bretag and Cunnington, 2005). Infected plants become stunted with a mild chlorosis. In cases of severe infection, most of the leaves turn completely yellow, wither and the taproot system becomes completely girdled with sharp lesions (Bretag and Cunnington, 2005). Control measures Bretag et al. (2006) suggested practising crop rotation, destruction of infected plant portions and chemical seed treatments to control primary infections by the disease efﬁciently. When the fungus was restricted effectively at the primary infection level, it did not spread to advanced stages in most disease trials conducted (Bretag et al., 2006), suggesting that restricting primary infection is the key to control of the disease. Powdery mildew Powdery mildew is one of the most common and serious fungal diseases of fenugreek, (a) Fig. 19.2. affecting both biomass and yield (Petropoulos, 2002; Basu et al., 2006a). Powdery mildew is most commonly found in hot and humid tropical and subtropical areas, as well as in temperate to subtemperate regions (Palti, 1959; Rouk and Mangesha, 1963; Prakash and Saharan, 2000; Basu et al., 2006a). On the basis of recent observations, Basu et al. (2006a) suggested that powdery mildew could become a serious disease problem in North America, where fenugreek is a recent crop introduction. Although some investigators have reported Oidiopsis sp. as the causal organism (Rouk and Mangesha, 1963; Petropoulos, 1973), the majority of investigators from across the globe consider E. polygoni (an Ascomycete) as the causal organism for the disease (Zimmer, 1984; Prakash and Saharan, 2000; Bretag and Cunnington, 2005; Basu et al., 2006a). The conidiophores of the fungus are simple and erect and the corresponding conidia are unicellular, hyaline in colour, ellipsoidal to cylindrical in shape (Agrios, 1997; Nyvall, 1999; Basu et al., 2006a) (Figs 19.2 and 19.3). The conidiophores vary in size from 32.5 to 65.6 µm × 9.7 to 13.6 µm, whereas dimensions of the conidia are 22.6–48.4 µm × 12.4– 20.8 µm (Basu et al., 2006a) (Fig. 19.2). The pathogen survives mostly by developing cleistothecia in diseased plant debris. They survive in soil until the next season. Ascospores are released after the disintegration of the wall of the asci. The ascospores ﬁrst infect the lower and older leaves in the next season. The spores are carried by the (b) Light microscopy images of Erysiphe polygoni conidiophores (a) and conidia (b). Diseases of Fenugreek (a) (b) (c) (d) 255 Fig. 19.3. Scanning electron microscopy (SEM) images of healthy fenugreek upper leaf surfaces [Top, (a) and (b)] compared to powdery mildew (caused by Erysiphe polygoni) infected upper leaf surface [Bottom, (c) and (d)]. Left images were magniﬁed 500×, while the right images were magniﬁed 1000×. wind to new hosts. The pathogen is also known to survive as a mycelium (Sharma, 2005). Powdery mildew is one of the easier diseases to identify on plants as its symptoms are quite distinctive. The disease can be identiﬁed easily by the presence of white to grey powdery masses or distinct circular to ellipsoidal patches on both the upper and lower surfaces of the leaves (Fig. 19.4), on pods but rarely on ﬂowers, and by the strong odour emitted by the infected plants. During the initial stages of an infection, fungal patches appear isolated or in scattered patches which coalesce as the infection progresses. At ﬁrst, leaves near ground level are infected, after which the whole plant can become covered with the fungus over a 256 S.N. Acharya et al. Fig. 19.4. Comparison of healthy fenugreek upper leaﬂet surfaces (centre) with infected leaﬂets from the same plant. short period of time (Fig. 19.5). The upper surface of the leaves typically bears more fungal structures and spores than the lower surface (Fig. 19.4). Severely infected leaves become irregular in shape, dry and shrivelled, resulting in stunted growth of the whole plant (Basu et al., 2006a) (Figs 19.4 and 19.5). Although Zimmer (1984) ﬁrst identiﬁed powdery mildew infecting fenugreek in North America, Basu et al. (2006a) reported the ﬁrst major in-depth investigation of powdery mildew as a major disease of fenugreek in North America based on trials that were conducted at different locations and under different physico-geographic conditions and variable climatic factors on the west and east coast of North America and the mid interior of Canada. The life cycle of the pathogen is presented in Fig. 19.6. Control measures Petropoulos (1973) and Avtar et al. (2003) reported variation in the sensitivity of fenugreek genotypes to powdery mildew; hence, use of resistant varieties has been strongly recommended to avoid disease infestation. Basu et al. (2006a) demonstrated that application of tilt 250E-propiconazole or milgo-ethrinol (28% at 2.5 ml/l) and captancaptane (50%) or benlate-benomyl (50% at 2.0 g\l) could control the disease at a satisfactory level, whereas Petropoulos (1973) showed that spraying with dinocap (8–10 oz a.i/acre in 100 gals) could also control the disease. Conclusions Fenugreek is affected mostly by seedborne fungal diseases. From our experience, and other reports of fenugreek disease, it is clear that powdery mildew and Cercospora leaf spot are the two most important diseases currently affecting this crop. These diseases can reduce the production and quality of fenugreek crops signiﬁcantly all across the globe. Other minor fungal diseases of fenugreek, namely collar rot, leaf spot, Fusarium Diseases of Fenugreek 257 Fig. 19.5. Spread of powdery mildew infection on a fenugreek potted plant in the greenhouse at Lethbridge Research Center, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada. wilt, pod spot, spring black stem and leaf spot, and downy mildew, have the potential to become major fenugreek diseases in many areas, including subtemperate climatic zones. Use of resistant cultivars and application of suitable chemical agents are suggested by most research groups as potential control measures against infection and spread of fungal diseases. Although certain Internet sites do make widely optimistic claims about effective biological control of fenugreek fungal diseases, they do not have strong evidence from multi-location and multi-year trials to support their claims and so are not included in this review. Fenugreek is being cultivated in many new areas as it becomes more widely recognized as a multiple-use crop. Development of new fenugreek cultivars and improvement of existing cultivars with disease resistance using conventional plant breeding methods (Acharya et al., 2007b) and advanced plant biotechnological approaches (Laroche, 2007) could be a good strategy to prevent emergence of new fungal diseases for this crop. Susceptibility of a plant to disease is determined by the genetic relationship between the plant and the pathogen. The relationship between genes of the host and the pathogen can determine disease expression in the host. Genetic resistance in plants is considered a major form of biological control of disease and is possibly the most cost-effective and environmentally friendly way to control crop diseases. Resistant cultivars have been used effectively to control diseases in many crops. However, development of resistant cultivars takes time and so work should continue in the interim to ﬁnd chemical and other biological control agents to protect the crop from disease and other pest damage. It should also be noted that disease control measures should not only be cost-effective but also need to be environment friendly and socially acceptable. 258 S.N. Acharya et al. Healthy green tissue infected, most prominent symptoms on leaves and shoots Ascospores and conidia disseminated by air Conidia Asci containing ascospores Cleistothecium Mycelia finally generates condiophores bearing conidia Infected buds giving rise to shoots and leaves completely covered by fungal mycelia Production of cleistothecia on plant parts Life cycle of powdery mildew on fenugreek host plant Fungus overwintering in non-treated dormant buds and seeds Young plant infected Fig. 19.6. The life cycle of Erysiphe polygoni on fenugreek host plant. Acknowledgements The authors express their sincere thanks to Mr Byron Lee, Research Technician, Electron Microscopy and Image Analysis Laboratory, Lethbridge Research Centre (LRC), Agriculture and Agri-Food Canada (AAFC) for his help in taking all the light microscopy and SEM images, Mr Doug Friebel, Technician, Forage Lab, AAFC, LRC for his help with ﬁeld trials. 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Adiver and Kumari Oilseeds Scheme, Main Agricultural Research Station, University of Agricultural Sciences, Dharwad, Karnataka, India Abstract During the past few years, pathogens in oilseed crops have been recognized as major forces causing economic losses, with identiﬁcation of certain important ones based on their symptoms, etiology and also ecological zones. Recent research has helped by developing new resistant varieties and other effective management strategies. This chapter describes the causal organisms, symptoms and management of diseases of oilseed crops like castor, groundnut, safﬂower, sesame and sunﬂower. Cultural practices for managing certain diseases have been pinpointed. Critical stages for growth of some foliar diseases, namely rust, early and late leaf spot of groundnut, blight and mildew of sunﬂower, fusarial wilt of safﬂower and castor, have been identiﬁed. Recommendations are given on controlling various diseases by chemical, botanical and other effective and eco-friendly methods. Oil is an essential household commodity required for food and daily use. Certain oils are used as therapeutic agents and are in much demand for their conversion into energy or potential biodiesel. Losses to the tune of 20% in certain oilseed crops need our utmost attention. Various fungal diseases of groundnut, sunﬂower, safﬂower, sesame and castor are described. Disease management with fungicides and other available methods are illustrated. Groundnut Groundnut, known as poor man’s almond, contributes about 38% to the oilseed pool of India. India is the second largest producer of groundnut after China. The crop is subjected to attack by numerous pests and pathogens. Among foliar fungal diseases, early and late leaf spots, commonly called ‘Tikka’ disease, and rust are economically important. Early leaf spot caused by Cercospora arachidicola Hori The perfect stage of the fungus is Mycosphaerella arachidis. In India, losses in yield due to leaf spots have been estimated to be in the range of 15–59%. Besides the loss in pod and kernel yield, the value of fodder is also affected adversely. Lesions are subcircular in shape and measure 1 to over 10 mm. On the upper surface of the quadrifoliate  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 263 264 S.S. Adiver and Kumari leaves, the lesions appear dark brown, while on the lower surface they are a lighter shade of brown. The early leaf spot usually has a light to dark brown centre and a yellow halo. These are oval to elongate in shape and have more distinct margins than the late leaf spot lesions. The early leaf spot pathogen survives through conidia on affected plant debris in soil, or through conidia being carried on the pod shell. Disease management Tolerant varieties like GPBD-4, ICGV-86590, ICGS-44, M-335, BG-3 and M-522 can be grown wherever early leaf spot is severe. Intercropping pearl millet/sorghum with groundnut (1:3) is useful in reducing the intensity of early leaf spot. Foliar spraying of carbendazim (0.05%) + mancozeb (0.2%), chlorothalonil (0.2%), difenaconazole (0.1%), tebuconazole (0.1%) or hexaconazole (0.1%) is recommended 2–3 times at 2- to 3-week intervals starting from the initiation of the disease (Adiver et al., 1995). Late leaf spot caused by Phaeoisariopsis personata (Burk. and Curt.) Van Arx The perfect stage of the fungus is M. berkeleyii W.A. Jenkins. Late leaf spot is more severe in the southern and central parts of India. Dark brown to black, circular to subcircular lesions, measuring 1–6 mm diameter appear on the lower surface of the quadrifoliate, where most sporulation occurs. The lesions are black in colour and fruiting structures occur in concentric rings on the lower leaﬂet surface, giving lesions a slightly rough appearance. The ambient temperature required is between 25 and 30°C. Prolonged leaf wetness hours and high relative humidity (> 80%) favour infection and disease development. Conidia are disseminated by the wind and insects, leading to secondary infection. Disease management Deep burying of crop residues in the soil and removal of volunteer groundnut plants are important measures in reducing the primary source of infection. Resistant/tolerant varieties like Girnar-1, RG-141, IGV-87160, ICGV-86590, ICGV-86325, R-8808, GPBD-4, Kadari-4, Co-3,4, M-335 and BG-3 can be grown wherever late leaf spot is severe. Foliar spraying of carbendazim (0.05%) + mancozeb (0.2%), chlorothalonil (0.2%), difenaconazole (0.1%) or hexaconazole (0.1%) is recommended 2–3 times at 2- to 3-week intervals, starting from the initiation of the disease (Adiver et al., 1995). Rust caused by Puccinia arachidis Speg. Rust of groundnut is prevalent throughout India; however, it is more severe in the southern states. In India, losses in yield due to rust alone have been reported in the range of 10–52%, depending on the variety. Rust can be recognized readily as orange-coloured pustules (uredinia) that appear on the lower leaﬂet surface and rupture to expose masses of reddish-brown urediniospores. Pustules appear ﬁrst on the lower surface and, in highly susceptible cultivars, the original pustules may be surrounded by colonies of secondary pustules. Disease management Early sowing in the ﬁrst fortnight of June is recommended to avoid incidence. Use of resistant/tolerant varieties like Girnar-1, ICGV-87160, ICGV-86590, DRG-12, ALR-2,3, Co-4, ALR-1, ICGS-5 and DRG-17 is recommended. Spraying mancozeb (0.2%), tridemefon (0.1%), chlorothalonil (0.2%), difenaconazole (0.1%), tebuconazole (0.1%), hexaconazole (0.1%) or cyproconazole (0.1%) 2–3 times at 2- to 3-week intervals starting from the initiation of the disease helps to control the disease. Seed and seedling diseases of groundnut Pre-emergence seed rot and post-emergence seedling mortality are of common occurrence. The disease develops either from the Fungal Diseases of Oilseed Crops fungi already present in the seed or result from direct invasion of seeds and seedlings by soil fungi. Among seedling diseases, collar rot, root rot and stem rot are of economic importance and are known to reduce yields by 25–50%. Collar rot caused by Aspergillus niger van Tiegh In India, collar rot, also known as crown rot or seedling blight, is prevalent in almost all groundnut-growing states, causing 28–50% losses. The diagnostic symptoms are preemergence rotting of seeds and rotting of hypocotyls, but the most common cause of loss is early post-emergence seedling blight. The ﬁrst symptom in emerged seedlings is usually a rapid withering of the entire plant or its branches. Lesions develop on the stem below the soil and spread upwards along the branches. The dead and dried branches are easily detached from the disintegrated collar region. 265 in both dry weight and oil content of groundnut kernels. The ﬁrst symptom is partial or complete wilting of the stem or branch that is in contact with the infected soil. The leaves turn brown and wilt, but remain attached to the plant. The pathogen has a wide host range. S. rolfsii can colonize either living plant tissue or plant debris. Deeply buried sclerotia survive a year or less, while those near the soil surface remain viable for many years. Disease development occurs when soil moisture is 40–50%. Generally, when the temperature remains between 29 and 32°C during the day and seldom drops below 25°C during the night, the disease develops more favourably. Disease management Deep ploughing, early sowing and close planting is recommended and rotation of groundnut with cotton, maize, sorghum and pearl millet. Seed treatment with T. viride/ T. harzianum at 4.0 g/kg seed or seed treatment with carbendazim/captan at 2–3 g/kg seed is suggested. Disease management Avoiding deep sowing (not more than 5 cm), mixed cropping with moth bean in alternate rows, deep tillage and early sowing of crop is recommended. Soil application of neem cake/castor cake at 500 kg/ha, seed treatment with Trichoderma harzianum/T. viride at 4.0 g/kg seed, bacterization of groundnut seeds with strains of ﬂuorescent pseudomonads or seed treatment with carbendazim (1.0 g/kg), mancozeb (2.0 g/kg seed) or chlorothalonil/captan (2.0 g/kg) is suggested. Stem rot caused by Sclerotium rolfsii Sacc. The stem rot pathogen has a very wide host range. In India, stem rot, also known as Sclerotium wilt, occurs in all groundnutgrowing states and is particularly severe in Maharashtra and Gujarat. In India, 27% or more yield loss has been reported. S. rolfsii also causes indirect losses such as reduction Sunﬂower Sunﬂower (Helianthus annuus var. macrocarpus (DC) Cockerell) is an important edible oilseed crop. It belongs to the family Asteraceae. The sunﬂower head is composed of about 1000–2000 individual ﬂowers. The fertile disc ﬂorets bear the seed, which is white, black or striped grey and black. The seeds contain 40–50% oil and 50–55% meal, which contain high protein (35%), calcium, phosphorus, iron, potassium and vitamin E. The sunﬂower is a native of North America, where it is used in dyes, food preparations and medicines. Alternaria leaf spots and blight of sunﬂower Different species of Alternaria, namely A. alternata, A. helianthi, A. zinniae, A. helianthiacola, A. leuconthemi and A. tenuissima 266 S.S. Adiver and Kumari have been reported to cause the disease. Among these, A. helianthi (Hansf.) Tubaki and Nishihara are economically important. The disease has been reported to cause a huge grain yield loss in Australia, where yield potential of 1.25 t/ha of the crop was reduced to 0.1 t/ha (Allen et al., 1981). In Karnataka, India, the disease occurred in epidemic form in 1987, with a disease incidence of 95–100% (Hiremath et al., 1990). The disease is caused by A. helianthi (Hansf.) Tubaki and Nishihara. The mycelium of the fungus is septate, rarely branched, brown and 2.5–5.0 µm in breadth. Conidiospores are cylindrical and yellow to black grey, with one to 11 transverse septa and a few longitudinal septa. The conidia measure in the range of 40–110 × 8–28 µm, with an average of 74 × 19 µm. Disease management Summer deep ploughing reduces the inoculum level in the soil, which is present in plant debris as dormant mycelium; altering the date of sowing in order to reduce disease pressure and sowing during August– September onwards is suggested. Following spacing 60 × 30 cm under irrigated and 45 × 20 cm under rainfed conditions is recommended. Use of resistant varieties like GP-145, AH-303, BSH-1 is suggested. Foliar sprays of mancozeb (0.2%), chlorothalonil (0.2%), difenaconazole (0.1%) or tebuconazole (0.1%) can prevent the crop from Alternaria blight. Rust of sunﬂower The pathogen Puccinia helianthi Schw. is a macrocyclic, autoecious fungus and it produces all the ﬁve stages on sunﬂower only. The disease has been reported to cause variable yield losses in the crop, depending on variety, environmental conditions and time of the outbreak of the disease in the crop season. Early infection of the variety ‘Sunrise’ and S-37-338 showed 17% and 68% less yield, respectively. Under ﬁeld conditions, the disease usually starts at ﬂowering stage, when the crop attains a dense canopy. The disease appears in the form of small cinnamon brown-coloured uredia on the lower surface of the lower leaves. In severe conditions, younger leaves, stems, petioles and ﬂoral parts are also infected. When the crop reaches physiological maturity, most of the uredia are converted to telia and are dark brown in colour. Disease management Altering the date of sowing reduces disease pressure. Removal of self-sown plants, crop rotation for at least 3 years and deep summer ploughing are recommended to reduce the inoculum level in the soil. Use of resistant varieties like SH-41, SH-187, PH-1, 2, 3, 4, 7 and 8, ICI-306, 331, PAC-36, 9128 and systemic fungicides containing triazoles, namely hexaconazole and cyproconazole (0.1%), are found suitable under ﬁeld conditions. Downy mildew of sunﬂower Downy mildew causes heavy yield losses in sunﬂower-growing countries of the world. A serious outbreak (80–90%) of the disease was recorded in the Red River Valley of North Dakota and Minnesota (USA) during 1970, resulting in a reduction of about 50% yield, with a loss of about US$0.5m. Later, it spread to many European countries, then to Asia. This spread was mainly through the seed trade. In India, the disease ﬁrst appeared during 1984, in experimental plots of the Regional Research Station, Latur, particularly during September–October. Later, it spread to many areas of Maharashtra (Mayee, 1989), Karnataka and Madhya Pradesh (Agarwal et al., 1991). Causal organisms are Plasmopara halstedii, P. perennis and P. patens. The sporangiophores, measuring 150–750 µm, are monopodially branched almost at right angles and bear zoosporangia singly at the tips of the branches. Zoosporangia produced from leaves are elliptical with an apical papilla and measure 17–30 × 15–21 µm. The zoosporangia from Fungal Diseases of Oilseed Crops 267 roots are uniform, pyriform to oval with 1–3 papillae and 36–66 × 39–40 µm. The sporangia germinate at 5–28°C, with the optimum temperature being 16–18°C. Zoosporangia formed at 27°C show 86–95% germination. Oospores are formed in the intercellular spaces of roots, stem and seeds and measure 27–32 µm. The fungus causes damping off, systemic infection and local lesions on leaves and basal root or stem galls, depending on the stage of infection during plant growth. Damping-off occurs either as pre- or post-emergence under damp and cool weather at seedling stage and gives poor plant stand (Goosen and Sackston, 1964). Systemically infected plants remain stunted with chlorotic leaves. soft and pulpy, with superﬁcial whitish to blackish mycelium on the head. Under severe conditions, rotting spreads to the ﬂower stalk and the head drops off. Sometimes, the seeds from the rotted head shed and those that remain on the head have a bitter taste. Disease management Regulatory measures: the pathogen is seedborne and exhibits races; therefore, a quarantine measure has been imposed to check the movement of virulent races from endemic areas to other countries. Use of the resistant variety LDMRSH-1 and seed treatment with metalaxyl MZ 72 WP at 5–6 g/kg or apron 35 SD at 5–6 g/kg seed is suggested. Safﬂower (Carthamus tinctorius L.) is one of the rabi season oilseed crops cultivated in medium to heavy textured soils, mainly in Maharashtra, Karnataka and Andhra Pradesh, India. Being a crop mostly of the poor smallholder, it came to be recognized as an edible oilseed crop because of its superior role over animal fats and other vegetable oils, resulting in a boom in the cultivated area under the crop. Rhizopus head rot of sunﬂower Alternaria leaf blight of safﬂower The disease is caused by three different Rhizopus spp., namely R. nigricans, R. arrhizus and R. oryzae. The fungal colony is cottony-white to brown in R. arrhizus, while it is cottonywhite turning brownish-grey to blackish-grey in R. oryzae and R. nigricans. The optimum temperature for the growth of R. arrhizus, R. oryzae and R. nigricans is reported to be 37°C (thermophyllic), 30°C and 22°C, respectively. The disease causes severe yield losses, particularly in wet weather conditions. The disease has no effect on seed size but it reduces seed weight. Affected seeds become scurfy with discoloration of the hull and partial to complete discoloration of the nut meal and the quality of the oil is affected because of off-ﬂavours. The disease ﬁrst appears as brown, water-soaked irregular spots on the back of the ripening head, usually adjacent to the ﬂower stalk. The spots enlarge and turn Leaf blight caused by A. carthami Choudhary is the most destructive disease of safﬂower in India, appearing in a severe form wherever the crop is grown and causing up to 90% reduction in crop yield and oil content of affected seeds. However, the pathogen is reported to increase signiﬁcantly the level of free fatty acids in the seeds (Heaton et al., 1978). Mycelium of the pathogen A. carthami is septate, inter and intracellular and dark coloured on maturity. Conidiophores are septate, unbranched, erect and brown to olivaceous brown, pale near the apex, measuring 15–85 µm × 6–10 µm, arising through the epidermis or stomata singly or in clusters. Conidia are light brown to translucent in shade, with/without a long beak, showing constrictions at the septa and borne singly or in short chains. The disease appears in seedlings on hypocotyls and on Disease management Management of insects by spraying endosulphan or diazinon at the onset of bloom and spraying of fungicide, i.e. carbendazim (0.1%), on completion of the ﬂowering stage is effective in controlling the disease. Safﬂower 268 S.S. Adiver and Kumari cotyledons as dark necrotic lesions up to 5 mm in diameter, which may sometimes result in damping-off. Spots, having concentric rings up to 2 cm in diameter, light to dark brown with the centre lighter in colour, are observed in mature plants on leaves and frequently coalesce into large irregular lesions. Disease management The disease can be managed by using seeds from early sown dry land crops and treating them with thiram and TPTH, captan 0.3% (Siddaramaiah et al., 1980). Hot water treatment at 50°C for 30 min is also found useful (Sastry, 1996). Bulb extract (1.0% w/v) of Allium sativum also shows promise in checking the disease. Varieties like EC-32012, JLA1753, C-2603, Co-1, C75-7218, HUS-524, 476, 305, 260, SSF-112, CTC-251, 248, 252, etc., are reported to exhibit a variable degree of tolerance to A. carthami infection. Fusarium wilt of safﬂower Wilt of safﬂower is caused by Fusarium oxysporum f.sp. carthami. Fusarial mycotoxins capable of causing mycotoxicoses have been reported as being produced in sufﬁcient quantities on infested seeds of safﬂower in storage (Ghosal et al., 1977). The disease manifests at all growth stages. It may cause pre-emergence death or delayed germination of seeds. Symptoms on seedlings during post-emergence are blackening at the collar region; chlorotic, small brown spots appear on cotyledonary leaves, which then shrivel, become brittle, sometimes get rolled and droop downwards; ﬁnally, the seedlings bend and die. Plants grown from infected seeds rarely survive beyond the seedling stage. In mature plants, lateral branches on one side may be killed, while the other half of the plant shows no disease symptoms. Such plants show partial recovery, but symptoms may reappear later. Sporodochial production on stems may also be visible. Flower head size is reduced in severely affected plants, less seeds are formed and many of them are small, distorted, black and chaffy (Chakrabarti, 1980; Sastry and Jayaraman, 1993). The mycelium of the pathogen F. oxysporum f.sp. carthami Klisiewicz and Houston is septate and branched conidia, straight or curved, often pointed at the tip with a rounded base and measure up to 10–36 µm × 3–6 µm. Microconidia are oval to elliptical, one-celled and measure up to 5–16 µm × 2.2–3.5 µm in size. Chlamydospores are single celled, smooth, faintly coloured, single or in chains and 5–13 µm × 10 µm in size. Four biotypes of the pathogen have been identiﬁed on the basis of the reaction of safﬂower differentials to its isolates (Sastry and Chattopadhyay, 1999). Disease management Seed treatment with carbendazim (1.0 g/kg), captan (2.0 g/kg), thiram (2.0 g/kg) or Trichoderma (4.0 g/kg) helps to avoid infection of the plant by the pathogen. Crop rotation with legumes like chickpea, cowpea and pigeon pea helps to manage the disease (Sastry and Jayaraman, 1993). Use of tolerant varieties HUS-3234, 3123, 305, BSF-3, CTV-53, etc., is recommended. Phytophthora root rot of safﬂower The mycelium of the pathogen Phytophthora drechsleri is hyaline, aseptate, branched and 4.5 µm wide. Sporangia are hyaline to faint in colour, thin-walled, non-papillate, pyriform to ovate, 34–38 µm × 15–24 µm in size and having zoospores measuring 10–20 µm in diameter. Oospores are spherical, smooth, thick-walled, yellow to bright brown and are 16–45 µm in diameter (Klisiewicz, 1977). Root rot of Safﬂower caused by P. drechsleri Tuck is reported to cause about 3% losses on average, although 80% losses have been observed in a few instances, particularly when grown under surface irrigation (Sastry, 1996). Safﬂower is affected by Phytophthora root rot at any stage from pre-emergence to maturity. Symptoms on seedlings of 2–3 weeks of age appear as water-soaked lesions with softening and collapse of cortical tissue of the lower stem, whereon the plants lodge, shrivel and die. Fungal Diseases of Oilseed Crops Disease management Draining out excess water from beds after irrigation and avoidance of monocropping may help to control the disease (Kolte, 1965). Use of resistant varieties US-10, Gila, Frio and VFR-1 is recommended. 269 in light soils during kharif and in heavy soils during the early rabi season. It occupies an area of 17.50 hundred thousand ha in India, with production of 587.1 thousand t. The overall productivity of this crop in India is 335 kg/ha. About 72 fungi have been reported on this plant in India (Vyas et al., 1984). Rust of safﬂower Uredosori of the obligate, autoecious, heterothallic, macrocyclic pathogenic fungus P. carthami (Hutz) Corda contain numerous globoid or broadly ellipsoid echinulate, light chestnut brown uredospores measuring 21–27 µm × 21–24 µm, thick-walled and 3–4 equatorial germpores (Singh, 1998). Safﬂower rust caused by P. carthami, is an important disease in India. It causes a stand loss of 55–97% in susceptible varieties with considerable yield loss, particularly if the infection starts early in the crop growth. The ﬁrst pathological phase of the safﬂower rust is seen in the seedling stage of the crop, when orange to yellow spots representing pycnia appear on cotyledons, which ultimately leads to drooping and wilting of the plants. With the development of uredospores and teliospores, the colour of the spots later changes to brownish black. The second pathological phase of the rust is uredia development on leaves, ﬂowers and fruits, where teliospores are formed later towards crop maturity when the atmospheric temperature rises (Schuster and Christiansen, 1952). Disease management Destruction of the infected host, crop debris and collateral host Carthamus oxycantha (Pohli weed) and crop rotation checks the disease to some extent. Three sprays of tridemorph (0.5%), thiophanate methyl (0.15%) or tridimefon (0.1%) are effective against safﬂower rust (Singh et al., 1997). Use of resistant varieties APPR-1 and APPR-3 is suggested. Sesamum Sesame (Sesamum indicum L.) is an important oilseed crop of India. It is grown mainly Phytophthora blight of sesame Phytophthora blight is caused by P. parasitica var. sesame. It was ﬁrst reported from India by Butler (1918). Now, it has become an important disease of sesame and has been reported from the Dominican Republic (Ciferri, 1930) and Argentina (Frezzi, 1950). In India, it was severe in Madhya Pradesh Rajasthan, Uttar Pradesh and Gujarat (Vasudeva, 1961; Verma, 2002). This disease has caused 66% losses in Gujarat (Kale and Prasad, 1957) and 79.8% in Central Madhya Pradesh (Singh et al., 1976). It may cause even 100% loss under the most favourable conditions for infection to occur severely at seedling stage. Disease occurs on all the aerial plant parts. The symptoms of the disease appear as brown, water-soaked spots on the leaves of seedlings at a very early stage. Gradually, the spots increase in size. Under favourable weather conditions, the whole leaf rots and becomes black. Rotting progresses further and the whole stem is rotted. Frequently, the attack on the seedling starts at the collar region and gives damping-off like symptoms. The cottonywhite growth of the fungal mycelia appears on the lower side of the leaves and on pods under humid condition. Disease management Intercropping with soybean, castor, maize, sorghum and pearl millet in the ratio of 1:3 or 3:1 shows a low incidence of the disease, with a higher yield. Application of FYM alone or neem cake with inorganic fertilizer (N60, P40, K20) reduces the disease as compared to without FYM. Application of the species of Pseudomonas, Bacillus and Streptomyces, which are most active at 25–27°C 270 S.S. Adiver and Kumari at ﬁeld capacity moisture level, can be suppressive to Phytophthora species in soil. Seed treatment with vitavax (1.0 g/kg) and captan (2.0 g/kg) controls seedling disease effectively. Captan 75D is the best fungicide for reducing the disease, followed by thiram 75D. Fusarium wilt of sesame Fusarium wilt of sesame is quite serious wherever the crop is grown. In India, it has been reported from all the sesame-growing areas, such as Madhya Pradesh, Maharashtra, Andra Pradesh, Rajasthan, Haryana, Punjab, etc. The disease is quite serious when it starts in the early stages of crop growth. The causal organism is F. oxysporum f.sp. sesami. The fungus produces profuse light pink mycelial growth on PDA. Microconidia are hyaline, ovoid to ellipsoid, unicellular and produce abundantly even on the medium and are about 8.5 × 3.25 µm in size. The macroconidia are produced abundantly in sporodochia and size ranges from 35 to 49 × 4.5 µm. The chlamydospores are globose to subglobose, smooth or wrinkled and about 7–16 µm in diameter. The pathogen grows at a temperature range of 10–25°C, with an optimum temperature of 26°C and a pH of 5.6. The initial symptoms of the disease appear as yellowing of the leaves, which later droop and desiccate. On the infected plant, the leaves may show inward rolling of the edge and eventually may dry up. If the disease appears at the later stages of crop growth, the symptoms may appear on one side of the plant, resulting in partial wilting. Discoloration of the vascular system is conspicuous in the roots. Disease management Seed treatment with benlate (1.0 g/kg) and vitavax (1.0 g/kg) is most effective against wilt. Application of conidial dust of Gliocladium virens gave better disease control. Similarly, application of T. harzianum and T. viride in the ﬁeld also reduced the incidence of wilt signiﬁcantly. Soil drenching with antibiotic KB-8A isolated from B. polymyxa at a concentration of 13 µm/ml inhibited F. oxysporum f.sp. sesami completely (Hyun et al., 1999). Alternaria leaf spot of sesame The pathogen is A. sesame (Kawamura) Mohanty and Behera. The conidiophores of the pathogen are pale brown, cylindrical, erect, not rigid and arise singly with a size of 30–54 × 4–7 µm. Conidiophores produce conidia at the apex, which are in chains of one to two. The conidia are straight or slightly curved, obclavate, yellowish brown to dark brown in colour and measure 30–120 × 9–30 µm. The disease affects all the aboveground plant parts. The initial symptoms appear as small, brown, round to irregular spots on the leaf blade. Later, the spots enlarge and turn dark with concentric rings. On the lower surface of the leaves, spots are light brown in colour. The appearance of the disease at the seedling stage can cause postemergence damping-off. On capsules, small, brown spots appear which result in the formation of shrivelled and deformed seeds. Disease management Application of Bordeaux mixture (0.1%) and zineb (0.1%) has been reported to be effective. Application of mancozeb (0.2%) at the time of disease initiation is effective in managing the disease. Powdery mildew of sesame This disease is common, especially in South India. It has been reported that powdery mildew of sesame is caused by Oidium erysiphoides, Leveillula taurica (Lav.) Trnaud, Sphaerotheca fuliginea (Schlecht) Pollacci and Erysiphe cichoracearum DC. The disease causes considerable losses in yield, depending on the time of its appearance, as well as the intensity of the disease. Powdery mildew causes a loss of 42%; every 1% increase in disease intensity results in a yield loss of 5.63 kg/ha. Four different fungi have been reported to cause powdery mildew, Fungal Diseases of Oilseed Crops but in India E. cichoracearum is predominantly prevalent. Both conidia and ascospores on germination give rise to an abundant superﬁcial mycelium of uninucleate cells, which form a white coating on the leaf and send haustoria into the host. The disease normally appears after 45–60 days. The initial symptoms appear as dirty whitish fungal patches on the upper surface of the leaves. Later, these specks coalesce to cover the entire leaf and result in premature defoliation. Generally, it affects the leaves but in severe cases, the disease spreads to petioles and other plant parts. In severe infection, pods or capsules are shrivelled and produce smaller seeds. Disease management Two sprays of wettable sulphur (0.3%), dinocap (0.1%) or hexaconazole (0.1%) at 15-day intervals can help to control the disease. Cercospora leaf spot/white leaf spot of sesame Mycelium of Cercospora sesami Zimmerman is yellowish-white in colour and produces profuse conidiophores in culture. The conidiophores are olivaceous, septate, usually single but sometimes up to 10, epiphyllous, nodulase, thickened towards the tip, conidia with 7–10 septa and measure about 90–135 × 3–4 µm. Generally, the symptom of the disease appears at the time of ﬂowering, but the disease may also appear after 30–40 days after sowing. The initial symptoms of the disease are circular spots scattered on both leaf surfaces. These spots enlarge rapidly and become up to 5 mm in diameter. The spots are initially brown in colour with a whitish centre, but later they may be brown to dark brown in colour. The symptoms on petioles are visible as elongated lesions, whereas on capsules they are more or less circular and brown to dark brown in colour. Disease management Three sprays either of carbendazim (0.05%) and topsin M-70 (0.2%) at 10-day intervals 271 were best in controlling the disease. Resistant varieties recommended are BIC-7-2, Sidhi-54, Rewa-114 and Seoni Malwa. Castor Castor (Ricinus communis L.), belonging to the family Euphorbiaceae, is the most important non-edible oilseed crop of arid and semi-arid regions of India. Castor oil ﬁnds its application in the manufacture of a wide range of ever expanding industrial products, such as nylon ﬁbres, jet engine lubricants, hydraulic ﬂuids, dyes, detergents, soaps, ointment, greases, paints, varnishes, cosmetics and perfumes, etc. (Pathak, 2003). Castor is grown in tropical and subtropical climates; the major growing countries are India, China and Brazil. India occupies about 57% of the world castor acreage, but produces about 62% of world production. The major castor-growing states in India are Gujarat, Andhra Pradesh, Tamil Nadu and Orissa. Productivity is highest in Gujarat state because more that 90% of the cultivated area is covered by castor hybrids under irrigation. There are a number of diseases occurring on castor and the important ones are explained below. Alternaria blight of castor This is caused by A. carthami (Yoshii) Hansford. The disease appears on leaves, stem, inﬂorescence and capsules. At seedling stage, light brown spots ﬁrst appear on cotyledonary leaves, which become angular with age. Severe infection results in the death of young seedlings or foliar blight. Symptoms on adult plant leaves are brown, zonate and variable in size and usually surrounded by yellow halos. In the case of severe infection, premature defoliation occurs. Sunken spots develop on capsules on one side, which gradually enlarge to cover the whole capsule with fungal growth. Such capsules are smaller in size and have underdeveloped or wrinkled seeds with little oil content. In heavily infected ﬁeld crop, all the young racemes and even ﬂower primordia are killed. 272 S.S. Adiver and Kumari Disease management Foliar application of mancozeb (0.2%) at intervals of 15 days starting from the appearance of the disease is beneﬁcial. Judicious use of nitrogenous fertilizers also reduces the development of the disease. Botrytis grey rot of castor This is a very serious disease of castor as it affects the ﬂowers and capsules directly and the entire crop may be lost if there are continuous rains during capsule formation. The disease is conﬁned to only a few states in India and is serious in Andhra Pradesh and Tamil Nadu. It is caused by Botrytis ricini Godfrey. The disease is conﬁned to spikes or racemes. Generally, pale to olive grey coloured woolly growth of the fungus is observed on ﬂowers or capsules. The disease appears initially as small blackish spots, exuding a drop of yellow liquid. Fungal infection from these spots further spreads to racemes. The infected ﬂowers appear soft due to the profuse growth and sporulation of the pathogen. This later turns to grey masses covered with dusty powder, resulting in the rotting of capsules. The unripe seed becomes soft and mature ones hollow, resulting in a discoloured seed coat and loss in seed weight. collar rot, root rot and twig blight. The disease appears at different phases as collar rot, stem blight and root rot. Initially, the infected plant shows signs of water shortage. Within a week, the leaves and petiole droop and ﬁnally, within a fortnight, the entire plant dries up and can be pulled up easily. Collar rot phase is observed 30–40 days after sowing. Dark black discolorations are seen at the collar region of the plant, which gets sunken and later becomes abnormal. The affected tissue becomes shredded and weak and ﬁnally shows sign of wilting. Stem blight symptoms appear slightly later, due to aerial infection, as straw-coloured or brown depressed small lesions on the stem, usually at the nodes. The lesions increase in size by both upward and downward extension of the infection, resulting in a 2–20 cm oval-shaped necrotic area. The surface of the infected stem shrinks at this region and the plant breaks easily at this point. The affected spikes are discoloured, turn black and dry up in the course of time. Infected capsules become discoloured and drop off easily. In the case of the root-rot phase, the taproot shows signs of drying and the root bark shreds off easily. Rotting sometimes spreads partly above the ground. At an advanced stage, sclerotial bodies may be seen as minute black dots on the surface of woody tissues and in the pith region. Disease management Disease management Adoption of wider spacing with varieties having open racemes reduces the severity of the disease. Two prophylactic sprays of carbendazim (0.05%), one at 50% ﬂowering and the other soon after the appearance of the disease, reduces incidence of the disease effectively. Crop rotation with non-host crops and mixed cropping with moth bean can be helpful in reducing the disease. Infected plant material should be collected and burnt. Application of thiram (2.0 g/kg) or carbendazim (1.0 g/kg) as seed dresser along with spray and soil drench is recommended. Topsin M-70 has also been found effective for controlling root-rot disease in castor. Macrophomina root rot of castor Wilt of castor Macrophomina phaseolina (Tassi) Goid is reported to cause different symptoms on castor, namely seedling blight, dieback due to aerial infection, spike blight, stem blight, Wilt of castor is caused by F. oxysporum f. sp. ricini Nanda and Prasad. The extent of disease incidence has been up to 80% in Fungal Diseases of Oilseed Crops Russia (Moshkin, 1986). Losses in yield were realized in all cultivated castor hybrids in Gujarat and up to 85% incidence of the disease has been reported in North Gujarat (Dange et al., 1997). Young seedlings at the two- to three-leaf stage exhibit discoloration of hypocotyls and loss of turgidity, with or without change in colour. The mycelium penetrates the vascular system of the roots, stems and leaves causing necrosis, which leads to wilting and ﬁnally death of the plant. At the time of ﬂowering and spike formation stages, the disease is characterized by a gradual yellowing and shrivelling, with marginal and interveinal necrosis of leaves. Infected plants rarely bear seeds and such seeds are deformed and light in weight. Roots of wilted plants show blackening and necrosis, while in the case of partial wilted 273 plants, only one side of the root system is observed as being blackish and necrotic; the other side of the root system remains healthy. When the stem of the wilted plant is split open, a white cottony fungal growth is observed in the pith region, which then becomes blackish. Disease management Use of healthy seeds, crop rotation, summer deep ploughing and ﬁeld sanitation reduce the incidence of the disease. Use of bioagents like T. harzianum and T. viride have been screened for their antagonistic activity against castor wilt pathogen. Seed treatment (1.0 g/kg) and pre-sowing soil application of carbendazim at 3.0 kg a.i./ha with thiram (3.0 g/kg seed) is recommended. References Adiver, S.S., Anahosur, K.H. and Giri Raj, K. (1995) Triazole for control of foliar diseases of groundnut (Arachis hypogaea L.). Karnataka Journal of Agricultural Science 8(1), 65–68. Agarwal, S.C., Gupta, R.K. and Prasad, K.U.V. (1991) A case of downy mildew of sunﬂower in Madhya Pradesh. Journal of Oilseeds Research 8, 12–13. Allen, S.J., Kochman, J.K. and Brown, J.F. (1981) Losses in sunﬂower yield caused by Alternaria helianthi in Southern Queensland. Australian Journal of Experimental Agriculture and Animal Husbndry 21, 98–100. Butler, E.J. (1918) Fungi and Diseases in Plants. Thacker Sprink and Co., Calcutta, India, 547 pp. Chakrabarti, D.K. (1980) Studies on the Fusarium wilt of safﬂower incited by Fusarium oxysporum f. sp. carthami. Indian National Science Academy B 46, 120–121. Ciferri, R. (1930) Phytopathological survey of Santo Damingo, 1925–1929. Journal of the Department of Agriculture of Porto Rico 14, 5–44. Dange, S.R.S., Desai, A.G. and Patel, D.B. (1997) Management of wilt of castor in Gujarat State of India. In: Proceedings of the International Conference on Integrated Plant Disease Management for Sustainable Agriculture, 10–15 November 1997. IARI, New Delhi, 107 pp. Frezzi, M.J. (1950) The species of Phytophthora in Argentina. Revista de Investigaciones Agricolas Buenos Aires 4, 47–133. Ghosal, S., Biswas, K., Chakrabarti, D.K. and Basuchoudhary, K.C. (1977) Control of Fusarium wilt of safﬂower by mangiferin. Phytopathology 67, 548–550. Goosen, P.G. and Sackston, W.E. (1964) Biology of Plasmopara halstedii on sunﬂowers. Proceedings of the Canadian Phytopathological Society 31, 12 (Abst.). Heaton, T.C., Knowles, P.F., Mikkelsen, D.S. and Ruckman, J.E. (1978) Production of free fatty acids in safﬂower seeds by fungi. Journal of the American Oil Chemists Society 55, 465–468. Hiremath, P.C., Kulkarni, M.S. and Lokesh, M.S. (1990) An epiphytotics of Alternaria blight of sunﬂower in Karnataka. Journal of Agricultural Science 3, 277–278. Hyun, J.W., Kim, Y.H., Lee, Y.S. and Park, W.M. (1999) Isolation and evaluation of protective effect against Fusarium wilt of sesame plants of antibiotic substance from Bacillus polymyxa KB-8. Plant Pathology 15, 152–157. Kale, G.B. and Prasad, N. (1957) Phytophthora blight of sesamum. Indian Phytopathology 10, 38–47. Klisiewicz, J.M. (1977) Identity and relative virulence of some heterothallic Phytophthora species associated with root and stem rot of safﬂower. Phytopathology 67, 1174–1177. 274 S.S. Adiver and Kumari Kolte, S.J. (1965) Diseases of Annual Edible Oilseed Crops. Volume III: Sunﬂower, Safﬂower, and Niger Seed Diseases. CRC Press, Inc, Florida, 135 pp. Mayee, C.D. (1989) Downy mildew of sunﬂower in India: problem and approaches. In: Raychaudhari, S.P. and Verma, J.P. (eds) Review of Tropical Plant Pathology Vol 5, 181–192. Moshkin, V.A. (1986) Castor. Amarind Publishing Co Pvt Ltd, New Delhi. Pathak, H.C. (2003) Emerging trends in castor seed development. In: Proceedings of National Seminar on Castor Seed, Castor Oil and Its Value Added Products, 22nd May 2003. Solvent Extract Association of India, Ahmedabad, India, pp. 54–62. Sastry, K.R. (1996) Symptoms of wilt disease – clues for use in resistance breeding. In: Hegde, D.M., Raghavaiah, C.V. and Patil, D. (eds) Proceedings of Training Programme on Breeding Approaches for Improving Productivity of Safﬂower and Group Meeting as Heterosis Breeding in Safﬂower. Directorate of Oilseeds Research, Hyderabad, India, pp. 25–32. Sastry, K.R. and Chattopdhyay, C. (1999) Development of Fusarium wilt resistant genotype in safﬂower. Journal of Mycology and Plant Pathology 29, 276–277. Sastry, K.R. and Jayaraman, J. (1993) Eradication of Fusarium oxysporum f. sp. carthami from heavily infected safﬂower seed. Journal of Oilseeds Research 10, 277–281. Schuster, M.L. and Christiansen, D.W. (1952) A foot and root disease of safﬂower caused by Puccinia carthami. Phytopathology 42, 211–212. Siddaramaiah, A.L., Desai, S.A., Bhat, R. and Hegde, R.K. (1980) Eradication of Alternaria carthami Chowdhary, a seed borne pathogen of safﬂower. Pesticides 14, 22–23. Singh, B.P. Shukla, B.N. and Kaushal, P.K. (1976) Evaluation of sesamum varieties for their susceptibility to Phytophthora parasitica Dastur at Jabalpur, (M.P.). Jawaharlal Nehru Krishi Vishwa Vidyalaya Research Journal 10, 76–77. Singh, R. (1998) Spore stages and disease cycle of safﬂower rust (Puccinia carthami). Journal of Mycology and Plant Pathology 28, 168–170. Singh, R., Khare, M.N., Vyas, S.C. and Niranjan, V.K. (1997) Chemical control of safﬂower rust. Indian Phytopathology 50, 69–75. Vasudeva, R.S. (1961) Disease of sesamum. In: Joshi, A.B. (ed.) Sesamum Monograph. Indian Central Oilseeds Committee, Hyderabad, India, 287–291. Verma, M.L. (2002) Fungal and Bacterial Disease of Sesame and Their Management Challenges for the Millenium. Jyoti Publishers, New Delhi, pp. 161–192. Vyas, S.C., Kotwal, I., Prasad, K.V.V. and Jain, A.C. (1984) Note on seed borne fungi of sesamum and their control. Seed Research 12, 93–94. 21 Occurrence of Pyrenophora tritici-repentis Causing Tan Spot in Argentina M.V. Moreno1,2 and A.E. Perelló2,3 1Laboratorio de Biología Funcional y Biotecnología, CEBB, Facultad de Agronomía de Azul, Universidad Nacional del Centro de la Provincia de Buenos Aires, Buenos Aires, Argentina; 2Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET); 3Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, La Plata, Provincia de Buenos Aires, Argentina Abstract Wheat (Triticum aestivum L.) is currently considered as one of the most important crops worldwide. It can be affected by several diseases. However, only a limited number of them, like ‘tan spot’ resulting from the fungus produced by Pyrenophora tritici–repentis, cause serious problems to the crop and may be given special attention. Tan spot has signiﬁcant economic consequences. In recent years, the incidence of the disease has increased in many areas where wheat is cultivated, becoming a serious problem by causing losses of up to 70%. It has been found in a lot of countries worldwide: North Dakota, Nebraska and Kansas (USA), Canada, Australia, Asia, Pakistan, Czech Republic, Poland, Ukraine, Hungary, France, Denmark and Belgium. This disease has increased its incidence, prevalence and severity, particularly in the whole of the South Cone region in the last few years: Argentina, Brazil, Bolivia, Colombia, Ecuador, Peru, Paraguay and Uruguay. Tan spot is one of the most destructive and widespread problems of wheat production in Argentina. In this chapter, we summarize the knowledge of many and diverse contributions and we highlight what is known and unknown about the disease. Introduction Wheat is considered one of the most important crops of the world, along with rice, maize and potato. Humans consume around 75% of worldwide production (Wiese, 1987; Rajaram, 2001). In the period between 1970 and 2000, wheat yields rose at an annual rate of 2.3%; however, the area cultivated remained the same. The volume of wheat traded is greater than any other grain (Eikboir and Morris, 2001). Wheat has been one of the most important crops for the past 100 years in Argentina. Between 2005 and 2006, 2m ha were cultivated, giving a yield of 70–74 Mt (Inf. Económico de Coyuntura No. 261, 2006). The future of this production and South America’s participation in the international market depended on how Argentina and  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 275 276 M.V. Moreno and A.E. Perelló Brazil developed the crop. In Argentina, planting in the ﬁeld to exportation of the crop was dependent on the introduction of new technological developments. These developments include new cultivar composition, management of the crop and the management of future areas to expand yield (Eikboir and Morris, 2001). Fungal pathogens are the result of a combination of these factors (Klein, 2001). Management of these diseases requires speciﬁc knowledge and an increased ability to identify the fungus and techniques to reduce crop losses to a minimum (Kohli, 1995). In the last few years, minimum tillage has been considered advantageous to soil conservation, but it leads to a loss of available nutrients and a potential increase in necrotic pathogens whose saprophytic stage lives in the straw of the crop (Annone, 1985). Establishment of the crop under this management can be affected by pathogens of this type (Table 21.1). In Argentina, the increased incidence in leaf spot since the application of minimum tillage has been a cause for concern (Annone and Kohli, 1996). Tan Spot The ﬁrst time that tan spot was observed on wheat was in the 1920s in Japan (Hosford, 1981). In 1954, the ﬁrst loss (75%) was reported in Kenya (Gilchrist et al., 1984). In 1940, tan spot was reported for the ﬁrst time in the USA (Barrus, 1942). At the present time, the name of the fungus causing tan spot is reported with high frequency in the wheat-growing areas of the world (Conners, 1939; Tekauz, 1976; Watkins, et al., 1978; Sim and Willis, 1982; Loughman et al., 1998, Postnifova and Khasanov, 1998; Ali and Francl, 2001a; Sarova et al., 2002). In South America, tan spot has been observed in Colombia, Ecuador and Peru (Dubin, 1983). It has recently gained predominance among wheat diseases in the Southern Cone region of South America, comprising Argentina, Brazil, Chile, Paraguay and Uruguay (Kohli et al., 1992; Linhares and da Luz, 1994). The disease began to affect wheat crops noticeably in the north-central region of the Buenos Aires Province in the early 1980s (Annone, 1985, 1996). Since then, tan spot symptoms have been detected in most growing areas of the country. The disease is particularly prevalent and intense in the northern area of the Argentine wheatproducing region (central and northern Buenos Aires, southern Santa Fe, southeastern Cordoba and Entre Rios Provinces), where highly conducive environmental conditions and increasing use of minimum tillage have created a disease hotspot. In the region, the pseudothecia of the pathogen are formed on wheat residue left on the soil surface at crop sowing and/or early growth stages. Conidia are formed and released soon after the development of the ﬁrst symptoms on leaves (Annone et al., 1994). Wright and Sutton (1990) observed that when P. tritici-repentis was introduced in an area of wheat, it was dominant over other leaf pathogens. In Argentina, tan spot is one of the most important diseases, along with rust and head blight (Annone, 2006). The massive expansion of minimum tillage in Argentina has encouraged the establishment and development of this disease (Annone and García, 2004). Importance Tan spot is frequently observed in most farmers’ ﬁelds, often affecting the upper leaves at ﬂowering to early grain ﬁlling stages. Yield losses of between 9 and 50% have been observed by several authors (Hosford and Busch, 1974; Sharp et al., 1976; Rees et al., 1982; Rees and Platz, 1983). In South America, yield losses of around 40% were observed by Mehta and Gaudencio (1991) in Brazil and Kohli et al. (1992) reported wheat yield losses of between 20 and 70% in Paraguay and Argentina. Estimates of losses (10–20%) caused by the disease have been made by comparing fungicides protected with non-protected wheat plots (unpublished data Annone, 1996). Similar results were obtained by Galich and Galich Tan Spot in Argentina 277 Table 21.1. Diseases of wheat and the agent cause (Annone and Kohli, 1996). Pathogen Disease Xanthomonas campestris pv. undulosa Septoria tritici Drechslera tritici-repentis Fusarium graminearum Gaeumannomyces graminis var. tritici Bacterial stripe Spot blotch Tan spot Head blight Take-all (1994) in Marcos Juarez (Cordoba Province). They determined that losses due to tan spot associated with Septoria tritici blotch ranged between 6 and 13.5%. Tan spot is a complex disease that is dependent on its geographic location and the environmental conditions prevailing (de Wolf and Francl, 1998). The pathogen The tan spot fungus is an Ascomycota currently known as P. tritici-repentis (Ptr) (Died.) Drechs. It is a facultative pathogen whose asexual stage is Drechslera triticirepentis (Dtr) (Died.). P. tritici-repentis was isolated for the ﬁrst time from Agropyron repens in Germany and it was named Pleospora trichostoma by Diecke. In 1928, it was isolated from wheat by Nisikado (Nisikado, 1928), when it was named Helminthosporium tritici-repentis (= Drechslera tritici-repentis) (Hosford, 1981). The genera Pyrenophora Fr. was used frequently for some ascomycota parasitic on cereals and other grasses (Diaz de Ackerman, 1987). It was described by Fries in 1849 and cited by Shoemaker in 1961 (Shoemaker, 1962). In 1869, Fuckel noted the tendency of P. phaecomes to mature only after overwintering and found a Drechslera conidial stage of P. phaecomes (Shoemaker, 1962). In 1883, Saccardo used the presence of setae on the ascocarp of Pyrenophora and the absence of setae on the ascocarp of Pleospora to separate these two genera. In 1885, Winter (Shoemaker, 1962) included the species of both genera in Pleospora and in 1934, Drechsler (Shoemaker, 1962) agreed with Winter that the presence or absence of setae was not an important enough characteristic to separate the two genera. He emphasized the connection between Pyrenophora and the conidial stage Drechslera, as found by Fuckel for P. phaecomes. Drechsler too determined the connection between P. teres and D. teres, P. tritici-repentis and D. tritici-repentis, and P. bromi and D. bromi (Shoemaker, 1962). At the same time, Ito and Kuribayaski (1931) connected ﬁve species of Pyrenophora with the conidial stage of Drechslera. In 1949, Wehmeyer worked on the distinction in form and size of the Pleospora and Pyrenophora ascospores (Wehmeyer, 1949). In 1930, Ito described the genera Drechslera. In 1809, Link described the genera Helminthosporium, where species of Drechslera were included (Ito, 1930). In 1902, Diedicke (Drechsler, 1923) determined H. tritici-repentis as formae of H. gramineum. In 1923, Drechsler recognized H. teres, H. bromi, H. gramineum and D. avenae as unique species. In 1959, Shoemaker (1962) made the distinction between two subgenera, CylindroHelminthosporium, in which all the species have conidia germinating from all cells and Eu-Helminthosporium, in which all the species have fusiform conidia germinating from end cells only. In 1930, Ito (Shoemaker, 1962) proposed the name Drechslera for those species with cylindric conidia germinating from all cells, using as a type D. tritici-repentis. He used the name Bipolaris for those species whose conidia were fusiform, germinating from end cells only. In 1962, Shoemaker considered D. tritici-vulgaris as D. triticirepentis. Currently, the teleomorphic nomenclature of the fungus is P. tritici-repentis 278 M.V. Moreno and A.E. Perelló and the anamorph of the fungus is unanimously accepted as D. tritici-repentis. Morphological data can be found in Drechler (1923), Shoemaker (1962) and Wehmeyer (1954). Host–Parasite Interactions Symptomatology. On susceptible wheat leaves, P. tritici-repentis(Ptr) produces characteristic oval to diamond-shaped lesions. However, newly formed tan spot lesions cannot be separated reliably from those caused by other necrotrophic pathogens. Later, lesions elongate and develop a tan colour with a chlorotic halo and a small dark brown infection site. Chlorotic areas tend to coalesce on heavily infected leaves, especially on young plants, a symptom which leads to the disease name, ‘yellow leaf spot’ (Fig. 21.1). On resistant and partially resistant wheat, lesion size is reduced and chlorosis and necrosis may be absent (de Wolf et al., 1998). Lamari and Bernier (1989a) identiﬁed two different types of symptoms produced by the pathogen: tan necrosis and extensive chlorosis. However, they reported that the pathogen isolates could be characterized by their ability to induce tan necrosis and/or chlorosis. They grouped the isolates into four pathotypes based on the production of different symptoms on different lines. In this system, an unlimited number of isolates were designated as races 1, 2, 3, 4, 5, 6, 7, 8, (a) Fig. 21.1. 9, 10, 11 and 12 (Lamari and Bernier, 1989a,b; Lamari et al., 1995, 1998, 2003, 2005; Lamari and Gilbert, 1998; Ali and Francl, 2001a,b, 2002a,b). Races 9 and 10 have been identiﬁed in South America, which indicates that the Ptr population is heterogeneous in this area (Ali and Francl, 2002b). In Argentina, the race population structure is unknown and in 2007, Moreno observed that isolates obtained from Argentina produced three reaction types on cultivars of local and international wheat (Moreno, 2007). Actually, the isolates were inoculated on different wheat sets to determine the races present in Argentina. Ptr can also infect wheat seed during the grain-ﬁlling period (Schilder and Bergstrom, 1994). This disorder is called red smudge, because infected seed has a reddish discoloration (Valder, 1954). Disease cycle Dispersal and infection by Ptr can develop between 10° and 30°C with moisture between 6 h and 48 h (Larez et al., 1986; Hosford et al., 1987; Sah, 1994). These conditions are the reason why tan spot can occur all year round and which distinguishes it from the white head disease, but they all depend on environmental conditions (Carmona, 2003). The disease cycle of tan spot (Fig. 21.2) provides a convenient framework on which (b) Pyrenophora tritici-repentis produces characteristic oval to diamond-shaped lesions. Tan Spot in Argentina 279 Symptoms on leaf tissues Primary infection Conidia Secondary host Secondary infection Seeds Primary infection Ascas Fruiting bodies on stem Fig. 21.2. Disease cycle of Pyrenophora tritici-repentis, agent cause of tan spot of wheat. to explain our current understanding of the progress of the disease. The rate of progression through the disease cycle depends on the host and on temporal and environmental components of the pathosystem (de Wolf et al., 1998). The seeds, straw and collateral hosts are the principal source of inoculum of tan spot. The primary inoculum can travel long distances through the wheat-growing areas and is introduced into new areas by seeds. In the seed, the pathogen lives in the pericarp as mycelium and transmission to the rest of the plant is non-systemic (Schilder and Bergstron, 1994). In Argentina, Barreto (1984, unpublished data) observed infection on 2% of wheat seed. Future investigations are required to establish the sanitatary management of seeds (Carmona, 2003). Another source of primary inoculum is wheat straw. Several authors consider straw as the principal source of the inoculum of Ptr (Rees and Platz, 1980). Collateral hosts of Ptr could play an important role as a source of primary inoculum between growing seasons, as a source of genetic variation and as a reservoir of a fungal population genetically different than that prevalent on wheat (de Wolf et al., 1998). The tan spot fungus has been reported on many grass species from different parts of the world, among which are Agropyron sp., Avena fatua, A. sativa, Echinochloa sp., Elymus innovatus, Andropogon gerardi, Alopecurus arundinaceus, Bromus inermis, Dactilys glomerata, Lolium perenne, Phalaris arundinaceae, Poa sp. and Secale cereale (Diedicke, 1902; Drechsler, 1923; Conners, 1939; Dennis and Wakeﬁeld, 1946; Sprague, 1950; Andersen, 1955; Dickson, 1956; Shoemaker, 1962; Hosford, 1971; Howard and Morral, 1975; Farr et al., 1989; Krupinsky, 1992c; Ali and Francl, 2002b). In Argentina, the host range is unknown. Ascospores are generated in the pseudothecia that live in the wheat straw. The conidia are formed in the straw containing the pseudothecia and on the leaves of infected plants or the leaves of collateral hosts. The ascospores of Ptr are dispersed primarily by wind, but the distance an ascospore 280 M.V. Moreno and A.E. Perelló can travel is limited (Schilder and Bergstrom, 1995). Limitations on ascospore dispersal distance have been attributed in part to short discharge distances from the pseudothecia. However, it is doubtful that the short discharge distance alone can account for these short dispersal distances. Schilder and Bergstrom (1992) proposed that movement was limited during periods of high relative humidity when ascospores were discharged from the ascocarps (de Wolf et al., 1998). Infested residue usually results in signiﬁcant disease severity at ﬂag leaf emergence and later growth stages due to secondary infections (McFadden and Harding, 1989; Wright and Sutton, 1990; McFadden, 1991). Following liberation from the host, the conidia of Ptr can be sampled readily during aerial dispersal and differentiated successfully from other fungi (Morral and Howard, 1975; Rees and Platz, 1980; Wright and Sutton, 1990; Krupinsky, 1992b; Maraite et al., 1992; Schilder and Bergstrom, 1992; Wolf and Hoffmann, 1993). Morrall and Howard (1975) reported that conidia numbers reached their highest levels late in the growing season and that the number of conidia has a clear diurnal periodicity. The numbers of conidia of the pathogen decline sharply with dispersal distance. Schilder and Bergstrom (1992) reported that the highest number of conidia occurred within 3 m of the inoculum source and that 60–100% of the recoverable conidia were sampled within 25 m. Only a few conidia could be recovered 100 m away from the inoculum source, but this suggested that longer dispersal distances were possible. When the conidia were deposited on the leaf, their germination was inﬂuenced by both temperature and the availability of free moisture (Mihtra, 1934). The conditions that contribute to infection by Ptr in an outdoor environment have also been studied (Ali, 1993; Francl, 1998; de Wolf and Francl, 1997). The precise range of temperatures optimal for disease development varies with cultivar (Luz and Bergstrom, 1986). Leaf age affects the severity of the disease caused by Ptr (Cox and Hosford, 1987; Lamari and Bernier, 1989a,b; Hosford et al., 1990; Perelló et al., 2003a). The growth stage seems to inﬂuence tan spot severity and expression of resistance (Hosford et al., 1990; Fernandez et al., 1994; Perelló et al., 2003a). Then, infecting the wheat leaves, conidia are produced and the pathogen’s asexual cycle life develops by infecting new plants of wheat. Even so, conidiogenesis continues in wheat straw. The production of conidia and the development of pseudothecia depend on temperature and water potential. Stem colonization appeared to be the result of the progressive colonization of the leaf sheath and upper internode. No differences in saprophytic colonization were observed among cultivars of varying resistance (de Wolf et al., 1998). Numerous researchers have investigated the factors affecting the initiation and development of pseudothecia in laboratory experiments (Odvody et al., 1982; Pfender and Wootke, 1987; Pfender et al., 1988; Summerell and Burgess, 1988a,b, 1989; Zhang and Pfender, 1993). The effects of water potential in wheat straw on pseudothecial development have also been studied in an outdoor environment (Fernandes et al., 1991; Zhang and Pfender, 1993). The number of ascocarps per gram of straw in near-soil straw was 32% and 42% of that found in mowed and no-till treatments, respectively. In addition, the number of ascocarps produced in the lower portion of standing stubble of no-till plots was 12% of the number found in the upper portion. Reports of pseudothecia maturation in an outdoor environment vary from region to region (Rees and Platz, 1980; Odvody et al., 1982; Summerell and Burgess, 1988b, 1989; Wright and Sutton, 1990; Wolf and Hoffmann, 1993). In most regions where wheat is grown, the pseudothecia of Ptr are initiated when the crop has reached full maturity and begins to senesce (Odvody et al., 1982; Wolf and Hoffmann, 1993). However, in colder climates, pseudothecia may not be initiated until the following growing season (Fernandez et al., 1998). In Argentina, the sexual stage of Ptr has been detected in wheat straw, but it is unknown in which regions and under what conditions development took place. Tan Spot in Argentina Physiological Specialization The terms ‘pathogenicity’ and ‘virulence’ are likely to be used to describe the ability of an organism to cause disease. Pathogenicity is regarded as a general attribute of a species, while virulence is an attribute reserved for a particular strain of a pathogen in relation to a particular host genotype (Day, 1960). There exist virulent races of Ptr that interact with wheat hosts in a highly speciﬁc manner. This suggests that host-speciﬁcity attributes are superimposed on the general pathogenic ability of Ptr. Variation in virulence in the population of this pathogen is essential in understanding the interaction of the genomes involved in tan spot. Studies of the diversity of virulence within a pathogen population should help in the development of a successful disease management programme, particularly resistant cultivars. Several investigators have described diversity among Ptr isolated from different areas around the world (Christensen and Graham, 1934; Misra and Singh, 1972; Luz and Hosford, 1980; Gilchrist et al., 1984; Krupinsky, 1987, 1992a,b; Diaz de Ackermann et al., 1988; Lamari and Bernier, 1989a; Schilder and Bergstrom, 1990; Ali and Buchenau, 1992; Sah and Ferhmann, 1992; Brown and Hunger, 1993; Moreno, 2007). In 1971, Hosford observed differences between the reaction type on wheat cultivars produced by isolates of Ptr. Misra and Singh (1972) tested isolates originating from India and they detected signiﬁcant differences in virulence, based on lesion size. Some results were observed by Gilchrist et al. (1984) when they tested isolates collected from Mexico on the wheat cultivar Morocco. Luz and Hosford (1980) grouped the isolates tested into 12 races based on statistical mean separation. However, Díaz de Ackermann et al. (1988) did not ﬁnd any difference in virulence among the isolates tested by Luz and Hosford (1980). Hunger and Brown (1987) tested nine isolates originating from the USA; these isolates showed signiﬁcant differences on the susceptible cultivar TAM 105. Krupinsky (1987) showed 281 differences in lesion length and percentage of severity among isolates of Ptr obtained from Bromus inermis. Lamari and Bernier (1989a) grouped the isolates of Ptr into three pathotypes on 11 cultivars of wheat based on the type of reaction. Schilder and Bergstrom (1990) tested 70 isolates obtained from Canada on 12 wheat cultivars and detected signiﬁcant differences among the interaction of isolate × cultivar. Some results were reported by Sah and Ferhmann in 1992 for isolates originating from Brazil, Germany, India, Nepal and the USA. However, Krupinsky (1992a,b) detected variation among levels of aggressiveness but he found no differences in levels of virulence. In 1992, Ali and Buchneau observed physiological specialization based on the reaction type for isolates obtained from the USA. Mehta et al. (2004) tested 40 isolates obtained from Parana (Brazil) on six wheat cultivars; they observed low interaction for isolate × cultivar. In 2007, Moreno detected signiﬁcant differences in isolate × cultivar for isolates of Ptr obtained from wheat-growing areas in Argentina. Races 1, 2, 3 and 4 of Ptr correspond with those determined by Lamari et al. (1995). Races 1 and 2 are predominant in North America (Ali and Francl, 2003). The greater part of isolates identiﬁed as race 5 originate from North Africa, North America and Azerbaijan (Ali et al., 1990; Lamari et al., 1995, 1998; Strelkov et al., 2002; Ali and Francl, 2003). Races 6, 7 and 8 were identiﬁed from collections originating from Algeria, Caucaso and South America (Ali and Francl, 2002a; Strelkov et al., 2002; Lamari et al., 2003). Finally, races 9 and 10 were identiﬁed from isolates originating from South America (Ali and Francl, 2002a,b). These studies indicate that variation in the pathogen population can be detected by using either quantitative or qualitative rating scales (Table 21.2). Research using quantitative scales generally detected variation in virulence on susceptible lines, but isolates in different studies produced an equal reaction on resistant cultivars (de Wolf et al., 1998). 282 Table 21.2. M.V. Moreno and A.E. Perelló Relationships between pathotypes, races and wheat cultivars. Cultivars/lines of wheat Races Glenlea Katepwa 6B662 6B365 Salomouni M3 1 2 3 4 5 6 7 8 N (Tox A) N (Tox A) R R R R N (Tox A) N (Tox A) N (Tox A) N (Tox A) R R Cl (Tox B) Cl (Tox B) N (Tox A) Cl (Tox B) N (Tox A) Cl (Tox B) R R R R Cl (Tox B) Cl (Tox B) Cl (Tox B) Cl (Tox B) Cl (Tox C) R Cl (Tox C) R R Cl (Tox C) R Cl (Tox C) R R R R R R R R R R R R R R R R Note: R, resistance; N, necrosis; CL, chlorosis; Tox A, presence of Tox A and production of Tox A; Tox B, presence of Tox B and production of Tox B. In Argentina, the race population structure is unknown. Future research studying physiological specialization in Ptr should consider collections originating in Argentina. Disease Management Strategies From the point of view of the disease’s development, its management is achieved in different ways: by reducing or delaying the disease early in the growing season or by reducing its rate of development during crop growth (Zadoks and Schein, 1979). This practice has helped to block the life cycle of the pathogens, preventing the introduction of inoculum and susceptible hosts, eliminating certain pathogens (Palti, 1981). Tan spot is one of a complex of necrotrophic leaf diseases of wheat which overwinter on infested crop residue (Hosford and Busch, 1974; Loughman et al., 1998; Carmona, 2003; Annone, 2006). The occurrence of tan spot with other leaf spots, such as septoria blotch, spot blotch and with rusts and mildews, can complicate disease management practices (de Wolf et al., 1998; Carmona, 2003; Annone, 2006). The management of tan spot is based on integrated management of diseases that use reasonable techniques and resources for sustainable agriculture (Carmona, 2006). Strategies used for the control of tan spot are the application of fungicides, cultural control and the search for new germplasms and their incorporation in Argentina (Carmona, 2003). Recently in Argentina, several biological antagonists of Ptr have been identiﬁed (Pfender et al., 1989; Li and Sutton, 1995; Perelló et al., 2003b; Annone, 2005). Genetic resistance Genetic resistance is complex for diseases such as head blight and leaf spot. The principal limitations are due to the changes made by pathogen populations over the years to challenge new cultivars (Carmona, 2006). Unfortunately, only a few of the currently grown cultivars have a high level of resistance, while somewhat larger numbers possess a moderate level of resistance (Rees and Platz, 1992). Kohli et al. (1992) reported the low presence in South America of cultivars resistant to Ptr. Several studies have been conducted in Argentina to screen breeding material for resistance (Galich and Galich, 1994; Annone, 1995). In Argentina, cultivars have either a moderate level of resistance or are susceptible to tan spot (Simón, 2006). Tan Spot in Argentina Chemical protection Fungicides offer a complementary tool to the genetic resistance available. Its use in direct seeding crops under-compensates for the lack of genetic protection to facultative parasites. Fungicides are used as seed protection and/or treatment coverage with ground or air equipment. The majority of literature regarding the use of fungicides to manage tan spot alone or in combination with other leaf diseases has focused on the timing of application and comparative efﬁciencies. Research results have been mixed, but it appears that in situations where disease pressure is high and conditions favour further development of foliar disease, a single well-timed application of an efﬁcacious fungicide can reduce disease severity, increase yield and improve product quality (Sutton and Roke, 1986; Bockus et al., 1992; Duczek and Jones-Flory, 1994; Stover et al., 1996). Fungicides such as tebuconazol, frutiafol, ﬂuzilazol propiconazol and prochloraz reduced the intensity of lesions and showed control of from 50 to 70%, depending on the cultivar and the density of wheat straw infested (Annone et al., 1994; Carmona, 1996). The most efﬁcient fungicides are systemic triazoles and estrobirulinas (Carmona, 2003). Cultural control Cultural practices alter the development of foliar diseases of wheat, particularly those caused by facultative pathogens. Tan spot, representative of the latter group of diseases, is affected by tillage practices in almost all wheat-growing regions of the world (Mehta and Gaudencio, 1991). Retention of wheat residue on the soil surface generally results in increased tan spot severity (Gough and Ghazanfani, 1982; Summerell and Burgess, 1988a,b; Schuh, 1990; Bockus and Claasen, 1992; Stover et al., 1996; Carmona and Reis, 1998). In areas where zero tillage is practised, tan spot and other debris-borne diseases 283 typically increase in incidence and severity (Rees and Platz, 1979; Mehta and Gaudencio, 1991; Kohli et al., 1992). The rotation of crops has a high impact on the sexual stage of this type of pathogen. Because sexual stage viability is minor or low when wheat straw is mineralized, the primary inoculum is therefore low and reduces the severity of tan spot (Carmona et al., 1999). In relation to crop rotation, of relevance was wheat as an antecessor of barley and oats as an antecessor of wheat and barley as an antecessor of oats (Carmona et al., 2001). Barley, wheat and oats are common hosts of Ptr and other pathogens, so there are therefore no alternative crops available for crop rotation. Oats were not hosts to leaf spots speciﬁc to wheat, so crop rotation is possible in Argentina. However, in Brazil, Paraguay and Uruguay, where B. sorokiniana is a relevant pathogen, rotation of these crops should not be authorized (Carmona, 2006). Biocontrol Biological control using antagonistic microbes alone or as supplements has become more important in recent years in order to minimize the use of chemicals (Annone, 2005). It is an additional tool available for the design of more sustainable control strategies of wheat diseases. While biological control is a widespread natural phenomenon, it is often inadequate, especially in agricultural ecosystems in which conditions strongly favour pathogens and disease epidemics. It can also fail in natural ecosystems, especially against aggressive alien pathogens that are able to overcome the natural biological buffering of these systems (Sutton, 2005). Several biological antagonists of Ptr have been identiﬁed (Pfender et al., 1989; Li and Sutton, 1995; Perelló et al., 2003b; Perelló et al., 2006, 2009). Luz et al. (1998) found that treatment with Paenibacillus maceruns or Pseudomonas putida reduced transmission of Ptr by seed to levels equivalent to that of a fungicide seed treatment. 284 M.V. Moreno and A.E. Perelló Some of the fungi found to be inhibitory to pseudothecia development by Ptr were Limonomyces roseipellis, Myrothecium roridum, Acremoniun terricola, Stachybotrys sp. and Laetisaria arvalis (Gough and Ghazanfani, 1982; Pfender et al., 1989). Assays in Argentina have demonstrated that some Trichoderma harzianum isolates are capable of suppressing growth, the mycelial development of Ptr and the severity of diseases on wheat plants (Perelló et al., 2003b, 2006, 2008, 2009). No previous records of antagonism between isolates of Trichoderma spp. and the necrotrophic foliar pathogen have been found. On the other hand, there are increasing economic and social pressures to develop usable biological control strategies in Argentina. References Ali, S. 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Cordo Comisión de Investigaciones Cientíﬁcas de la Provincia de Buenos Aires, Centro de Investigaciones de Fitopatología (CIDEFI) – Facultad de Ciencias Agrarias y Forestales, La Plata, Argentina Abstract This chapter introduces the detailed and novel contributions on the epidemiological spread of Mycosphaerella graminicola over the wheat ﬁeld and over time. The within-season and between-crop methods of multiplication, survival and their environmental relations are reviewed. Genetic arguments are given to demonstrate the inﬂuence of ascospores as the major source of movement of the pathogen into new ﬁelds. Coupled with the evidence that populations worldwide are genetically very similar, it does seem possible that a novel form of the pathogen has been spreading worldwide. This would raise the interesting question as to what epidemiological characteristic confers the new form’s invasiveness. There is a clear association between the evolution of the disease and weather conditions. Wheat cultivars exhibit differential responses to infection by M. graminicola. Breeding for disease resistance is an important tool in the integrated management of disease. Also, fungicide application and the use of biocontrol organisms alone or in combination with fungicides is mentioned as other integrated action. Introduction Cereals and the processed foods derived from them are still the principal sources of nutrition in many parts of the world. Bread wheat (Triticum aestivum L.) is the most widely grown and consumed food crop. It is the staple food of nearly 35% of the world’s population and the demand for wheat will grow faster than for any other major crop (Rajaram, 1999). The forecasted global demand for wheat in the year 2020 varies between 840 (Rosegrant et al., 1995) to 1050 Mt (Kronstad, 1998). To meet this demand, global production will need to increase by 1.6–2.6% annually from the present production level of 560 Mt. For wheat, the global average yield must increase from the current 2.5 t/ha to 3.8 t/ha. In 1995, only 18 countries worldwide had an average annual rate of growth over 2% between 1961 and 1994 (INTA-CIMMYT, 1996). In Western Europe and North America, the annual growth rate for yield was 2.7% from 1977 to 1985, falling to 1.5% from 1986 to 1995 (Rajaram, 1999). Argentina, with a production of 16.11 Mt in the 2006/07 campaign (Encuesta Agrícola, DIEA-MGAP) and 17.47 Mt in the 2007/08 campaign (OPYPA, 2008), is an important wheat exporter with a volume of 300.00 t and an average yield of 2800 kg/ha (data from OPYPA yield and production). The increased need for wheat export forces producers to improve soil  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 291 292 C.A. Cordo management and plant cultivation strategies, including the application of crop protection agents to reduce losses, and is associated with the effective use of measures designed to increase yield and ensure quality. With the increased proportion of conservation tillage practice and with the striving for optimal exploitation of the yield and quality potential of cultivars with the aid of appropriate cultivation and fertilization measures, the importance of certain fungal diseases as yield-limiting factors has increased considerably. The occurrence of fungal pathogens can not only limit cereal production in temperate climates, but can also jeopardize the requisite return on capital under conditions of intensive farming. The epidemic development of pathogens, which vary greatly in their ecological requirements, is strongly dependent on the weather and, together with the slightly different cultivation systems, this leads to differences in the type of infection (pathogen species) and in the level of infection (severity of the disease) from one year to the next. Given these circumstances, the level of application of fungicides in cereal cultivation in Argentina has increased since 1996. Its application produced an increased yield of 20–32% in relation to the control test with respect to time of application, fungicide molecular type and wheat variety (Annone et al., 1995). Chemical crop protection generally has been accepted in Argentina because it should only be used when circumstances make it necessary to achieve the production target and when all other options have been considered. Crop protection measures should be harmonized with the actual infection situation prevailing in the crop and targeted countermeasures must be initiated only where there is a real risk. Cortese et al. (1998) and Carmona et al. (1999) have ﬁxed the economic damage threshold (UDE) and an action threshold (UDA) for different diseases on wheat. UDA represents the incidence value of the disease to decide the fungicide application on the crop to reduce the cost of application. UDE is based on the formula of Munford and Norton (1984) and represents the value of the disease when the yield losses produced by the pathogen would be equivalent to the cost of fungicide application. The threshold values established should represent infection limits beyond which economic losses are highly likely in the shorter or longer term, and the point at which the pathogen population reach this limits in the ﬁeld crop determines the time at which fungicide should be used. The pathogen-speciﬁc thresholds must be worked out within the framework of exact scientiﬁc investigations in relation to the crop management methods used and the environmental conditions prevailing during the growing period. Their development requires extensive case studies (under outdoor conditions) in order to establish how the population dynamics and detrimental effects are inﬂuenced by the weather, wheat varieties, use of fertilizer, the preceding crops and the level of inoculum. The purpose of collecting all this information is to control individual pathogens with appropriate products at appropriate application rates, and to do this at a time at which one application is most likely with regard to pathogen development and limitation of damage, using the lowest possible input. Epidemiology and population genetics are different but related subsets of population biology. Epidemiology focuses on disease progression, the increase in pathogen populations through time and the movement of pathogen populations through space (usually from plant to plant). Most epidemiology studies deal with a short timescale (e.g. 1–2 growing seasons) and small spatial scales (e.g. disease development in a ﬁeld or a plantation). Epidemiology involves mainly physical processes such as distances of spore movement or effects of weather variables on latent periods. It does not take account of the differences in behaviour or genetically distinct individuals in a collection of individuals. Population genetics focuses on the processes that lead to genetic changes, or evolution, in populations over time and space. Population genetics deals mainly with genetic processes such as genetic drift, gene ﬂow, mating system, natural selection and mutation. Present study records that epidemiological investigations, based on disease Septoria Leaf Blotch of Wheat in Argentina evolution, resistance supply, early detection of the disease, biological crop protection and genetic studies of the pathogen, can be used to orientate the management of disease under natural conditions. The Disease as a Problem Septoria tritici blotch (STB) is caused by M. graminicola (Fuckel) Schroeter, in Cohn, which is the teleomorphic stage of S. tritici Roberger and Desmazieres (anamorph stage). Sanderson (1972) proved the connection between the two stages and the sexual (teleomorph) form has been reported in several countries (Hunter et al., 1999). Cordo and Arriaga (1990) reported the sexual stage in Argentina. It is also known to play a role in the disease’s cycle. It causes most of the initial infection in winter wheat crops, during the autumn in the UK (Shaw and Royle, 1989) and USA (Schuh, 1990). In Argentina, an increase in ascospores at harvest time has been reported, suggesting that the sexual stage may be important in initiating the infection in the next growing season (Cordo et al., 1999). Another possible means of spread within a crop during summer is by airborne ascospores, which may play a more major role than previously recognized (Hunter et al., 1999; Cordo et al., 2005). Studies on the Disease’s Evolution Several control methods, including the use of fungicides and other cultural practices, may reduce the effect of STB, but genetic resistance is the most cost-effective and environmentally safe technique to manage the disease. In Argentina, an inoculation technique using oat grains covered with the stromatic mycelia of S. tritici were presented to check the resistance of the Septoria Monitoring Nursery (SMN) set. The international set created by the CIMMYT provides information on the interactions of pathogen × cultivar on different regions of the world. The use of this set allows the generation of extensive 293 information on resistance sources for diverse pathogen populations under variable environmental conditions. Differences among accession reaction were signiﬁcant due to the rich composition of the selected sources of resistance identiﬁed by CIMMYT and a tentative group of differentials proposed by Eyal (Gilchrist et al., 1999) that were incorporated into the SMNs. The inoculum for grain application was prepared in sterilized 500-ml ﬂasks with 100 g of oat grains and 50 ml of a liquid extract malt medium (Perelló et al., 1997). The grains were soaked with 10 ml of an inoculum suspension (107 conidia/ml) of S. tritici isolate and incubated for 15–21 days at 23 ± 2°C in darkness and shaken daily to promote good fungal growth. After the incubation, the grains were colonized by a stromatic mycelium and were spread and dried on trays under laboratory conditions. The covered grains were spread on to the soil next to the plants during the tillering growth stage (GS23, Zadoks et al., 1974). Plants in the plots were assessed for S. tritici infection at anthesis (GS60) and at the medium milk (GS75) stages. The accessions (1-BOBWHITE S; 2-TIA. 2/4/CS/TH.CU//GLEN/3/ALD/PVN; 3-CHIRYA.1; 4-CHIRYA 4; 5-CS7TH.CU// GLEN/3/ALD/PVN/4/NANJING; 6-EG-A/H56 7.71//4#EG-A/3/2#CMH79.243; 7-MH86.540A-1Y-3B-2Y-1B-1B-1B-1Y-1M-1Y; ALD/PVN// YMI#6; 9-SHA5/BOW; 10-ENCOY 1582–1B; 11-BOBWHITE S as the other derivative line; 12-DON ERNESTO INTA; 13-SERI M82; 14BETHLEHEM; 15-LAKHISH; 16-KAUZ; 17PENJAMO; 18-ETIT 38; 19-GLENNSON M81) were sown in a factorial design experiment. The pulverization inoculum was produced using the same isolate as in the previous year. The conidial concentration of the suspension was adjusted to 1 × 107 conidia/ ml. A comparison between the pulverization and the grain application methods was made in the ﬁeld in 2000. The inoculum suspension was sprayed on to the leaves at the tillering stage (GS23). After inoculation, plants were kept moist by sprinkling water several times a day over 3 days. The severity of the infection was registered on the ﬂag leaf at the beginning of the ﬂowering (GS60) 294 C.A. Cordo and medium milk (GS75) stages using a modiﬁed double digit Saari–Prescott scale (Saari and Prescott, 1975). The cut for resistant behaviour was estimated as 5.3 (Gilchrist et al., 1999). Weather variables (daily temperature, relative humidity and rainfall) were recorded from the date of inoculation to anthesis. Plant height was evaluated. To compare the inoculation techniques, both the necrotic coverage percentage (NCP) and pycnidial coverage percentage (PCP) were scored on the upper three leaves of 15 plants, 21 days after inoculation. The cutoff point between resistant and susceptible response classes was 16.8% NCP following Eyal et al. (1985). The comparison between pulverization and grain application showed that, except for the variety Bobwhite ‘S’ CM 33203-K-10M-7Y-3M-2Y-1M-OM and the line Tia.2/4/CSTH.CU//GLEN/3/ALD/PVN CIGM88.734-1B-3PR-0PR-1M which reacted as in the observations of Gilchrist et al. (1999), all genotypes were more susceptible under Argentine conditions. The higher level of virulence of the Argentine isolates and frequency of variation could explain this behaviour (Eyal et al., 1985; Gilchrist et al., 1999; Cordo et al., 2006). The results of the severity for NLP and PCP in this study are in agreement with previous research (Eyal, 1985; Gilchrist et al., 1999). The advanced resistant lines coming from the crosses with a group of resistant Chinese lines did not show a high level of resistance (Ald/Pvn/YM#6, Milan/Sha#7, Catbird, Talhuen INIA, Sha3/Seri/PSV/Bow and the cultivar with Kavkaz/K4500 sources). The resistant check Bethlehem was not resistant at CIMMYT or in our conditions. The bread wheat checks SeriM82 and Glennson M81 (with Veery ‘S’ germplasm) and Lakhish were susceptible, as was expected (Gilchrist et al., 1999). The durum wheat ETIT 38 and the resistant check Bethlehem had the same level of susceptibility as bread wheat checks (SeriM82, Lakhish) and as was scored by Kohli (1995). The disease resistance introduced from Brazilian germplasm was detected on a short, earlymaturing resistant line derived from IAS 20 spring wheat and a more susceptible reaction on lines derived from IAS 58. Bobwhite ‘S’ germplasm and its derivative lines (in Argentina represented by Don Ernesto INTA) showed variable levels of resistance caused by its background with more than one genetic source and the presence of a low number of major genes (Cordo et al., 1994). Plant height was not associated with the resistant reaction. The negative associations were present when weather conditions were less conducive to the development of the disease. Non-conducive conditions and the further distance between leaves in tall cultivars could have reduced the rainsplash dispersal of pycnidiospores, thus causing this negative association (Arama et al., 1999; Simón et al., 2005; Arraiano and Brown, 2006); it could also depend on the presence of ascospores, which could reduce the effect of plant height on the expression of the disease. In Argentina, the presence of the teleomorphic stage during the whole growing period has been reported (Cordo and Arriaga, 1990; Cordo et al., 1999, 2005). The modiﬁed double digit Saari–Prescott scale was adopted for evaluation of this set (CIMMYT, rules for evaluation, Eyal et al., 1987). The separate analysis of digit 1 and 2 allowed the relative height reached by the disease to be shown simultaneously with the severity of the damage (PCP) (Table 22.1). The differences observed for the ﬁrst digit in the accession response to the inoculum concentration were attributed to the maximum level of attacked leaf (8th leaf) that was reached with the highest concentration of inoculum: 280 g. In contrast, the second digit did not show differences for either concentration. The lesions were restricted in extension, reaching only a maximum of 20% more of the PCP in the cases of highest susceptibility. This result conﬁrmed that the level of resistance of tested materials was adequate to maintain a low intensity of infection according to the objectives proposed by Eyal and Gilchrist at the beginning of this project (Gilchrist et al., 1999). Two factors were inﬂuencing the expression of the disease on the leaves: the concentration of the grain inoculum (120 g/ m2 was optimum for differentiation between susceptible and resistant accessions) and Septoria Leaf Blotch of Wheat in Argentina 295 Table 22.1. S. tritici infection average (digit 1 and 2) for different concentrations of inoculum and different years. Inoculum concentration Accession Digit 11 Digit Years 21 Digit 11 Digit 2 n C1 C2 C1 C2 1997 1999 1997 1999 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 6.00 b2 5.50 b 2.50 a 7.25 c 6.75 b 6.25 b 6.50 b 6.50 b 6.50 b 6.00 b 6.50 b 6.25 b 6.75 b 7.00 c 6.25 b 7.00 c 6.75 b 6.50 b 6.50 b 6.50 b 7.25 d 7.50 e 7.25 c 7.25 d 6.75 b 8.00 g 6.75 b 5.75 b 7.75 f 7.00 c 7.00 c 7.25 d 7.25 c 6.50 b 7.25 c 7.75 f 7.75 f 7.75 f 2.50 b 2.25 a 2.75 c 0.50 a 2.00 a 1.25 a 1.00 a 2.75 c 1.50 a 1.00 a 0.75 a 1.00 a 0.75 a 1.25 a 4.00 f 3.00 d 2.25 a 2.00 a 2.61 c 1.00 a 0.75 a 1.75 a 1.50 a 2.25 a 1.75 a 0.75 a 0.50 a 2.00 a 1.75 a 2.00 a 5.00 g 1.50 a 2.25 a 3.00 d 3.50 e 2.25 a 0.50 a 1.50 a 6.50 b2 6.75 b 5.50 a 8.00 f 7.00 c 6.75 b 7.75 e 7.00 c 6.25 b 7.25 d 7.75 e 7.25 d 7.75 e 8.00 f 6.75 b 8.00 f 8.00 f 7.750e 7.50 d 6.00 b 6.00 b 4.50 a 6.50 b 6.00 b 6.25 b 6.75 b 6.25 b 6.00 b 6.50 b 5.75 a 6.00 b 6.25 b 6.25 b 6.00 b 6.25 b 6.50 b 6.50 b 6.75 b 2.50 b 2.50 b 3.00 d 1.75 a 2.75 c 1.75 a 1.00 a 2.75 c 2.50 b 1.75 a 1.00 a 3.25 d 0.50 a 2.75 c 3.25 d 2.50 b 3.25 d 1.25 a 3.25 d 1.00 a 0.50 a 1.50 a 0.25 a 1.50 a 1.25 a 0.75 a 0.50 a 1.00 a 1.00 a 1.75 a 2.75 c 1.75 a 0.75 a 3.75 e 4.00 e 1.25 a 1.25 a 0.86 a Note: 1As assessed by a modiﬁed double-digit Saari–Prescott scale (1975). 2Mean values followed by the same letter are not statistically different. LSD test (P < 0.01); C1 = 120 g/m2; C2 = 280 g/m2. the wet environment. For the grain application treatment, the density of the plants was too important for rainfall to produce the infection. If the rain regime was not frequent and intensive, the pycnidiospores could not reach the higher leaves, making it difﬁcult for the inoculum to ascend. In the pulverization treatment, the surface covered by the inoculum included more than one leaf stratum. A simultaneous proliferation of the pathogen was obtained in all foliage levels, which, in addition to the beneﬁcial structure of the canopy, produced the highest values of severity. The most susceptible varieties at the GS75 stage were those that had a longer period of green leaf during the growth cycle; but something different occurred with ETIT 38 that did not show any difference on NCP and PCP for both growth stages. This could be explained by the quick senescence of the leaves that practically stopped the development of the fungus at the end of GS60. The higher values of the disease in 1997 compared with those of 1999 were caused by the inﬂuence of the climatic conditions. The temperature was not an important factor because there was no statistical difference in 3 years of experiments. In 1998, high humidity (30% more than the following year) and increased rainfall (425.29 mm more than the following year) were responsible for the rapid increase of the disease compared with results of 1999 (data not shown). Both inoculation techniques were appropriated to monitor the behaviour of the accessions of the SMN set. If the experimental ﬁeld is under a good rain regimen from tillering to ﬂowering, grain application is recommended. But if it is on a dry irrigated area, pulverization with extra irrigation as a humidity chamber is suggested. 296 C.A. Cordo Comparing the efﬁcacy of each inoculation technique, different symptoms produced by each treatment at the beginning of the disease have been associated with environmental conditions (Table 22.2). In relation to the grain application treatment, the generalized necrosis and pycnidial development on the lower leaves could have risen to the upper leaves if irrigation or rainfall had been present at this early stage of infection. The density of the plants was too important for rainfall to produce the infection. The more compact density of the canopy helps to maintain a microclimate for the progress of the disease. The lack of germination that affected seeds of some accessions could have modiﬁed the canopy structure and the inner microclimate; consequently, it could have delayed the movement of the pathogen to the upper leaves of the plant (Lovell et al., 1997). It could explain the absence of relation between the increase of inoculum concentration and the decrease of the severity on digit 2. In the pulverization treatment, the surface covered by the inoculum included more than one leaf stratum. A simultaneous proliferation of the pathogen was obtained in all foliage levels, which, in addition to the beneﬁcial structure of the canopy, produced the observed reactions. The strong differences observed between treatments (pulverization and grain application) could be explained by the different rates in the progress of the disease for each treatment. In pulverization, the inoculum included more canopy levels. In agreement with Lovell et al. (1997), the ascending movement of the disease was facilitated, especially in cultivars that, because of their compressed canopy, maintained a more favourable microclimate. On the contrary, the delayed attack with grain application could be explained because only the rain dispersed the conidia from the infected grains. If the rain regimen was not frequent and intensive, the spores could not reach the leaves, making it difﬁcult for the inoculum to ascend. Although the pulverization and grain application techniques were shown to be effective and could be recommended for ﬁeld trials, each one demonstrated different advantages. The grain application treatment had the advantage that the incubation period of the disease was maintained with irrigation. In this case, a known weight of inoculated grains was spread on to the soil, between the rows, as primary inoculum. In this technique, pycnidia on stromatic mycelia formed on grains could release pycnidiospores over a long period if wetted. This process may be repeated several times if the grains are dried and wetted again. The splash dispersal effect can increase spore transport from a low to a high level of the crop and from plant to plant. There was a general correlation between the reaction of some lines and the differential varieties in relation to NCP and PCP. The most susceptible varieties at the GS75 stage were those that had a longer period of green leaf (8 days more); therefore, the pathogen had a higher probability of proliferating in the leaf. Something different occurred with Lakhish. In this differential variety, NLC and PCP did not show any differences for both growth stages. This could be explained by the quick senescence of the leaves that practically stopped the development of the fungus at the end of GS60. The technique of grain inoculation presented in this research has the advantage of being simple to handle compared with the installation of a barrier to infect wheat plants artiﬁcially, as is necessary in the pulverization methods (Sanderson et al., 1986). In the latter, it is necessary to plan the exact date of the previous sowing and the direction of this barrier in relation to the tested wheat rows. Comparing infectivity of different inoculum concentrations in the grain application treatment, a gradient of infection was obtained and its effect was related to the variation of humidity and rain regime. The differentiation between susceptible and resistant entries of this set was possible using 120 g of oat grains/m2 covered with stromatic mycelia of S. tritici. With this, it is not necessary to use the highest concentration of oat grains. This type of inoculum has a long-lasting effect next to the plants, but the most important characteristics are that the incubation period is produced without the installation of a wet chamber; it is simple to Table 22.2. Necrotic and pycnidial coverage percentages caused by Septoria tritici. Inoculum type Pulverizationa Nb Pc Grain applicationa N Growth Stage a Accessions Pc GS75d N P 51.28 cde2+ 67.03 ijk 64.63 hij 54.88 efgh 52.71 def 43.67 abc 42.55 abc 61.99 ghij 88.58 m 75.84 l 45.29 bcd 60.93 fghi 43.58 abc 69.22 jkl 86.95 m 73.82 kl 74.97 l 39.23 ab 32.57 a 59.46 50.66 cd2+ 67.24 f 60.00 e 37.93 a 51.37 d 36.66 a 38.15 a 51.83 d 85.49 h 70.82 fg 44.86 bc 56.90 de 43.10 b 75.26 g 82.70 h 73.48 g 65.77 f 39.30 a 41.03 ab 56.45 % 41.60 abc1+ 42.59 bc 62.45 fg 27.96 a 31.92 a 46.52 bcd 38.89 ab 42.73 bcd 70.62 gh 51.60 de 48.73 cd 46.77 bcd 50.64 cde 58.97 ef 76.43 hi 83.23 i 62.21 f 66.24 fg 33.79 a 51.78 40.94 1+e 42.68 de 61.91 g 11.67 a 26.98 b 39.60 d 33.34 c 37.68 cd 67.48 g 47.35 ef 41.15 de 47.16 ef 39.80 d 51.89 f 63.16 g 83.55 h 61.88 f 65.99 g 38.58 cd 47.42 28.91 abcd1+ 37.33 defg 32.26 bcde 25.32 ab 40.91 efg 18.99 a 26.14 abc 36.93 def 47.88 g 35.07 cde 45.73 fg 25.63 ab 25.32 ab 34.75 cde 44.35 fg 45.79 g 40.54 efg 19.39 a 20.97 a 33.27 28.02 def1+ 37.43 i 28.17 ef 25.32 cd 37.14 hi 18.76 ab 17.97 ab 30.61 fg 48.56 j 34.66 ghi 40.59 ij 21.81 bc 20.23 abc 31.52 fgh 37.24 hi 35.10 ghi 38.19 i 14.87 a 25.48 cdf 30.09 3.21 a2+ 12.89 b 30.08 bcd 1.59 a 20.12 b 21.84 b 22.48 bc 17.67 b 29.93 bcd 10.83 b 49.17 e 11.48 b 32.39 cd 24.49 bc 33.84 d 55.20 e 27.77 bcd 46.40 e 22.18 bc 25.60 18.31 cde2+ 12.88 bc 30.08 fg 0.93 12.74 bc 21.70 de 13.16 bc 16.47 bcd 30.55 fg 11.18 b 36.89 gh 42.07 h 16.93 bcd 17.64 cd 17.70 cd 45.17 h 24.81 ef 41.56 e 23.02 de 21.15 Septoria Leaf Blotch of Wheat in Argentina 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Mean GS60d Nb P Note: atwo types of inoculum; bmean of necrotic coverage percentage; cmean of pycnidial coverage percentage; dtwo growth stages. 1+Each value is the average of the three upper leaves with two inoculation methods; 2+each value is the average of the upper three leaves in two growth stages. LSD test (P < 0.01). 297 298 C.A. Cordo transport over a long distance and to store for a long period of time (5 days at 5°C). Climate Inﬂuences % Relative humidity After the initial infection or inoculation, the environment is one of the factors conducive to the development of the disease. Different experiments have demonstrated which are the weather conditions for a more favourable expression of the disease (Simón et al., 2005, Cordo et al., 2006). In one experiment conducted in Argentina in 1998 (Simón et al., 2005), the severity of the disease was highest in the early cultivars because precipitation was higher and radiation lower for these cultivars. Precipitation was 53.4 and 18.8 mm and radiation 3511 and 5127 Watt/m2 for a period of 15 days before evaluation for the earliest and the latest cultivars, respectively. Also, these differences in weather variables in 1998 produced a negative asso- ciation between pycnidial coverage and days to heading. In another experiment, Cordo et al. (2007) related that the higher values of the disease in 1997 compared with those of 1999 were caused by the inﬂuence of the climatic conditions from boot (GS 43) to hard ripening (GS 87) stages, following Zadoks et al. (1974). In 1997, high humidity (30% more than the following year) (Fig. 22.1) and increased rainfall (425.29 mm more than the following year) (Fig. 22.2) were responsible for the rapid increase of the disease compared with the results of 1999. For this experiment, temperature was not an important factor in the development of the disease since in 3 years of experiments there were no statistical differences in temperature (mean temperature from the inoculation to the end of the experiment was 17.10°C for the ﬁrst 2 years and 16.69°C for 1999). Related to the inoculation process that is under discussion, if the experiment is to be carried 100 80 60 1997 40 1999 20 0 7Oct 14Oct 21Oct 28Oct 4Nov 11Nov 18Nov 25Nov 2Dec Weeks Rainfall (mm) Fig. 22.1. Histogram showing relative humidity during October–December of 1997 and 1999. 60 50 40 30 20 10 0 1997 1999 7Oct 14Oct 21Oct 28Oct 4Nov 11Nov 18Nov 25Nov 2Dec Weeks Fig. 22.2. Histogram showing rainfall during October–December of 1997 and 1999. Septoria Leaf Blotch of Wheat in Argentina out on an artiﬁcially irrigated area, pulverization with an appropriate suspension of spores and 48 h of extra irrigation in a wet chamber are suggested. However, if the area is under a good rain regime from tillering to ﬂowering stages, the application of grains covered with sporulated mycelia is a feasible option. Early Detection of the Disease At the beginning of the wheat-growing season in 1997, Adgen Phytodiagnostic invited the author to participate in a pilot project to identify and quantify S. tritici and S. nodorum by antibody-based immunoassays and also to compare with the visual method and the sensitive methods for the early detection of S. tritici. The objective of this work has been to test the Adgen ELISA kit in a monitoring process on lower to higher leaves during the wheat season in Argentina. A randomized complete block design with four replicates and a 1.4 × 1 m size subplot was used. Treatment consisted of either an inoculated plot or a control treated with a foliar fungicide spray programme. Plantvax and Tilt were applied at 500 cm3/ ha. Ten main tillers were collected per subplot using a uniform, randomized sampling pattern at GS10.1 (ﬁrst spikelets just visible 28 October); GS10.3 (heading process 14 November); GS10.5 (ﬂowering 27 November); GS11 (ripening 8 December). Samples for testing consisted of ten leaves bulked for each layer of leaves, each replication and each data of collection. At the same time, the severity of the lesions on the sampled leaves was noted. Samples were homogenized in 50 ml buffer and testing following the protocols described for the DU PONT enzyme-linked immunosorbent assays (ELISA) for S. tritici. ELISA results were expressed as the number of S. tritici antigen units/ml of homogeneized plant tissue (AgU/ml). AgU/ml values were averaged. A good correlation was observed between ELISA readings from infected leaves coming from different growth stages (GS10.1, GS10.3, GS10.5) and the visual development on the respective foliar level (C. coefﬁcient = FL 299 0.68; FL-1 = 0.69). This indicates that the infection increased (correlated with AgU/ ml) throughout the different growth stages (Table 22.3). With a variance analysis, the antigenic units registered and the severity of the infection on three wheat cultivars coming from inoculated and protected treatments were compared (Table 22.4). Highly signiﬁcant differences were observed between inoculated and protected treatments for severity and antigenic units into Flag leaf and Flag leaf-1. A high correlation was calculated (C. coefﬁcient = 0.56) between the average per cent visual attack of a sample and the measured antigenic units. Despite the good correlation, the lower interval of the level of attack scale gave the most conﬁrmable antigenic unit values. This immunoassay has demonstrated to be highly sensitive and quantitative, with antigenic unit concentrations being correlated with the severity of the disease. The infection levels of S. tritici in Los Hornos samples could be determined with signiﬁcant precision. In addition, the speciﬁcity of the assay allowed accurate identiﬁcation of this pathogen, despite the presence of other foliar pathogens. In this assay, only the presence of Alternaria triticimaculans gave a cross-reaction. The Adgen Phytodiagnostic Septoria ELISA kit detected and quantiﬁed the amount of S. tritici antigen in infected plant tissues. As our experience indicates, the use of this kit can be recommended in a monitoring process for earlier reports of S. tritici infection. Checking the Ascendant Movement of the Inoculum The ascendant movement of the inoculum was also checked with the diagnostic immunoassay kit for S. tritici from the Adgen Company. Increased severity on different levels of the canopy was registered from the latent period of the infection produced by two types of inoculum application (grains covered by pathogen mycelium and pulverization) during 4 weeks from inoculation at tillering stage (GS23). 300 C.A. Cordo Table 22.3. Correlation between percentage of lesions covered by pychnidia and antigenic units during the 4 weeks of study. Grain application Susceptible cultivars Canopy level Y1 B2 Y B Y B Y B 1°a 1°a 2°b 2°b 3°c 3°c 4°d 4°d Resistant cultivars 1°a 1°a 2°b 2°b 3°c 3°c 4°d 4°d Y B Y B Y B Y B Grain application Pulverization AU3 PCP4 AU 42 b 140 c 5.5 a 34 b 3.6 a 34 b 5.5 a 54 b 0.0 a 0.0 a 0.0 a 3.91 b 0.0 a 3.91 b 0.0 a 6.5 b AU3 PCP4 AU PCP 2.45 a 65 b 9.30 a 8.5 a 65 b 140 c 42 b 80 c 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 4.80 b 0.0 a 1.28 a 3.10 a 115 b 3.6 a 180 c 130 b 195 c 20 b 80 c 0.0 a 0.0 a 0.0 a 15 b 0.0 a 10 b 0.0 a 1.28 a 42 c 300 d 1.0 a 120 c 4.2 a 82 b 22 b 54 a Pulverization PCP 0.0 a 19.16 c 0.0 a 20.8 c 0.0 a 25 c 0.0 a 6.5 b Note: 1youngest leaf; 2the leaf below; 3antigenic units/ml; 4pycnidial coverage percentage; a, 14 October; b, 9 November; c, 16 November; d, 23 November. Table 22.4. Information summary for two populations of Septoria tritici from Argentina. Total isolates No. of genotypes No. of alleles Isolates having ﬁngerprint data No. of ﬁngerprint patterns Fingerprint pattern types Los Hornos population Balcarce population 58 35 24 55 14 A,E,F,G,M,N,O,P,R,S,U,V,W,X 62 39 22 58 13 A,B,D,E,H,I,K,L,M,P,Q,R,V Six leaves per canopy level were sampled per week. Two levels of asymptomatic leaves (the youngest and the leaf below) were chosen from two varieties (Chirya 1 as resistant and Bethlehem as susceptible) belonging to the 8th SMN set. The ﬁrst sample was taken at GS30 (ﬁrst node) stage and the following were taken one per week for 3 more weeks. Samples were homogenized in 50 ml buffer and tested following the protocols described for the Adgen ELISA. An analysis of variance was performed with the dates of percentage of lesion covered by pycnidia and antigen units/ml for each week. An LSD test was used to compare treatment means. Correlation between per cent of lesions covered by pycnidia and antigenic units were performed throughout the treatments and during the 4 weeks. According to the analysis of variance (Table 22.3), highly signiﬁcant differences were found between each level of the canopy for AU and PCP throughout the weeks and with the two inoculation techniques. There were signiﬁcant differences for cultivars. A signiﬁcant correlation was found Septoria Leaf Blotch of Wheat in Argentina between PCP and antigen units (C. coefﬁcient = 0.56***) (calculated on 67 dates). The youngest leaves almost had the lowest values of AU. On the leaves below, it had increased. The AU and PCP values were higher in susceptible than in resistant cultivars. In general, on the 2nd or 3rd week after inoculation, the infection was detected in the youngest symptomatic leaf, indicating that the infection was installed in an ascendant level by splashing. Population Studies of the Pathogen The population structure and genotypic diversity of S. tritici from two crop ﬁeld populations in Buenos Aires Province separated by 500 km were studied with DNA restriction fragment length polymorphism. From of the 137 isolates from different areas of the Argentine wheat-growing region, only 120 were characterized using the RFLP technique with P32 labelled probes. The pSTL70 ﬁngerprinting probe hybridized many DNA fragments of different sizes in isolates from ﬁeld populations of both locations. All leaf samples were processed for isolation of the fungus, followed by fungus culture, DNA extraction, Pst1 enzyme digestion, radioactive hybridization and X-ray ﬁlm detection. Some of the isolates did not yield good quality DNA for the restriction enzyme digestion process. This explains the loss of 17 isolates in the samples of the populations. In total, 24 alleles were found for the Los Hornos population and 22 alleles for the Balcarce population at the eight RFPL loci (Table 22.4). Despite the difference in the number of alleles, Nei’s measure of genetic diversity across all loci was different for both populations (0.2619 for Los Hornos and 0.3161 for Balcarce). Among the 58 isolates of Los Hornos and 62 of Balcarce with complete data from individual RFLP loci, 35 multilocus haplotypes for the ﬁrst locality and 39 for the second locality were registered. Seven new haplotypes (3a, 20a, 71a, 37a, 47a, 52a, 58a) were added to the list published on the Internet (S. tritici RFLP alleles). The haplotype frequency in 301 per cent varied from 1 to 21 times for the Los Hornos population and from 1 to 9 times for the Balcarce population. Genotype diversity was greater in the Balcarce population (Ĝ = 31.61 or 26.34% of the theoretical maximum of 120) than in the Los Hornos population (Ĝ = 26.19 or 21.82% of the theoretical maximum of 120). As the mean genetic diversity between populations was high for the 8 loci of RFLP, a signiﬁcant difference existed between the populations of the two localities. Fifty-eight multilocus haplotypes and 13 ﬁngerprint patterns were registered for the Los Hornos population and 55 multilocus haplotypes and 14 ﬁngerprint patterns for the Balcarce population when they were hybridized with pSTL70. Many isolates of both populations had from one to several haplotypes for each ﬁngerprint pattern. In the Los Hornos population, the E ﬁngerprint pattern was present on 14 different haplotypes, but it corresponded 3 times with the same 11112110611 haplotype. In the Balcarce population, the same ﬁngerprint was present on 11 different haplotypes, but it corresponded 8 times with the 11101010211 haplotype. This last result showed that there were clones in both populations. Some genotypes were detected as shared across the populations. In other cases, several individuals in the two populations had the same multilocus haplotypes but different DNA ﬁngerprints, indicating that they were not the same clone. The alleles’ frequencies were signiﬁcantly different from the 8 loci of RFLP. The Argentine population must be compared with other continental populations – Swiss and USA (Oregon) – as independent populations. Over a total of 834 individuals, there was a 40% gene diversity between native populations and the total population differentiation was 11%, showing that differentiation between native and foreign populations exists. The average number of migrants was 3.68. This number meant that 3–4 individuals would need to be exchanged across populations of each generation to maintain the observed level of genetic similarity. Moreover, the amount of gene ﬂow 302 C.A. Cordo between populations was high when all the populations were compared. The genetic distance was small when comparing the population of Los Hornos with the other populations, showing a high level of similarity, but the genetic distance of the Los Hornos and Balcarce populations was major compared with the Oregon and Swiss populations. Salamati et al. (2000) suggested that the similarity among populations on a regional basis was explained because the gene ﬂow was signiﬁcant over spatial scales of at least several hundred kilometres. It was found that genetic distances among ﬁelds within a region were small, while genetic distances among different continents were larger for the Rhynchosporium secale populations. Genotypic diversity within populations and similarity over regional spatial scale was explained because regular sexual recombination was occurring in S. tritici rather than in R. secalis (Salamati et al., 2000), Stagonospora nodorum (McDonald et al., 1994) and Phaeosphaeria nodorum (Keller et al., 1997) populations. This was explained because the ascospores from the teleomorph were dispersed over distances of up to 100 km (Shaw and Royle, 1989; Cordo et al., 1990/1991). The ﬁeld populations of the fungus exhibited high degrees of gene and genotype diversity distributed on very small spatial scales. Microgeographical-level observations showed a higher variation of type and number of genotypes for the Balcarce than for the Los Hornos population. In general, different genotypes were often found within a single lesion and most lesions on the same leaf also had different genotypes. This result demonstrated, in coincidence with Boerger et al. (1993), that a lesion might result due to coinfection by two or more genotypes. The genetic distance, for native populations, was very small considering that the geographic distances between them was 500 km; the North American and European populations, separated by to 7000 km, had a low increase of this genetic distance. Then, the high degree of similarity could be caused by the gene ﬂow on a regional scale and between continents (Boerger et al., 1993; Zhan et al 2003; Banke et al., 2004; Banke and McDonald, 2005). The results of this contribution are in agreement with Keller et al. (1997), who demonstrated that ascospores were the primary agent for unifying geographically separated populations on a regional scale. Added to this, Cordo et al. (2005) showed that ascospores were the most signiﬁcant component of the M. graminicola life cycle in the wheat-producing areas in Argentina. Their release was registered in the vegetative and debris wheat stages for the periods analysed. According to these experiments, the high degree of gene ﬂow among populations would be associated neither with the pycnidiospores presence as dominant in the life cycle of the pathogen nor the infected seeds that could act as a human dispersal mechanism (Keller et al., 1997). The Los Hornos population result was different because the clonal lineages of S. tritici probably originated from the inoculations applied for the resistance tests. If it is assumed that S. tritici had not colonized Argentina recently, the high degree of similarity could be explained from the most likely centre of origin for this pathogen. Banke et al. (2004) demonstrated that the New World areas (where the South Cone is located) appeared less likely to represent ancestral populations because they had lower diversity, whereas Israel and Europe appeared to be the ancestral populations because they showed the highest genetic diversity. This pattern is related to the fact that wheat has been grown in the Old World for thousands of years, but in the New World for only hundreds of years. Movement of the fungus from Israel into Europe could have been from windblown ascospores or via transport on infected seed or straw. Ascospore movement produced a natural gene ﬂow out of the possible centre of origin and into European populations, which could explain the ﬁnding that more haplotypes were found in European than in New World populations. Another way of dispersion could be an alternate host of S. tritici producing pycnidia, which constitute a continuous host population where ascospores (Boerger et al., 1993; Linde et al., 2002) would maintain a uniform source of inoculum that infects the wheat Septoria Leaf Blotch of Wheat in Argentina ﬁeld each autumn. This way of transmission was not demonstrated in Argentina. Disease Control with Alternative Techniques The most common approach to biological control consists of selecting antagonistic microorganisms, studying their modes of action and developing a biological control product. Despite progress made in the knowledge of the modes of action of these biological control agents, practical applications often fail to control diseases in the ﬁeld. One of the reasons for this failure is that biocontrol products are used in the same way as chemical products. Other methods include the choice of an appropriate crop rotation with the management of crop residues, added to organic amendments and biological disinfestations of soils. In that sense, Cordo et al. (2007) evaluated the efﬁcacy and mechanisms of action of Trichoderma sp. for controlling leaf blotch in wheat grown under greenhouse conditions. Because of their capacity to act as biocontrol agents, members of the fungal genus Trichoderma have been broadly studied (Barnett and Lilly, 1962; Tronsmo, 1986; Melo, 1991; Harman, 2000; Monte, 2001). Thus, T. harzianum and T. aureoviride are known to be effective antagonists against phylloplane pathogens (Perelló et al., 1997, 2001, 2003, 2006). There were signiﬁcant differences for necrosis and pycnidial coverage percentages for 2 years of experiment and for the behaviour of the 14 antagonists, each treated with 303 pulverization and undercoated seed treatments. Two strains of Trichoderma sp. (Th5 and Tk11) were selected. Conversely, trials performed during 2005 examined only plants produced by seeds coated with Trichoderma Th5 and Tk11 isolates. The T. koningii 11 strain was selected for the third experiment, instead of the Th2 strain, because the necrotic coverage percentage of Tk11 was statistically different with respect to the control and with a higher value than the others (Table 22.5). Moreover, the value of pycnidial coverage percentage was also one of the highest that was statistically different from the control. This work shows that T. harzianum, T. koningi and T. aureoviride reduce the leaf blotch caused by S. tritici in greenhousegrown wheat. The effect of T. aureoviride was considered similar to that of T. harzianum in reducing the leaf blotch caused by S. tritici because, under the most effective application method (seed coating), the pycnidial coverage percentage was statistically different to the control, but not to that of T. harzianum. The positive result of the immunochemical test applied on all asymptomatic leaf intercellular ﬂuid samples demonstrated the presence of S. tritici on plants free of Trichoderma and plants coming from Trichoderma-coated seeds, both inoculated with S. tritici. Effect of Trichoderma on Leaf Proteolysis Plants pretreated with Trichoderma Th5 and Tk11 isolates were selected to assess the balance between leaf apoplast proteolysis and protease inhibitory capacity. Table 22.5. Severity of necrosis and pycnidial coverage percentage in leaves with different Trichoderma spp. isolates in 2005. Trichoderma spp. isolates Th5 Tk11 Control Necrotic coverage (%)* 34.23 a 41.62 a 46.17 a Pycnidial coverage (%)* 46.91 b 57.50 a 59.14 a Note: *Each value is the mean of two replicates for necrotic and pycnidial coverage percentage. Means followed by the same letter are not signiﬁcantly different (P = 0.05) according to the LSD test. 304 C.A. Cordo Compared to controls, leaf proteolytic activity decreased by 40% 12 days after S. tritici inoculation. Conversely, it increased in plants produced by Th5-coated seeds. This was visible within 15–22 days after sowing (Table 22.6). Moreover, the increased proteolytic activity coincided with a decreased protease inhibitory capacity. Furthermore, the proteolytic activity remained higher in plants produced by Th5-coated seeds challenged with S. tritici. Proteolytic activity did not increase when comparing wheat plants grown without inoculation and plants produced by Tk11-coated seeds. The genus Trichoderma is a soilborne fungus whose survival on the phylloplane environment is difﬁcult (Perelló et al., 1997, 2003). Thus, a Trichoderma spp. population applied over wheat leaf decreases rapidly. Conversely, coating wheat seeds with T. harzianum is the ﬁnest application technique to control the leaf blotch caused by S. tritici. To understand how T. harzianum acts, the general features of the biological control set up by Viterbo et al. (2002) were tested. Leaves of plants formed by pre-coated seeds did not contain T. harzianum. This suggests that its biocontrol of leaf blotch is indirect and able to produce morphological or biochemical changes. As mentioned, inoculation with S. tritici decreases the apoplastic serine protease action in plants of susceptible cultivars (Segarra et al., 2002). Conversely, leaf blotch symptoms decrease in susceptible cultivar PRO INTA Molinero plants pretreated with some Trichoderma isolates after challenging with S. tritici. For this, the protease action in plants pretreated with isolates Th5 and Tk11 with high and low biocontrol capacity, respectively, was tested. The apoplastic protease activity increased only after treatment with Th5. In order to know if this increase ocurred independently of the inoculation with the pathogenic fungus, the kinetics of this phenomenon were analysed in plants pretreated with this isolate. The proteolytic activity was controlled by the leaf germin-like protease inhibitor already described (Segarra et al., 2003). In plants, the apoplast forms a space the pathogens necessarily must cross to colonize tissues. Therefore, it plays a central role in defence strategies, being a place where not only signals for plant response originate, but also where the proteins for defence mechanisms accumulate: glucanases, chitinases and proteases among others (Bowles, 1990). Within proteases must be mentioned the tomato P-69 induced by the citrus viroid (Vera and Conejero, 1988; Tornero et al., 1996), the tomato aspartil protease that degrades proteins related to pathogenesis (Rodrigo et al., 1988), the speciﬁc race join protease Table 22.6. Effect of S. tritici and Trichoderma spp. on leaf apoplast proteolytic and inhibitor activity. Treatment Days after sowing Protease activity (%) Inhibitor activity (%) T1 T2 T4 (Th5) T4 (Th5) T4 (Th5 T6 (Th5) T4 (Th11) T4 (Th11) 22 22 7 15 22 22 22 22 100 61 +/– 15 95 +/– 4 167 +/– 25 140 +/– 20 128 +/– 10 70 +/– 10 98 +/– 12 100 150 98 87 33 60 – – Note: In T1 (wheat plants without inoculum), the IWF (leaf intercellular washing ﬂuid) was obtained 22 days after sowing. In T2 (wheat plants inoculated with the pathogen) and T6 (wheat plants grown from Trichoderma spp. pre-coated wheat seeds and inoculated with the pathogen), the plants were inoculated with S. tritici 10 days after sowing and the IWF was examined 12 days after inoculation. In the case of plants grown from Trichoderma spp. pre-coated wheat seeds (T4), the IWF was examined 7, 15 or 22 days after sowing. For all treatments, the proteolytic and the protease inhibition activity was considered 100% in non-inoculated plants. Each value is the mean of two replicates. Septoria Leaf Blotch of Wheat in Argentina that processes the AVR9 of the compatible reaction tomato–Cladosporium fulvum (de Witt et al., 1985; Schaller and Ryan, 1996), two closely related subtilisin-like proteases that are associated with the defence response of tomato and encoded by the P69B and P69C genes (Jordá and Vera, 2000) and a unique 33-kDa cysteine protease mobilized in response to caterpillar feeding in maize lines that are resistant to feeding by several lepidopteran species (Pechan et al., 2002). Conclusions The relevant advances for Septoria leaf blotch of wheat fall into two classes. First, qualitative, as the conditions that allow inoculum transfer, permit infection and encourage sporulation; second, quantitative as, in a given agroecosystem, what factors in practice control pathogen regulation size. It was demonstrated that the distance between leaf layers with and without infection varied greatly according to both the architecture of the wheat cultivar and the latent period of the pathogen on the cultivar. The interaction of these factors, as was observed on the SMN collection, caused great variation in the potential for the spread of pathogens to the upper part of the crop. DNA restriction fragment polymorphism (RFLP) markers labelled with radioactive compounds were used to assess the potential for gene and genetic diversity and for gene ﬂow between geographically separated populations. The results on the genetic composition of two populations separated by 500 km show shared haplotypes. This has signiﬁcant implications for wheat-breeding programmes 305 that seek to incorporate resistance to S. tritici. In coincidence with Boerger et al. (1993), our evidence of gene ﬂow suggests that plant breeders in Argentina are driving the breeding process well. They are testing the resistance of their cultivars at many locations away from the area of local adaptation. The ﬁne scale of patterns with genetic variability suggests that plant breeders should use a wide spectrum of pathogen genotypes when testing wheat cultivars resistant to this pathogen in any location. Throughout the genetic evidence on the gene ﬂow between continents, it is possible to afﬁrm that lesions of leaf blotch of wheat could arise from seed transmission or could be attributed to a failure of isolation and a stray ascospore. In the face of the high environmental contamination produced by agrochemical products, new ecological alternatives are applied to control diseases in extensive plant cultures. So, biological control is a complementary strategy in the ecological management of wheat cultivation. These results suggest that the saprophytic fungus T. harzianum provokes a biochemical plant defence response, as has been reported previously. 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Zhan, J., Pettway, R.E. and McDonald, B.A. (2003) The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity, low mitochondrial diversity, regular recombination, and gene ﬂow. Fungal Genetics and Biology 38, 286–297. Part VI Alternative Control Strategies This page intentionally left blank 23 Review of Thecaphora amaranthicola M. Piepenbr., Casual Agent of Smut on Amaranthus mantegazzianus Pass. M.C.I. Noelting,1 M.C. Sandoval,2 M.M.A. Gassó1 and M.C. Molina1,3 1Instituto Fitotécnico de Santa Catalina, Facultad de Ciencas Agrarias y Forestales, UNLP, Llavallol, Buenos Aires, Argentina; 2Facultad de Ciencas Agrarias, UNLZ, Llavallol, Buenos Aires, Argentina; 3CONICET (Consejo Nacional de Investigaciones Cientiﬁcas Tecnicas) Abstract The amaranth (Amaranthus spp.) is becoming a socially and economically important crop due to the high level of quality proteins in its seeds and leaves. Among the factors that could limit the expansion of this crop are the smuts (Ustilaginales) that prevent normal development of the seeds. The objectives of the project were to: (i) characterize the pathogen which is responsible for smut on A. mantegazzianus, taking into account the cultural and morphobiometrical characters, and the germination of its teliospores; (ii) assess the incidence of smut in two amaranth cultivars; (iii) analyse fast techniques for the detection of the inoculum in seeds and plants cultivated in the ﬁeld; and (iv) identify any possible reservoirs of the pathogen on wild amaranths. The results obtained allowed the authors to determine that: (i) Thecaphora amaranthicola is the causal agent of smut on A. mantegazzianus; (ii) the cultivar Don Manuel was affected most by the smut (36% incidence); (iii) the residue and plastic card techniques are fast and efﬁcient for the detection of the pathogen inoculum; and (iv) the wild species A. hybridus and A. retroﬂexus are hosts of the pathogen. This is the ﬁrst report in the world of Thecaphora amaranthicola as a pathogen of the A. mantegazzianus cultivated species. Introduction The amaranth is an ancestral precolumbine crop that was cultivated by the Aztecs, Mayas and Incas and which remained relegated for a long time after being banned by the Spanish conquistadors. However, in the past few decades it has been subjected to numerous investigations with the objective of studying its nutritional value, improvement and adaptation to new areas of cultivation (Afolabi et al., 1981; Kulakow, 1987; Bressani, 1989; Espitia, 1991). The seeds and leaves of this plant have a very high level of quality proteins. This property makes the amaranth a valuable resource, especially appropriate for a population which lives in areas that are considered marginal for the cultivation of traditional cereals. The amaranth can be affected by pests and diseases. Among the diseases of fungal etiology which affect the normal development of its seeds are two species of smut, T. amaranthi (Hirschh) Vanky (syn. Glomosporium amaranthi) (Vánky, 1994) and  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 311 312 M.C.I. Noelting et al. T. amaranthicola M. Piepenbr. (Piepembring, 2000). The spores of both types of smut infect the ovaries, preventing normal development of the seeds. On the inside of the affected ovaries, hypertrophied sorus development takes place and these contain a mass of spores (teliospores). The species T. amaranthi was described and reported in wild species of amaranth (Vánky, 1985; Hirschhorn, 1986) and in cultivated species (Alcalde, 1995; Noelting and Sandoval, 2003). On the other hand, T. amaranthicola was described in only one wild species of amaranth collected in Ecuador (Piepembring, 2000). In Argentina, the data on this species are in a report where the symptoms caused by this smut on a cultivated species of amaranth are described (Noelting et al., 2005a). There are no records of T. amaranthicola on cultivated species of amaranth in the rest of the world. Owing to the little information available on the biological and epidemiological aspects of this smut, and that its spreading could have a negative effect on the propagation of the amaranth crop, it was decided to start the present study, the objectives of which were: 1. To characterize the pathogen which is responsible for smut on A. mantegazzianus, taking into account the cultural and morphobiometrical characters, and the germination of its teliospores; 2. To assess the incidence of smut in two amaranth cultivars; 3. To analyse fast techniques for the detection of the inoculum in seeds and plants; and 4. To identify any possible reservoirs of the pathogen on wild amaranths. Materials and Methods Morphological and cultural characterization of pathogen The taxonomic identiﬁcation of the fungus was carried out by observing the teliospores, the evolution of the germination process (Piepembring, 2000) and the characteristics of the cultivated colonies in vitro which developed in potato dextrose agar (PDA 2%). Samples of teliospores obtained from infected seeds of amaranth (A. mantegazzianus) were used in order to study these characters. The seeds were harvested in an experimental ﬁeld of the Instituto Fitotécnico de Santa Catalina, situated in the locality of Llavallol, in the south of the Buenos Aires Province, Argentina, in 2003. The teliospores were observed in an optical microscope and measured with a micrometric objective (Carl Zeiss Jena 7×) (n = 50). In order to observe the germination, the teliospores were disinfected with a solution of sodium hypochlorite (2%) for 5 min, rinsed three times with sterile distillate water and dried between sterile paper ﬁlters. To inoculate Petri plates, teliospores were streaked on the surface of the agar media to dilute the inoculum. The plates were incubated at 25 ± 2°C with a 16 h photoperiod. To assess the colony characteristics, sections 6 mm in diameter, obtained from a colony in active growth, were transferred to dishes with PDA. The material was incubated in a chamber at 25 ± 2°C with a 16 h photoperiod. After 14 days of incubation, the cultural and morphological characteristics (colour, edge, diameter) of the developed colonies were described. Incidence assessment The incidence assessment was carried out in seeds of two cultivars of A. mantegazzianus (cvs. Don Juan and Don Manuel) which had been harvested in 2004 and presented spontaneous infections with smut. Each panicle was harvested and threshed by hand. Once the material had been threshed, samples of 200 seeds/panicle were taken, determining the number of seeds infected with T. amaranthicola. The incidence was calculated by using the formula: Incidence (%) = Number of infected seeds Total number of analysed seeds × 100 Review of Thecaphora amaranthicola M. Piepenbr. Techniques for Detection of Inoculum Results and Discussion In seeds Morphological and cultural characterization of pathogen Samples of four different cultivated species of amaranth were used: A. mantegazzianus, A. caudatus, A. hypochondriacus and A. cruentus from Argentina, Bolivia and Mexico. The technique applied consisted in depositing 10 g of seed samples from the different countries between pieces of reticulated cellulose paper and then submitting them to three cycles of manual pressure. Next, the seeds were taken out of the paper and the residue contained in the reticules was observed with a stereoscopic magnifying glass (10×). The observations that tested positive for the presence of teliospores were conﬁrmed by means of a preparation, with a solution of lactophenol and cotton blue, and observed with an optical microscope (450×). In plants cultivated in ﬁelds In order to determine the infection in the crop, 4 cm × 6 cm plastic cards were used, with lithium grease as an adhesive on one side of the cards. Eighteen cards were distributed randomly, 9 in each of the cultivars of A. mantezzagianus (cvs. Don Juan and Don Manuel), hanging from the plants for a month. Each card was later analysed with a stereoscopic magnifying glass to detect the teliospores. Wild hosts of T. amaranthicola To detect natural reservoirs of the inoculum of this smut, a sampling that involved three wild species of amaranth, A. retroﬂexus L., A. hybridus L. and A. viridis L., was carried out in 2006. The plants of these species were located around a crop of A. mantegazzianus, as well as areas further away at a distance of 1.5 km. The panicles of the collected material were taken to the laboratory and observed with a stereoscopic magnifying glass and an optical microscope. 313 Morphobiometrical characteristics of teliospores The observations carried out in samples of infected A. mantegazzianus seeds allowed determination of the presence of spore balls with the following characteristics: ochre colour, globose to subglobose (40.78 µm × 34.23 µm) (Fig. 23.1a). Each spore ball was formed by 8–23 teliospores, polyhedral to cuneiform: spore walls were deeply corrugated on the central parts of the teliospores, as seen by SEM (Fig. 23.1b). No individual teliospores were observed in any of the samples analysed. This phenomenon coincides with the one observed in smuts that have grouped spores and which develop on different kinds of plants (Barrus and Muller, 1943; Andrade et al., 2004). The germination of the teliospores plated on PDA was initiated after 24 h of incubation generating phragmobasidia, followed by the development of lateral and terminal basidiospores. Variations in the number of germinated teliospores in each spore ball were observed (Fig. 23.2a,b,c). In addition to this, multiple germinations occurred simultaneously (Fig. 23.2b,c). The cultural characteristics of the colonies grown in laboratory conditions were as follows: velvety surface at the expense of the development of the mycelium, light beige colour, slightly serrated edges and softly lobated outline of the colony (Fig.23.2f). The growth of the colonies was slow, reaching a maximum diameter of 34 mm after 14 days of incubation. In the colonies analysed, no yeasty type of development characteristic of this type of fungus was found. According to analysis, T. amaranthicola, belonging to the Basidiomycota Subkingdom, Ustilaginomycetes Class, Ustilaginomycetidae Subclass, Ustilaginales Order, Glomosporiaceae Family, was identiﬁed as the causal agent of smut in A. mantegazzianus (Fig. 23.3). The morphological and cultural data shown complement the 314 M.C.I. Noelting et al. (a) (b) Fig. 23.1. Teliospore balls of Thecaphora amaranthicola: (a) under the light microscope (scale bar 10 µm); (b) SEM (scale bar 10 µm). pr (a) bl (b) (c) Coil (d) (e) (f) Fig. 23.2. Culture of Thecaphora amaranthicola on PDA: (a) single germination of a teliospore after 24 h; pr = probasidium; (b) and (c) multiple germination of teliospores, bl = lateral basidiospores; (d) hyaline basidiospores; (e) mycelia formation, coiling of hyphae (coil); (f) colony of T. amaranthicola developed on PDA after 17 days of incubation. Review of Thecaphora amaranthicola M. Piepenbr. 315 cb (a) (b) (c) Fig. 23.3. (a) Panicle of Amaranthus mantegazzianus; (b) healthy seeds (12×); (c) seeds of A. mantegazzianus infected with T. amaranthicola (12×), seed coats show irregularities (cb). preliminary information about the pathogen (Noelting et al., 2005a). Incidence The percentage of incidence varied between 10 and 36.66% (average rates) for the Don Juan and Don Manuel cultivars, respectively. These results apparently indicate the existence of resistance mechanisms, especially in the Don Juan cultivar. More studies would have to be carried out in the future in order to learn more about said mechanisms. Nevertheless, it cannot be discarded that the relatively high rates of incidence which were detected may be so because the pathogen could have been introduced by contaminated germplasms in the region. With respect to this, it can be stated that the growing interest in amaranth cultivation crop has originated the incorporation of seeds from several countries and, since this pathology had not been reported previously in cultivated species of amaranth, there are no controls for it in Argentina. Inoculum detection in seed samples Teliospores of T. amaranthicola were found in 50% of the analysed samples (Table 23.1). The technique applied is effective, fast and simple (Noelting et al., 2005b) compared with the test which consists of washing and ﬁltering seeds and is used for the detection of smuts in the seeds of many crops (ISTA, 1985). The inoculum (teliospores) was detected on and among the seeds; therefore, individualization of the teliospores with this technique offered information about infection as well as contamination in amaranth seeds. Furthermore, the retrospective character of the analysis led to the conclusion that T. amaranthicola was already present in cultivated species of amaranth in Argentina prior to its ﬁrst report (Noelting et al., 2005a). In plants cultivated in the ﬁeld The employment of cards to detect aerial inoculum in plants cultivated in the ﬁeld allowed the detection of teliospores (T. amaranthicola) in 55% of the cards analysed, as well as other fungal propagules. This technique is a sampling method by deposition or capture similar to those which use slides covered by an adhesive substance (Bugiani and Govoni, 1991). The results obtained from both A. mantegazzianus cultivars indicate that the T. amaranthicola teliospores are spread by the wind. Wild host of T. amaranthicola Seeds infected by T. amaranthicola from panicles of A. hybridus and A. retroﬂexus 316 M.C.I. Noelting et al. Table 23.1. Results of the analysis of amaranth seeds samples. Seed sample Locality Province Country Year Thecaphora amaranthicola A. cruentus cv. Don Armando A. cruentus cv. Don Guiem A. hypochondriacus cv. G. Covas A. hypochondriacus Se143 A. caudatus A. hypochondriacus cv. G. Covas A. hypochondriacus A. caudatus A. hypochondriacus A. cruentus cv. Se1MC A. mantegazzianus cv. Don Manuel A. mantegazzianus cv. Don Manuel Anguil Anguil Anguil Anguil Llavallol Luis Guillon – – Llavallol Luis Guillon Santa Rosa Colonia 25 de Mayo Llavallol Llavallol La Pampa La Pampa La Pampa La Pampa Buenos Aires Buenos Aires – – Buenos Aires Buenos Aires La Pampa La Pampa Argentina Argentina Argentina Argentina Argentina Argentina Mexico Bolivia Argentina Argentina Argentina Argentina 1995 1997 1999 1999 2003 2003 2004 2004 2004 2004 2005 2005 x x x x – – – – – x – – Buenos Aires Buenos Aires Argentina Argentina 2005 2005 x x A. mantegazzianus cv. Don Juan A. mantegazzianus cv. Don Juan plants were detected (Noelting et al., 2006). The infections of a spontaneous nature which were found in the two species of wild amaranth that affect many crops and which grow in a vast region of Argentina are thought to be of epidemiological interest. This is due to the fact that they may turn into ‘bridge’ species for the entrance of the inoculum and the spreading of smut to cultivated amaranth species. Conclusions The presence of T. amaranthicola in A. mantegazzianus (cultivated crop) is the ﬁrst report, not only in Argentina but also in the world, of smut as a pathogen. The inoculum detection techniques applied to samples of seeds and plants cultivated in the ﬁeld are appropriate as a fast method for identifying the presence of smut. Two species of wild amaranth, A. hybridus and A. retroﬂexus, are hosts of T. amaranthicola. The interest in the amaranth crop has originated an intense interchange of germplasms among several countries of America, Asia and Europe. This situation suggests the need to undertake a major study of the biological and epidemiological characteristics of the seedborne pathogens that as the smuts (T. amaranthi and T. amaranthicola) have negative incidence in the crop. References Afolabi, A.O., Oke, O.L. and Umoh, I.B. (1981) Preliminary studies on the nutritive value of some cereal-like grains. Nutrition Reports International 24, 389–394. Alcalde de L, M.A. (1995) Patógenos del amaranto (Amaranthus sp.) en el sur de la provincia de Córdoba, Argentina. Resúmenes IX Jornadas Fitosanitarias Argentinas 95. Andrade, O., Muñoz, G., Galdames, R., Durán, P. and Honorato, R. (2004) Characterization, in vitro culture, and molecular analysis of Thecaphora solani, the causal agent of potato smut. Phytopathology 94, 875–882. Barrus, M.F. and Muller, S.S. (1943) An Andean disease of potato tubers. Phytopathology 33, 1086–1089. Bressani, R. (1989) The proteins of grain amaranth. Foods Reviews International 51, 1338. Bugiani, R. and Govoni, P. (1991) Aerobiología e difusa delle pianta. Informatore Fitopatologico 11, 9–15. Espitia, R.E. (1991) Revancha: variedad mejorada de amaranto para los valles altos de México. In: Primer Congreso Internacional del Amaranto. INIFAP, Chapingo, Mexico, 64 pp. Review of Thecaphora amaranthicola M. Piepenbr. 317 Hirschhorn, E. (1986) Las Ustilaginales de la ﬂora argentina. Edition CIC, La Plata, Buenos Aires, 530 pp. International Seed Testing Association (ISTA) (1985) International rules for seed testing. Seed Science and Technology 13, 299–513. Kulakow, P.A. (1987) Genetics of grain amaranths. Journal of Heredity 78, 293–297. Noelting, M.C. and Sandoval, M.C. (2003) Patógenos fúngicos en amarantos cultivados. Boletin Sociedade Argentine de Botanica 38 (Suppl), 273. Noelting, M.C., Sandoval, M.C. and Astiz Gassó, M.M. (2005) Primer reporte en Argentina de Thecaphora amaranthicola como agente responsable del carbón en Amaranthus mantegazzianus (Res). Fitopatología 40, 76. Noelting, M.C., Sandoval, M.C. and Astiz Gassó, M.M. (2005) Técnica rápida para detección de carbones (Ustilaginales) en semillas de amaranto (Amaranthus spp.) (Res). Boletin Sociedade Argentine de Botanica 40 (Suppl), 164. Noelting, M.C., Astiz Gassó, M.M. and Sandoval, M.C. (2006) Especies silvestres de amarantos (Amaranthus spp.) hospedantes de Thecaphora amaranthi y T. amaranthicola (Ustilaginales) en la pcia de Buenos Aires (Res) XII Jornadas Fitosanitarias Argentinas. Catamarca, Argentina, pp. 312–313. Piepembring, M. (2000) New neotropical smut fungi. Mycological Research 105, 762–763. Vánky, K. (1985) Carpathian Ustilaginales. Symbolae Botanicae Upsalienses 24(2), 1–309. Vánky, K. (1994) New combinations proposed. Mycotaxon 51, 170. 24 Population Biology and Management Strategies of Phytophthora sojae Causing Phytophthora Root and Stem Rots of Soybean Shuzhen Zhang1 and Allen G. Xue2 1Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese Education Ministry, Northeast Agricultural University, Harbin, Heilongjiang, China; 2Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada Abstract Soybean is an important oilseed crop; it is also the richest source of protein. Root and stem rot pathogen Phytophthora sojae can reduce yield by up to 40%. Symptoms, disease cycle and genetic diversity of P. sojae are described. The paper is a valuable document listing molecular markers and the role of 14 resistance genes located at eight genomic loci in the development of disease-resistant soybean varieties. There have been only a few fungicides available for the control of P. sojae and their effects are limited. The development of pathogen resistance to these fungicides is not known. Little information is available on cultural and biological controls of P. sojae. More effective management of P. sojae will require integration of all available strategies to address all stages of the disease cycle. The integrated approach may prove a boon to growers using a variety of susceptible cultivars. Suitable cultural, chemical and biological methods are recommended as alternative control strategies. Introduction Phytophthora root and stem rot of soybean (Glycine max (L.) Merr.), caused by P. sojae Kaufmann and Gerdemann, is a destructive disease throughout the soybean-planting regions of the world (Schmitthenner, 1985). The symptoms were ﬁrst discovered as an unknown etiology in the state of Indiana in the USA in 1948, and subsequently in Ohio in 1951, but the causal agent was not described until 1958 (Kaufmann and Gerdemann, 1958). 318 The disease has since been reported from many countries, including Canada (Hildebrand, 1959), Australia (Pegg et al., 1980; Ryley et al., 1998), Argentina and Brazil (Wrather et al., 1997), China (Shen and Su, 1991) and the Republic of Korea (Jee et al., 1998). The disease is more prevalent when soil is saturated for prolonged periods of time and susceptible cultivars are planted (Grau et al., 2004). Diseased plants reduce yield by 10–40%, or a total crop loss when infection is severe (Anderson and Tenuta, 2003).  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Phytophthora Root and Stem Rots of Soybean Pathogen and Disease Symptoms P. sojae traditionally has been classiﬁed as a fungus due to its outward resemblance of growth habits and nutritional requirements. In fact, it is very distant from ‘true’ fungi evolutionarily and falls within the Kingdom Stramenopila (Förster et al., 1990; Harper et al., 2005), which constitutes a distinct branch of the eukaryotic evolutionary tree (Tyler, 2007). The pathogen may infect soybean at any stage of plant development and cause seed rot, seedling pre- and post-emergence damping-off and root and stem rots of soybean (Kittle and Gray, 1979; Athow, 1987). The seeds could be rotted by infection of P. sojae in both heavy and light sandy soils, after periods of cool and rainy weather. The damping-off, stem and root rot symptoms often appear shortly after emergence and during early ﬂowering when plants are under stress (Anderson and Tenuta, 2003). The infected seedlings have dull grey leaves and reddish, water-soaked lesions that occur from the base of the stem and slowly advance up the plant, and may collapse if the infection is severe. Symptoms on older plants are characterized by chocolate-brown discoloration extending from the soil line to the third or fourth node and into lower branches at 319 any time from the ﬁrst trifoliate to late R9 stage (Grau et al., 2004). Severely infected plants have few lateral roots, with almost no nodules and only a short portion of taproots left, and brown and drooping leaves remaining attached to the stem, even though the plants die. Disease Cycle P. sojae has a narrow host range and is restricted primarily to soybean, but there are reports that lupin, lucerne, bean and sweetclover could be infected using artiﬁcial inoculation in controlled environments (Erwin and Ribeiro, 1996). P. sojae has both asexual and sexual stages in the life cycle and produces sporangia, zoospore and chlamydospore in the asexual stage and oospore in the sexual stage, as shown in Fig. 24.1 (Tyler, 2007). Oospores are produced by the fusion of a female organ, called oogonium, and a male organ, antheridium. Chlamydospores are thick-walled spores that protect the organisms surviving through periods of abiotic stress. P. sojae can survive for many years in soil, mainly as oospores that are formed in the roots and stems of infected soybeans Secondary zoospore Motile zoospore Zoosporangium Cyst Mycelium Sporangium (attached or detached) Sexual reproduction Germination Oospore Fig. 24.1. Phytophthora sojae life cycle. Oogonium Antheridium Germinated cyst INFECTED PLANT 320 S. Zhang and A.G. Xue in large quantity and are released into the soil when these tissues decompose (Anderson and Tenuta, 2003). Oospores serve as the primary inoculum and germinate to produce sporangia under ﬂooded conditions or infective hyphae (Anderson and Tenuta, 2003). Zoospores do not have a cell wall and each has two ﬂagella and are released by ﬂooding. They can swim a short distance (1.0 cm or less) in saturated soil, but are disseminated primarily by moving ﬂood water. At the end of the motile period, which may last up to several days, zoospore movement becomes sluggish and jerky and encystment occurs (Schmitthenner, 2000). Zoospores can be attracted towards the compounds excreted by soybean root tips (Morris and Ward, 1992; Tyler et al., 1996). On reaching the root surface, the zoospores begin to encyst and germinate, and the hyphae penetrate directly between the cell walls of the epidermis (BeagleRistaino and Rissler, 1983). The infection process can be completed in 30 min in optimum conditions. On resistant cultivars, hypersensitive response (HR) may occur, the pathogen is contained in numerous necrotic or dead cells and there is no development of haustoria in the resistance interaction. However, there is no early HR reaction occurring in susceptible cultivar; the hyphae initially grow intracellularly and then form many haustoria in root cells, which remain alive in direct contact with the pathogen after infection for around 10 h (Enkerli et al., 1997) or approximately 12 h (Ward, 1990), when P. sojae is able to colonize host cells in an initial biotrophic phase of growth without triggering any response from the plant. After the initial infection, the pathogen begins to enter a necrotrophic growth mode and causes many host cells to die. The hypha penetrates from the epidermal cells of the root into the deep layers and vascular tissues. Isolation and Identiﬁcation of Physiologic Races of P. sojae P. sojae is known for the difﬁculty in isolation due to its slow growth. As a result, the pathogen is often covered by bacteria and saprophytic fungi. The commonly used methods of isolating P. sojae are the plant stem lesion and soil methods described by Dorrance et al. (2008). The protocol for plant stem lesion isolation is by surfacesterilizing cut-off tissues of symptomatic stems ﬁrst and incubating on the selective medium, e.g. PBNIC agar, to control bacteria and other fungi, such as Pythium. P. sojae has a distinct growth pattern on PBNIC agar, showing white mycelium 2–3 days after incubation. The mycelium is coenocytic and have branches almost at right angles, with curved tips. The asexual sporangium looks like an inverted pear and the round oospores on solid culture media can be seen 8–10 days later. Soil isolation is usually done by grinding the soil to ﬁne particles, ﬂooding it for 24 h, then draining and air-drying the soil until it cracks or pulls away from the side of the container, although it is still damp. The process is to break dormancy and induce germination of oospores in the infested soil. The soil is then used for planting a susceptible cultivar and P. sojae can be isolated readily from collapsed hypocotyls of emerging seedlings 5–6 days later using the same procedure described for plant stem lesion isolation. The common way to store P. sojae is to grow the pathogen on V8 juice agar slants for 2 weeks, then cover the culture with 2 ml of sterile deionized water and store the culture at 15°C. The fungus may be stored in such conditions for up to 3 years without losing its virulence and aggressiveness. For a longer-term preservation, P. sojae can be stored in liquid nitrogen for at least 4 years (Dorrance et al., 2008). Pathogenic and genetic diversity of P. sojae The pathogenetic variation of P. sojae was ﬁrst reported in 1958 (Kaufmann and Gerdemann, 1958). During the interaction process with the soybean varieties, the pathogenicity and virulence of P. sojae evolved rapidly and 55 physiologic races were identiﬁed Phytophthora Root and Stem Rots of Soybean based on their differential reaction on a set of 8 or 13 differentials with a single resistance gene. Of the 55 races, races 1 to 45 were identiﬁed using a set of 8 differentials (Bernard et al., 1957; Morgan and Hartwig, 1965; Schmitthenner, 1972; Schwenk and Sim, 1974; Haas and Buzzel, 1976; Laviolette and Athow, 1977; Keeling, 1979, 1982; Laviolette, 1983; White, 1983; Layton, 1986; Wagner and Wilkinson, 1992; Henry and Kirkpatrick, 1995; Abney et al., 1997) and races 46 to 55 on a set of 13 differentials (Ryley et al., 1998; Leitz et al., 2000). Several DNA-based molecular markers such as SSR (simple sequence repeat), rDNAITS (rDNA-internal transcribed spacer), RFLP (restriction fragment length polymorphism) and RAPD (random ampliﬁed polymorphism DNA) have been used successfully to identify the genetic variation and diversity of P. sojae. Whisson et al. (1992) used RFLP to conﬁrm sexual recombination of P. sojae in vitro and studied the segregation of avirulence genes. Föster et al. (1994) proposed that occasional outcrosses had been a major contributor to the origin of new physiological races of P. sojae, in addition to clonal evolution. Meng et al. (1999) used the RAPD method to study populations of P. sojae from Indiana, Iowa and Minnesota (USA) and found no correlation of populations with a geographic origin. Wang et al. (2003) analysed genetic diversity of 75 P. sojae isolates from China using the RAPD method and distinguished 12 genetic groups, but most of the isolates were clustered into one group and no relationship between clustering and geographic origin was found. Wang et al. (2006) studied the genetic variation among P. sojae in the USA and China and found that there existed higher genetic variations in populations in the USA compared to the Chinese populations based on the RAPD analysis. Gally et al. (2007) examined by RAPD analysis the diversity of 32 P. sojae isolates of different geographic origins from Argentina and detected intraspeciﬁc variability even among isolates of the same geographic origin. Xu et al. (2007) detected 17 P. sojae isolates from three locations in Heilongjiang Province, China, and demonstrated by sequence analysis the difference 321 between the base constitution of ITS1 and ITS2 among isolates. The 17 isolates were classiﬁed into three groups based on the ITS sequence and those isolated from the same region belonged to the same group, which showed the variation in geography. These studies demonstrated that molecular tools could be used to disclose the intraspeciﬁc diversity of P. sojae isolates both within and among geographic origins. Management Strategies The management strategies for prevention against Phytophthora root and stem rot at present are mainly by deployments of cultivars with race-speciﬁc or race non-speciﬁc resistance, or a combination of the two. Chemical methods and cultural practices like crop rotation and tillage and integrated management are used to a lesser extent. Screening for resources of race-speciﬁc and race non-speciﬁc resistance Race-speciﬁc resistance (Rps) genes in soybean have been used extensively to manage P. sojae (Dorrance et al., 2003). New sources of resistance to P. sojae have been reported, mainly from soybean varieties and germplasm in China, where soybean was originated. Lohnes et al. (1996) reported that the Rps1d gene was common in accessions from Anhui and Jiangsu Provinces after they evaluated 517 soybean germplasms collected from several provinces in central China. Kyle et al. (1998) investigated soybean accessions from southern China in response to several races of P. sojae and demonstrated that germplasm from Hubei, Jiangsu and Sichuan Provinces appeared to be valuable multi-gene resistance sources. Lv et al. (2001) screened 956 soybean accessions from north-east China (Heilongjiang, Jilin and Liaoning Provinces) and identiﬁed 23 varieties with resistance to both race 1 and race 25, the predominant and the most virulent races of P. sojae in the region, respectively. Zhang et al. (2007) evaluated 322 S. Zhang and A.G. Xue 530 soybean germplasms including 280 native soybean accessions and 250 commercial cultivars and found that the percentage of resistance in native soybean varieties was higher than that of the commercial cultivars. Similarly, Zhu et al. (2000, 2004), Li et al. (2001), Huo et al. (2005) and Jin and Zhang (2007) reported the identiﬁcation of P. sojae resistant germplasm with a number of Rps genes from wild soybean accessions and soybean varieties from China. In addition, Dorrance and Schmitthenner (2000) identiﬁed several soybean accessions with multi-gene resistance after evaluating 1015 plant introductions originated from the Republic of Korea. These single Rps genes, however, have often been short-lived, with an effective ‘life’ of 8–15 years, due to the emergence of new virulent races in response to selection pressure exerted by the continuous use of speciﬁc resistant cultivars (Schmitthenner and Van Doren, 1985; Ferguson, 1987; Schmitthenner et al., 1994; Abney et al., 1997; Ryley et al., 1998). With the known Rps genes defeated, race non-speciﬁc resistance or partial resistance, described as the ability of plants to survive root infection without displaying severe disease symptoms such as death, stunting or yield loss, is of great interest and gains more and more attention from soybean breeders (Buzzell and Anderson, 1982; Tooley and Grau, 1984; Schmitthenner and Van Doren, 1985). The strategy of the combination of race non-speciﬁc resistance with race-speciﬁc resistance is brought forward to provide long-term management of Phytophthora root and stem rot, as well as to avoid the boom-and-bust cycle of single gene deployment, since it reduces the severity of root rot and slows the rate of disease development (Buzzell and Anderson, 1982; Burnham et al., 2003b). Race non-speciﬁc resistance commonly had been evaluated under natural infection in the ﬁeld until recently, owing to the unavailability of a suitable laboratory procedure. Jimenez and Lockwood (1980) ﬁrst described a laboratory procedure for screening race non-speciﬁc resistance by growing soybean seedlings in cups that were placed in plastic trays containing a speciﬁed number of zoospores of P. sojae. Irwin et al. (1982) reported a laboratory assay by inoculating soybean seedlings with dry P. sojae mycelium for rapid determination of relative levels of race non-speciﬁc resistance. Dorrance et al. (2008) described a layer test and a tray test for screening soybeans for race nonspeciﬁc resistance to P. sojae. The layer test is done by placing inoculum of a 14-day-old P. sojae culture 5 cm below the seeds in cups containing coarse vermiculite and the amount of root rot and seedling death is rated 3 weeks after planting. The tray test is assessed by wound inoculation of a mycelial slurry on the root of 7-day-old seedlings and root rot is rated after 7 days. Using the layer test, Jia and James (2008) identiﬁed several accessions with high levels of race non-speciﬁc resistance compared with Conrade, the common known race non-speciﬁc resistant soybean variety to P. sojae. Resistance genes and marker-assisted selection A single dominant resistance gene has been widely explored since the ﬁrst resistance gene (Rps1a) was identiﬁed by Bernard et al. (1957). With the Rps1a defeated and the emergence of new races in response to selection pressure exerted by the continuous use of Rps1a, new Rps genes are identiﬁed. A total of 14 Rps genes including Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps7 and Rps8 at eight genomic loci have been reported so far (Bernard et al., 1957; Kilen et al., 1974; Laviolette and Athow, 1977; Mueller et al., 1978; Athow et al., 1980; Bernard and Cremeens, 1981; Athow and Laviolette, 1982; Ploper et al., 1985; Anderson and Buzzell, 1992; Burnham et al., 2003a). All of these loci have been placed on the soybean genetic map. Rps1 and Rps3 are mapped on molecular linkage groups (MLG) N and F, respectively (Diers et al., 1992; Demirbas et al., 2001; Burnham et al., 2003a). Rps2 (MLG J), Rps4 (MLG G), Rps5 (MLG G), Rps6 (MLG G), Rps7 (MLG N) and Rps8 (MLG A2) have also been mapped (Diers et al., 1992; Phytophthora Root and Stem Rots of Soybean Lohnes and Schmitthenner, 1997; Demirbas et al., 2001; Burnham et al., 2003a). Molecular markers have been used to facilitate selection for both single and multiresistance genes (Bent and Yu, 1999; Kumar, 1999). SSR and RFLP markers have been identiﬁed for Rps1 (Diers et al., 1992), Rps1a (Weng et al., 2001), Rps1b and Rps1c (Demirbas et al., 2001), Rps1d (Sugimoto et al., 2008), Rps1k (Kasuga et al., 1997; Bhattacharyya et al., 2005), Rps2, Rps3, Rps4, Rps5, Rps6 (Diers et al., 1992; Cregan et al., 1999) and Rps7 (Lohnes and Schmitthenner, 1997). With the development of the molecular biotechnique, more and more markers closer to the resistance genes will be used in markerassisted selection (MAS), which has been complementary to conventional breeding programmes and to shortening the breeding period. In some cases, genes for complete racespeciﬁc resistance that have already been defeated by new races of P. sojae may contribute to race non-speciﬁc resistance (Gebhardt and Valkonen, 2001), which appears to be controlled by several genes (Walker and Schmitthenner, 1984; Glover and Scott, 1998) and is more durable (Tooley and Grau, 1984). Several QTLs have been mapped to linkage groups for race non-speciﬁc resistance to P. sojae. Burnham et al. (2003b) used three recombinant inbred line (RIL) populations with the cultivar Conrad as the race non-speciﬁc resistance parent and identiﬁed two putative QTLs on MLG F and D1b + W from Conrade in all three populations. Han et al. (2008) used the RILs population of Conrade and OX760-6-1 as the race non-speciﬁc resistance and susceptible parent, respectively, and detected three QTLs, i.e. QGP1, QGP2 and QGP3, for Phytophthora root and stem rot tolerance. They further conﬁrmed that QGP1 was located on linkage group F and QGP2 in a different interval on linkage group F and QGP3 on linkage group D1b + W. Furthermore, an RIL population of a cross between Conrade and Hefeng 25 was constructed and four markers on three linkage groups, MLG D1b + W, MLG F and MLG A2, were identiﬁed as being associated signiﬁcantly with race non-speciﬁc resistance (Li et al., 2008). 323 Chemical, cultural, biological and integrated control The most commonly used chemical in the prevention of P. sojae is metalaxyl, which is an acylalanine fungicide speciﬁc to oomycetes. The fungicide is commonly applied as seed treatment (apron fungicide) and infurrow, spray or granule (ridomil fungicide) to reduce plant emergence loss and increase yields of susceptible varieties (Anderson and Buzzell, 1982; Guy et al., 1989). Since soybean is the only host of P. sojae in the ﬁeld, crop rotation is an effective means of reducing the severity of the disease. Although a short-term crop rotation may not allow for reduction of inoculum, it does prevent the immediate build-up of P. sojae populations (Schmitthenner, 1985). Because saturated soil favours the occurrence of P. sojae, tillage that could promote soil drainage is proven to be effective in reducing the infection period (Grau et al., 2004). Oospores could also be buried deeper in the soil by tillage (Workneh et al., 1998). Biological control has been considered a more natural and environmentally acceptable alternative to the existing chemical treatment methods (Cook and Baker, 1983; Baker and Paulitz, 1996). Several bacteria and fungi have been identiﬁed as potential bioagents in dualculture and greenhouse experiments. There are inevitable shortcomings for each of the prevention measurements, that is, the Rps genes in cultivars can be defeated by new races of P. sojae, and the varieties with race non-speciﬁc resistance, seed treatment, rotation and tillage cannot provide an effective control when disease pressure is high. A combination of two or more strategies to prevent the infection of P. sojae is very essential. The disease could be best managed with integrated strategies in a combination of deployment of cultivars incorporated into race-speciﬁc and race non-speciﬁc resistance genes, fungicide treatments, improved soil drainage and biocontrol. These management tactics can reduce inoculum in ﬁelds and limit the amount of water available for the pathogen to germinate and infect, therefore minimizing disease damage and increasing soybean production efﬁcacy. 324 S. Zhang and A.G. Xue Conclusions Owing to the shift of P. sojae races, use of race-speciﬁc resistance may quickly become inefﬁcient in management of the disease. More efforts are needed in the identiﬁcation and incorporation of multi-gene resistance and race non-speciﬁc or partial resistance into new soybean cultivars in the future for prevention against Phytophthora root rot and stem rot. Soybean cultivars with a combination of two types of resistance would be long-lived and more desirable by the soybean industry. Conventional breeding will still be the main method of resistance breeding in the foreseeable future but, with the rapid development of molecular technology, there will be more and more molecular markers closely linked to the Rps genes mapped, which could be useful in MAS to hasten the breeding and cultivar development process. To our knowledge, limited research has been carried out on the expression of genes which are possibly involved in soybean resistance to P. sojae, notwithstanding that 14 dominant Rps genes at 8 loci have been identiﬁed and resistance gene mapping and quantitative trait loci have been explored. Moy et al. (2004) reported the patterns of gene expression on infection of soybean plants by P. sojae race 2 and demonstrated that genes identiﬁed as strongly upregulated during infection included those encoding enzymes of phytoalexin biosynthesis and defence and pathogenesis-related proteins. A better understanding of the resistance mechanism in the P. sojae–soybean interaction at the molecular level is needed for effective gene deployments and resistance breeding. Research into these new disease management strategies is required with transgenic soybean cultivars, in order to provide solutions for future needs when transgenic technologies will be more acceptable. 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Journal of Plant Genetic Resources 5(1), 22–25. 25 Management of Fungal Pathogens – A Prerequisite for Maintenance of Seed Quality During Storage Anuja Gupta Indian Agricultural Research Institute, Regional Station, Karnal, Haryana, India Abstract Storage of seed is essential for any seed programme to sow the crop in the next season, to maintain buffer stock as an insurance against crop failure in times of drought, excessive rainfall or other natural calamities, to maintain parental lines for the production of hybrid seed, to conserve germplasm for breeding purposes and for seed trade at national and international levels. Availability of good quality seed at the right time and place is a basic prerequisite for sustaining agriculture. While maintenance of seed germination is of utmost importance to any seed person, preservation of seed quality in terms of its health status is equally important. A quality seed should have high genetic purity, physical purity, seed germination, seed vigour and good health status. Introduction Seed health is being recognized as one of the important criteria in evaluating seed quality. Seed health refers primarily to the presence or absence of disease-causing organisms such as fungi, bacteria and viruses, or animal pests such as nematodes and insects, or physiological disorders due to deﬁciency of trace elements. One of the major problems associated with crop production in India is the maintenance of the prescribed level of seed vigour and viability from seed harvest till the next sowing season. In our plans for attaining self-sufﬁciency in food grains, preventing their loss in storage is as important as the various measures to increase production. About 60–70% of the annual output is retained by the farmer. Storage facilities with the farmer, as well as with the traders, are far from satisfactory. Farmers and traders are not fully aware of the large savings that can be obtained by proper storage and preservation techniques. The important factors that determine the longevity of seeds are seed moisture, the type of storage container and storage environment. These factors generally interact, leading to a number of physiological and biochemical changes in the stored seeds, which result in deterioration of seed both in quality and quantity, especially in tropical and subtropical countries. According to a current estimate, 10% of food grain is lost in storage due to microbial spoilage and insect attack. The damage caused by rodents and insects is visible and therefore remedial measures are adopted for their control, but microbial spoilage of seeds/grains cannot be seen easily.  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 329 330 A. Gupta Microbial Spoilage of Seed during Storage Seeds are the end product of a series of steps that include sowing, growing, harvesting and threshing, wherein it becomes vulnerable to various pathogens/saprophytes. Among the different microbes, fungi form a major group of organisms that infest seeds. Nearly 150 species of fungi have been found associated with grains and seed in storage (Dharam Vir, 1974). Mechanical damage in the seeds, cracks, breaks or scratches in the pericarp or seed coat developed during threshing and processing substantially facilitate invasion by fungi, which ﬁnd their way to the storage warehouses. The fungi found associated with seeds during storage are known as storage fungi. They can grow without free water, on media with high osmotic pressure, at RH = 70–90%. Some common storage fungi include species of Aspergillus, Penicillium, Rhizopus, Fusarium, Cladosporium, Alternaria, Mucor, Chaetomium, Epicoccum, etc. Discoloration and distortion of seeds is a major degrading factor because of seedborne infection. Other common manifestations are reduction in seed size, seed rots, shrivelling of seeds, seedling decay and pre- and postemergence mortality and abnormalities. The fungi primarily invade the embryo and in the early stages of infection, the seed may appear normal but, due to well-established infections, these embryos are killed and the seeds appear darkened. These fungi are responsible for a decrease in market value, germinability and nutrition of the produce, making the grains unﬁt for human consumption and reducing the viability of the seed. Excessive fungal growth may also result in heating, caking and decay. The seed may thus become totally spoiled, darkened or charred by prolonged exposure to the heat generated during storage, which brings about biochemical changes leading to the production of toxins and loss in seed weight. The invasion by fungi leads to physical and chemical changes in the seeds. Prasad et al. (1990) observed changes in the amount of fatty acids, glycerol, sugar and amino acids in radish seeds infected with Aspergillus ﬂavus. Dube et al. (1988) reported changes in starch, fatty acids and sugars in wheat grains infected with A. ﬂavus and A. niger. Mishra and Dharam Vir (1991) observed higher milling losses ranging from 34.0 to 58.6% in discoloured rice grains. Joshi et al. (1988) reported 73% reduction in starch content in stored pearl millet seeds infected with storage fungi and an increase in the amount of reducing sugars and phenolic contents. Bilgrami and Sinha (1983) have reported aﬂatoxin contamination in maize, groundnut and a variety of agricultural foods and feeds. Vaidehi (1997) showed that storage fungi lowered the quality of maize grains due to the biochemical changes they brought about. Storage fungi may be present as dormant spores or mycelium on the seed surface or below the pericarp, which activate and multiply at a phenomenal rate under favourable storage conditions. Seed Mycoﬂora The initial mycoﬂora of the seeds can give an idea of the type of fungi that can initiate the process of deterioration in storage. With an increase in the storage period, there is an increase in the incidence of storage fungi and a decrease in seed germination (Gupta and Singh, 1990, 1993). Loss in germination due to storage fungi may be attributed to several factors. A toxin produced by A. ruber kills the tissues in the embryonic axes of pea seeds in advance of infection (Harman and Nash, 1972). Wheat seed infected with Aspergillus spp. and imbibed with water becomes a jelly-like mass, suggesting that cell wall degrading enzymes may be involved. In contrast, pea and squash embryos are killed without physical invasion by fungi, indicating the involvement of diffusible toxins (Harman and Pﬂeger, 1974). Mitochondria isolated from the embryonic axes of A. ruberinfected pea seeds were less active than those from non-infected seeds, suggesting Management of Fungal Pathogens that mitochondria damaged by fungi play a role in seed deterioration (Harman and Drury, 1973). Gupta et al. (1989) observed a decrease in the amount of volatile aldehyde compounds with increased levels of fungi on the seed and treatment with benomyl increased these compounds, indicating control of fungi and increased germination. An increase in seed mycoﬂora is correlated directly to an increase in FFA content and leaching of solutes (especially electrolytes and water-soluble sugars) with advancing storage period (Gupta, 2003). Agarwal (1980) demonstrated that seed deterioration in okra, carrot and onion seeds was accompanied with leakage of sugars. Analysis of exudates from the seeds showed that the permeability of the membrane increased with the deterioration of seeds during ageing (Dadlani and Agarwal, 1983). According to Chen et al. (1998), with an increase in the fatty acid contents of different seeds like wheat and brassicas, storage potential decreased. Ramamoorthy and Karivaratharaju (1986) also found that with an increase in the storage period, the oil and protein content in groundnut seeds decreased gradually, while free fatty acid content increased, accompanied by a loss in seed viability under ambient storage conditions. Thus, during storage, especially under an ambient environment, seeds produce changes due to fungal activity, resulting in deterioration of their quality (Zagrebenyer and Bern, 1998; Gupta and Aneja, 2004). 331 Seed Mycoﬂora and Seed Viability The incidence of fungal ﬂora associated with different seeds is low initially, but it increases with an increase in the duration of storage and subsequently there is a decrease in seed viability. With the advancing storage period, the ﬁeld fungi become limited and the produce becomes infested with storage fungi. A signiﬁcant negative correlation (r = –0.793) between seed viability and seed mycoﬂora has been observed with advancing storage period (Table 25.1), and consequently a decrease in seed viability. Treatments and seed mycoﬂora Seed treatments, especially with fungicides like captan, thiram or mancozeb, restrict the growth of mycoﬂora on the seeds and maintain better seed viability (Gupta, 2003). Moreno et al. (1985) suggested the use of fungicides to protect the viability of corn seeds. In another study, Moreno and Ramirez (1983) recorded that after 330 days of storage at 26°C with 75% RH, the germination of untreated corn seed was only 61%, while it ranged from 68 to 90% in seeds treated with different fungicides either singly or in combination. The incidence of storage fungi was also very low in treated seeds. Kushwaha and Raut (1994) reported that seeds treated with thiram and stored in poly-lined bags suppressed most of the fungi. Asalmol and Zade (1998) also observed Table 25.1. Inﬂuence of seed mycoﬂora on seed viability and seed moisture during storage. Storage period (months after seed treatment) Seed germination* (%) Seed mycoﬂora* (%) Seed moisture* (%) 0 2 4 6 8 10 12 Correlation coefﬁcient (r) 92.0 86.5 92.8 85.3 50.8 44.3 38.3 –0.872 1.05 2.4 3.4 2.7 5.9 4.6 5.0 7.8 5.9 6.6 9.9 8.6 8.9 8.8 0.35 Note: *Average of 20 treatments. 332 A. Gupta Fungal inhibition (%) that pre-storage seed treatment helped to improve the shelf life of seeds and checked seed mycoﬂora during storage. Fungicide seed treatments were found to restrict the growth of mycoﬂora on different vegetable seeds (Gupta and Singh, 1993). The incidence of Colletotrichum dematium associated with chilli seed at the time of storage was 5%. Seed treatment with captan controlled the pathogen just after its application. Other fungicides like thiride and carbendazim controlled the pathogen after 5 months of storage, whereas in the untreated control the pathogen persisted up to 7 months in a cloth bag and up to 15 months in an airtight container (Gupta et al., 1992). Thiram, bavistin and captan could control more than 96, 93 and 90% of the fungi associated with paddy seed as against 72% and 65% in hinosan and emisan + streptocycline treatments, respectively, after 17 months of storage under ambient conditions (Fig. 25.1). Mancozeb (78.6%) was most effective in the control of seed mycoﬂora on soybean seed during storage, followed by thiram (65.1%), bleaching powder (13.1%) and nimbecidine (10.1%) during storage (Fig. 25.2). Onion seed variety Phule Safed dried at 6–7% moisture content and treated with 0.2% carbendazim could be stored safely in 700 gauge polyethylene bags for 32 months, as against 24 months in untreated seed (Mahajan et al., 2001). Pumpkin seed treated with iodine-based halogen mixture at 3 g/kg seed and stored in 700 gauge polythene bags maintained seed quality. The disease, ginger yellow, was controlled effectively by seed treatment with 0.1% carbendazim (Rana and Sharma, 1995). Sandhu (1989) reported seed dressing in the form of slurry with benlate at 1 g/kg and dry seed treatment with brassicol at 2.5 g/kg resulted in 100% and 93.9% inhibition of germination of pea seeds after their storage for 1 year. However, captan (0.25%) and bayletan (0.1%) treatments enhanced germination by 5.21 and 11.88%, respectively. He also found that in steam-sterilized soil, germination of poor quality seeds of pea variety Punjab-87 was enhanced from 17 to 56%. They were further enhanced up to 69% in seeds treated with captan. Van Toai et al. (1986) observed that only reduced quality seed of soybean responded to fungicide seed treatment under prolonged storage. The accelerated ageing germination (AAG) results of the 24-month-old methanolwashed seeds were lower than the AAG results of the unwashed seeds (the fungicides were removed from the treated seeds by methanol prior to the AAG test), but signiﬁcantly higher for all cultivars than the AAG values of the untreated seeds. The fungicidal seed treatments, in addition to protecting the seeds and seedlings during imbibition and germination, also helped to maintain the seed quality of soybean during storage. 100 80 60 40 20 0 Thiram Bavistin Captan Hinosan Emisan + streptocycline Seed treatments Fig. 25.1. Effect of different seed dressings on the control of seed mycoﬂora on paddy seed during storage. Management of Fungal Pathogens Occurrence (%) 333 Inhibition (%) Occurrence/inhibition (%) 100 80 60 40 20 Untreated Bleaching powder Neembicidine Thiram Mancozeb 0 Seed treatments Fig. 25.2. Inﬂuence of seed dressings on the occurrence and inhibition of seed mycoﬂora on soybean seed during storage for 15 months. Treatments and seed viability The inﬂuence of seed treatments on seed viability and vigour is not apparent during the early period of storage, but becomes signiﬁcant on prolonged storage. In some crops like mung bean, mustard, muskmelon, etc., the effect of seed treatments on seed germination is insigniﬁcant during storage, but in other crops like cowpea, sorghum, chillies, fenugreek, spinach and soybean, etc., seed treatments have a signiﬁcant effect on maintaining seed viability for longer duration under ambient storage conditions. Mung bean cv. PS-16 retained seed dormancy for up to 6 months of storage and viability for more than 36 months of storage (Table 25.2). Chickpea cv. P-256 treated with fungicides like thiram and ABC dust retained more than 85% germination for up to 24 months of storage. Thiram-treated seed had better germination compared to ABC dust treatment, and re-treatment with thiram further enhanced seed germination. The germination of soybean seeds, cv. P-16, treated with mancozeb or thiram stored in poly-lined cloth bags (polythene bags of 400 gauge kept inside a cloth bag) remained above the prescribed standard of certiﬁcation (70%), even on the 15th month of storage after seed harvest, whereas germination of both treated and untreated seeds stored in cloth bags fell below the certiﬁcation standards on the 11th month of storage. Seed treatment with mancozeb and thiram resulted in signiﬁcantly better root and shoot lengths of seedlings as compared to other treatments. Seedling vigour measured in terms of seedling dry weights and/or seedling lengths closely follow the pattern of seed germination. The results of treating mustard seeds with different seed dressings are shown in Table 25.3. The seed germination of wheat variety HD-2329 remained above the prescribed standard of certiﬁcation (85%) for up to 20 months of storage under ambient conditions when treated with carboxin, as against 16 months in untreated seeds. The stacking of seed bags (8 bags of 40 kg each stacked one above the other) in the seed warehouse had an insigniﬁcant effect on the viability 334 Table 25.2 A. Gupta Treatments to enhance storability of legume seeds under ambient storage. Crop seeds Effective seed treatment at 2 g/kg seed Cowpea Mung bean Chickpea Captan/thiram*/mancozeb *** Thiram* Storability in months after seed harvest** 28 > 36 References Gupta and Singh, 1990 Gupta and Singh, 1990 Gupta and Singh, 1990 Note: *Seed treatment at 2.5 g/kg seed; **period that seed viability remained above the prescribed standards of certiﬁcation; ***germination in treated and untreated seeds on a par. Table 25.3. Treatments to enhance storability of oilseeds under ambient storage. Effective treatments Crop seeds Seed treatment at 2 g/kg seed Storage container Soybean Mustard Mancozeb/thiram* *** Poly-lined bag Poly-lined bag Storability in months after seed harvest** 15 24 Note: *Seed treatment at 2.5 g/kg seed; **period that seed viability remained above the prescribed standards of certiﬁcation; ***germination in treated and untreated seeds on a par. of both treated and untreated seeds. However, varietal differences were observed with respect to the storability of the seeds. Wheat seed of the HD-1553 variety maintained more than 85% seed germination for up to 15 months of storage, irrespective of seed treatments. Germination in untreated seeds of the HD-2285 variety fell below the certiﬁcation standard on 9 months of storage as against seeds treated with captan, where germination remained above 85% for up to 15 months of storage. In the HD-2009 variety, the germination of seeds treated with carbendazim, captan or thiram was above the prescribed standard, even after 21 months of storage as against 15 months in untreated seeds. The decline in germination started at a faster rate after 21 months of storage and reached zero level after 39 months, irrespective of variety or fungicide seed treatment when stored under ambient conditions. In paddy, seed germination remained above the prescribed standard of certiﬁcation (80%) for up to 20 months after seed harvest and both the seed treatments and storage containers had insigniﬁcant effect on the viability of the stored seeds. The results of fungicidal seed treatments of sorghum cv. PC-9 are shown in Table 25.4. Among vegetable seeds, fungicidal seed treatments also inﬂuenced germination signiﬁcantly in spinach cultivar Pusa jyothi and fenugreek (Pusa kasuri) seeds (Table 25.5). However, the effect of seed treatments was insigniﬁcant in brinjal (Pusa kranti) and muskmelon (Pusa madhuras) seeds. The germination of brinjal and palak seeds remained above the certiﬁcation standards (70% and 60%, respectively) for up to 18 months of storage after seed harvest. Spinach seeds treated with fungicides like captan or mancozeb had higher seed germination. Muskmelon seeds were stored for 42 months after seed harvest without any substantial loss in seed viability under ambient storage conditions. Fenugreek seeds retained viability for up to 30 months and treatment improved seed germination as against untreated seeds. Fungicide treatments improved seed germination by about 5–7% on the 30th month of storage after seed harvest, but thereafter their inﬂuence on seed germination was negligible. The germination of chilli seeds Management of Fungal Pathogens 335 Table 25.4. Treatments to enhance storability of cereal seeds under ambient storage. Crop seeds Effective seed treatment at 2 g/kg seed Storability in months after seed harvest** Wheat Sorghum Paddy Carboxin* Captan/thiram*/brassicol/carbendazim/mancozeb *** 20 21 20 Note: *Seed treatment at 2.5 g/kg seed; **period that seed viability remained above the prescribed standards of certiﬁcation; ***germination in treated and untreated seeds on a par. Table 25.5. Treatments to enhance storability of vegetable seeds under ambient storage. Effective treatments Crop seeds Seed treatment at 2 g/kg seed Spinach Brinjal Chilli Muskmelon Fenugreek Mancozeb/brassicol *** Thiram* *** Thiram*/captan/carbendazim Storage container Airtight Storability in months after seed harvest** 18 18 19 42 30 Note: *Seed treatment at 2.5 g/kg seed; **period that seed viability remained above the prescribed standards of certiﬁcation; ***germination in treated and untreated seeds on a par. remained above the minimum prescribed standard (60%) for 19 months in airtight containers as against 10 months when stored in cloth bags, irrespective of fungicide treatments. However, thiram gave higher seed germination. Adverse effects of copper-oxychloride fungicide (CuO) have been reported on vegetable seeds during storage (Gupta et al., 1996). Fenugreek, brinjal and muskmelon seeds treated with CuO recorded 37, 50 and 36% germination as against 69, 83 and 80% in untreated seeds after 18, 30 and 24 months of storage, respectively. However, in spinach seed, the CuO treatment retained germination on a par with other seed dressings. Paddy seeds of parental lines of paddy IR58025A and IR58025B retained seed longevity above the prescribed standards (80%) for up to 5 years after seed harvest when stored under controlled conditions (temperature = 15°C; RH = 30%), as against 2 years under ambient storage conditions in both the parental lines (Fig. 25.3). The germination of paddy seeds of both the parental lines stored in poly-lined bags (76.62%) was signiﬁcantly higher than seeds stored in cloth bags (72.05%). Seed treatment with thiram and captan also improved seed germination as against the untreated control under both storage conditions (Fig. 25. 4). The different treatments also inﬂuenced the germination of parental lines of pearl millet (MS841A, MS841B and D23) during storage (Gupta, 2007). Seeds of MS841A, MS841B and D23 retained germination above minimum seed certiﬁcation standards (MSCS) (75%) for up to 16, 16 and 20 months after seed harvest, respectively. As in paddy, the germination of pearl millet seeds (Fig. 25.3) stored under controlled conditions (82.25%) was signiﬁcantly higher than seeds stored under ambient conditions (66.43%). Thus, storage under low temperature can prolong the longevity of the precious seeds of the inbred parental lines of paddy and pearl millet. 336 A. Gupta Controlled conditions A Controlled conditions B Ambient conditions A Ambient conditions B 100 Germination (%) 80 60 40 20 0 0 Fig. 25.3. 8 12 16 24 28 Storage months 36 48 60 Effect of storage conditions on seed germination in parental lines of paddy during storage. Thiram Captan Untreated 100 90 Germination (%) 80 70 60 50 40 30 20 10 0 0 8 12 16 24 28 36 48 60 Storage months Fig. 25.4. Effect of seed treatments on seed germination in parental lines of paddy during storage. Treatment of pearl millet seeds of different parental lines with bioagent T. viride maintained 78.30% germination during storage that was on a par with thiram (78.34%), captan (78.16%) and carbendazim (77.37%), as against 74.27% in the untreated control and 59.58% in P. ﬂuorescence treatment (Fig. 25.6). Also, the seeds stored in polythenelined cloth bags maintained higher seed germination in both paddy and pearl millet than the seeds stored in cloth bags (Fig. 25.7). Role of Storage Environment in the Perpetuation of Fungi The lifespan of seed is highly inﬂuenced by storage conditions, especially temperature Management of Fungal Pathogens 337 Effect of storage conditions on germination in bajra seeds Controlled storage Ambient storage Germination (%) 100 80 60 40 20 0 0 8 16 20 24 32 Storage months Effect of storage conditions on seed germination in pearl millet. Germination (%) Fig. 25.5. Bavistin Captan Thiram Trichoderma viride Pseudomonas fluorescence Untreated 90 80 70 60 50 40 30 20 10 0 0 8 16 20 24 32 Storage months Fig. 25.6. Effect of seed dressings on seed germination in pearl millet. and relative humidity (RH). The effects of temperature and RH (and its subsequent effect on seed moisture) of the storage environment are highly interdependent. Most crop seeds lose their viability when RH is about 80% and the temperature varies from 25 to 30°C. This hot and humid environment is congenial for the activity and growth of microorganisms, which leads to deterioration in seed quality. The expression of the mycoﬂora depends essentially on the temperature and humidity conditions of seed warehouses and also on the intergranular atmosphere of the seed. The moisture in the seed is present either on the seed surface or in the internal tissues, from where it moves to the surface and evaporates and is dependent on the RH of the atmosphere. At different RH levels, the equilibrium moisture content (MC) varies with different seeds. The MC of seeds ranges from 3 to 7, 6 to 10 and 9 to 14% at 20, 45 and 75% RH, respectively. According to Harrington’s rule of thumb (Harrington and Douglas, 1970): ● ● For each 1% decrease in seed moisture content, the storage life of the seed is doubled. For each 10°F (5.6°C) decrease in seed storage temperature, the storage life of the seed is doubled. 338 A. Gupta Germination (%) Poly-lined bag Cloth bag 90 80 70 60 50 40 30 20 10 0 0 8 16 20 Storage months (a) Jute bag 24 32 Poly bag Germination (%) 100 80 60 40 20 0 0 (b) 8 12 16 24 28 36 48 60 Storage months Fig. 25.7. Effect of storage containers on seed germination in pearl millet (a) and paddy (b) seed during storage. ● The arithmetic sum of the storage temperature in degrees F and the per cent RH should not exceed 100, with no more than half the sum contributed by the temperature. However, this rule is valid only when the seed moisture varies from 5 to 14%. Storage fungi are unable to grow and multiply if the MC of the stored produce is 12% or less. The optimum moisture for many types of seed is 6–8%, at which even damage by insects reduces (Dharam Vir, 1996). It was observed that the per cent of seed viability was highest at low temperatures and RH and short storage periods, but it decreased with increased storage period. Temperatures above 35°C are reported to cause rapid dete- rioration of soybean seeds in 6 months of storage (Nkang and Umoh, 1996). Under normal storage conditions, the temperatures and relative humidity of the seed warehouse ﬂuctuates with the environment. The temperature range in a seed warehouse located in Karnal varies from 13 to 33°C and 18 to 38°C (Fig. 25.8). The relative humidity of the seed warehouse varies from 51 to 75%. Since seed moisture is a function of RH, so it changes with variations in the RH of the seed warehouse. Seed dressings do not affect the MC of seeds appreciably, but storage containers do seem to affect it. It is higher in seeds stored in cloth bags as against those in poly-lined cloth bags because cloth bags, being pervious, allow a free ﬂow of air from the surrounding atmosphere. Management of Fungal Pathogens Max. temp. (°C) RH (%) 40 80 35 70 30 60 25 50 20 40 15 30 10 20 5 10 0 January April June September Relative humidity (%) Temperature (°C) Min. temp. (°C) 339 0 December Months Fig. 25.8. Ambient conditions in a seed warehouse at Karnal during the year. Although seed MC, type of storage container and storage temperature are interrelated, high temperatures hasten the deterioration of high-moisture seeds by increasing the metabolic activity of hydrolysed substrates and enzymes. Hence, maintaining these factors at low levels in the seed warehouses can improve the longevity of the seeds. Persistence of seed dressings during storage Suitable dressing at proper dosage and its uniform distribution on the seed is equally important for proper effect of seed treatments. This also goes a long way in enhancing the storage life of seeds, especially under ambient conditions in the seed warehouses. Dharam Vir (1977) observed that organomercurials retained their bioefﬁcacy for a longer period as compared to antibiotics and dithiocarbamates, which degrade and become biologically ineffective after storage of treated paddy seeds for 1 year. The persistence of fungicides on the seed is of paramount importance as it determines the longevity of effective seed treatment during storage. Certain fungicides, irrespective of the amount of dressing pres- ent on the seed, inhibit the fungi strongly, while others exhibit weak or almost nil inhibition. The storage container also seems to inﬂuence the residual activity of the chemical on treated seeds during storage. Vyas and Nene (1971) reported minimum loss of thiram on the seeds of cowpea, maize, paddy and soybean in tin boxes as compared to polyethylene bags, polyethylenelined cotton bags or hessian bags. Gupta and Chatrath (1983) found that the quantity of thiram on soybean seeds decreased gradually with increase in the storage period and fungicide degradation was maximum when the seeds were stored in cloth bags, followed by paper and alkathene-lined jute bags. Sastry and Chatrath (1984) correlated the persistence of carbendazim on wheat seeds with storage conditions and type of container. The quantity of fungicide on seed decreased with the increased storage period, but the least loss was of seed stored in polythenelined jute bags followed by polypropylene polyethylene, cloth and jute bags, and storage at 30°C resulted in more degradation as compared with lower temperatures. Lakshmi and Gupta (1997) reported a signiﬁcant reduction in the quantity of thiophanate methyl on soybean seed with an extended period of storage. Maximum persistence of fungicide was found on seeds 340 A. Gupta stored in polythene-lined jute bags, followed by polypropylene polyethylene bags. Gupta (2002) observed a loss of 10–100% in the activity of different chemicals during storage. The loss was more in treated seeds stored in cloth bag packaging compared to treated seed stored in a polythene bag inside a cloth bag. The loss in the activity of the chemicals apparently may be due partly to evaporation of the active compound of the chemical and partly to diffusion of the compound into the seed. Raju and Chatrath (1978) reported that the process of the degradation of fungicides was inﬂuenced by storage conditions. Besides, other factors such as environmental changes or physiological changes may also be responsible for the depletion of the activity of the chemicals. However, this loss in the activity of the chemical can also be correlated to the presence of seed mycoﬂora during storage. The loss of activity is lower, the incidence of fungal ﬂora is lower and seed germination is higher. Management of Storage Fungi to Preserve Seed Longevity Seed longevity can be maintained either by reducing or preventing the fungal inoculum, or by creating unfavourable conditions for their growth. Management strategies should include practices both at preharvest and postharvest stages. Proper storage and application of safe chemicals as postharvest treatments can control seed mycoﬂora effectively and reduce losses due to storage fungi considerably. Seed treatment is one of the most effective, safe and economic technologies which protects the seed from microbial deterioration, and thus improves its health status during storage and also ensures better ﬁeld emergence and seed yield. before the crop is harvested. It is essential to keep the crop healthy and disease free. Use of pathogen-free/certiﬁed seed material is the most effective method of disease-free seed production. Disease-free seed production should be planned in safe areas and seasons where disease development is restricted or absent. Preharvest sprayings with suitable chemicals, namely fungicide or biocontrol agents, and harvesting the crop at proper maturity also help to maintain seed quality during storage. Stress conditions during plant growth also inﬂuence seed longevity. Discoloration of seeds by fungi occurs when the crop is in the ﬁeld. Govindrajan and Kannaiyan (1982) observed reduction in grain discoloration of rice through preharvest spraying with copper oxychloride. Seed discoloration in paddy increased with higher levels of nitrogen and phosphorus and decreased with larger spacing in the ﬁeld (Misra and Dharam Vir, 1992). According to Deka et al. (1996), application of maneb at boot leaf stage, followed by spraying with common salt, was highly effective in reducing discoloration in paddy grains. The association of fungi is likely to be greater in regions where the produce is harvested in the wet season. Indira and Rao (1968) observed higher association of storage fungi in samples obtained from areas with high humidity. Misra and Kanaujia (1973) considered the presence of antifungal substances in the seed coat of some oilseeds to be the reason for less storage fungi. Nair (1982) reported fewer fungi on seeds of Luffa acutangula because of their thick and hard seed coat, which has a low moisture-holding capacity. Varietal differences with regard to their susceptibility to fungal attack during storage has been observed by Sheeba and Ahmed (1994), who found higher fungal incidence on seeds of high-yielding varieties of paddy as compared to local cultivars. Preharvest management strategies Postharvest management strategies For seed to remain healthy during storage, management strategies need to be followed from the time the crop is in the ﬁeld, i.e. Initial seed quality, seed moisture, storage temperature and RH play an important role Management of Fungal Pathogens in determining seed longevity. It is essential to avoid mechanical injuries to seed during harvesting/threshing. The produce needs to be dried properly to safe moisture levels before storage. The maximum drying temperature recommended for vegetable crop seed is 35°C. Sun drying of seeds can be practised at the farmer’s level. The seeds are usually packed in gunny bags or cloth bags. Moisture-proof containers, hermetically sealed cans, polyethylene pouches or polylined aluminium foil packets are usually used for high-value, low-volume seeds, but it is essential to dry the seed to 5–6% moisture level before packing in these containers because moist seeds tend to deteriorate faster in sealed containers in comparison to ordinary containers. Pre-storage seed treatment improves the shelf life of the seed, protects it from microbial deterioration and ensures better seed germination and better ﬁeld stand. Many horticultural crops are propagated by stems, roots, leaves, tubers, corms, rhizomes, suckers, grafts and other vegetative stocks besides seed. This propagative material may carry several pathogens which cause different diseases, thereby affecting their ﬁeld establishment. The pathogens present in the soil may also hamper ﬁeld establishment of these propagules. Adopting proper seed treatment technology can reduce most of these problems. This technology is beneﬁcial as it involves less wastage of chemicals, greater control over application, less environmental pollution, low risk for operators, minimum man power, is independent of weather conditions and has less deleterious effects on the treated material. Lal (1975) reported propionic acid and potassium metabisulphite as effective against A. niger, A. ﬂavus, P. oxalicum and A. alternata on wheat and maize grains. Acetic acid and propionic acid has proved effective against A. ﬂavus and C. lunata on groundnut kernels. According to Vaidya and Dharam Vir (1986, 1987), sodium metabisulphite and propionic acid checked the growth of Aspergillus and Penicillium sp. on groundnut kernels. After ensuring the quality of seed material meant for storage, it is also essential to ensure that the seed warehouses 341 where the material has to be kept is clean, dry, cool and properly aerated. The seed material should be packed in clean and, if possible, new containers. If old containers are being used, they should be disinfected or fumigated properly to avoid any carryover pathogens. Storage structures should not permit entry of water by seepage from the ground or walls. Low temperature retards the development of storage fungi on seeds and so is advisable, especially for low-volume, highvalue seeds. It is also essential to ensure that the seed material meant for storage should be of high quality. The material should be stacked on wooden pallets, maintaining a proper distance from the walls and ceilings. The material should be checked regularly for the development of any pests and efﬁcient remedial measures must be employed immediately to keep them under control. Thus, disease management of stored grains requires optimum storage conditions and deployment of treatments that do not pose any health hazards to consumers. Conclusions Seed storage is highly inﬂuenced by several intrinsic and extrinsic factors. Among them, genotypes, seed treatment and storage containers assume a prime role in successful seed storage. Studies are required to identify disease-free and disease-prone areas for seed production, as healthy seed produces healthy crops. Certiﬁcation standards for many seedborne diseases need to be developed. The development of suitable cost-effective packaging material for safe and prolonged storage of seeds is also needed. Identiﬁcation of suitable pre-storage treatments with suitable agrochemicals and botanicals for the seeds is another important ﬁeld which needs further work. Some research has been initiated on an eco-friendly approach against diseases in the ﬁeld, but their efﬁcacy during storage needs elucidation. The methods available for the proper application of seed/soil dressings also need further reﬁnement. The application of microorganisms as agents for 342 A. Gupta the biocontrol of plant diseases in agriculture is an important alternative to chemical fungicides. Halogenation of seeds has also proved a better seed storage treatment for prolonging seed viability (Dharmalingam et al., 2000). Invigoration treatments, namely the effect of hydration–dehydration or invigoration with different salt solutions on substandard seed lots for microbial growth, need elucidation. Mid storage hydration– dehydration treatment helps to maintain vigour, viability and productivity of crop seeds (Basu, 1994; Mandal et al., 2000). Priming improves biological seed treatment, where the primed seeds of pea and French bean show superiority over other treatments (Rawat and Kumar, 2003). Pelleting of seeds is also advantageous, as the application of pesticides, micronutrients, biofertilizers or plant leaf powders can be incorporated into the seed for improvement in germination. Some biotechnological approaches may also be exploited by incorporating genes for better storability of seeds. The production of artiﬁcial seeds has unravelled new vistas in plant biotechnology. These synthetic seeds are artiﬁcially encapsulated propagules used for sowing as a seed and they possess the ability to convert into a plant under in vitro or in vivo conditions. The preservation of viability and vigour of somatic embryos and synthetic seeds is one of the problems which has to be solved prior to applying synthetic seed technology practically. One of the future uses of synthetic seeds would be in germplasm conservation through cryopreservation. With the introduction of transgenic crops, it has become all the more important to ensure the quality of seeds that are traded across borders. The genetically modiﬁed seeds need to be assessed very carefully for any contamination with seedborne pathogens, which can be accomplished by using the modern tools of biotechnology. Although Bt genes have proved to be quite effective in short-term protection against insect damage, there are concerns that widespread use of Bt varieties will accelerate the development of resistance to Bt in target pests. Thus, to facilitate seed trade, strict quarantine measures at national and international level are necessary in restricting high-risk diseases. References Agarwal, P.K. (1980) Relative storability of seeds of ten species under ambient conditions. Seed Research 8, 94–99. Asalmol, M.N. and Zade, V.R. (1998) Effect of seed treatment on storability of seeds of different crops. Seed Research 26, 53–56. Basu, R.N. (1994) An appraisal of research on wet and dry physiological seed treatment and their applicability with special reference to tropical and subtropical countries. Seed Science and Technology 22, 107–126. Bilgrami, K.S. and Sinha, K.K. (1983) Aﬂatoxin contamination in agricultural commodities with reference to maize and groundnut. Phytopathology 73, 253–263. Chen, X.L., Chen, S.P. and Lu, X.X. (1998) Studies on the seed storability of different types within the crop. Scientia Agricultura Sinica 31, 89–91. Dadlani, M. and Agarwal, P.K. (1983) Mechanism of soybean seed deterioration. Plant Physiology and Biochemistry 10, 23–30. Deka, B., Ali, M.S. and Chandra, K.C. (1996) Management of grain discoloration of rice. Indian Journal of Mycology and Plant Pathology 26, 105–106. Dharam Vir (1974) Study of some problems associated with post-harvest spoilage of seeds and grains. In: Raychaudhri, S.P. and Verma, J.P. (eds) Current Trends in Plant Pathology. Botany Department, Lucknow University, Lucknow, India, pp. 221–226. Dharam Vir (1977) Efﬁcacy of fungicides. xx. Studies on biological activities of some antibiotics and fungicides during storage. Pesticides 11, 39–40. Dharam Vir (1996) Seed treatment with fungicides for control of diseases in ﬁeld and storage. In: Agnihotri, V.P., Prakash, O., Kishen, R. and Mishra, A.K. (eds) Diseases Scenario in Crop Plants, Volume II. International Books and Periodicals Supply Services, New Delhi, pp. 199–216. Management of Fungal Pathogens 343 Dharmalingam, C., Vasantha, R., Malarkodi, K. and Lakshmi, S. (2000) Halogenation treatment to safeguard pulse seeds in storage under ambient conditions. Seed Research 28, 42–46. Dube, S., Shukla, H.S. and Tripathi, S.C. (1988) Changes in sugar and protein content of wheat due to Aspergili. Indian Phytopathology 41, 633–635. Govindrajan, K. and Kannaiyan, S. (1982) Fungicidal control of grain infection. International Rice Research Newsletter 7, 1. Gupta, A. (2002) Residual efﬁcacy of seed dressings during storage using bioassay technique. Indian Phytopathology 55(1), 99–101. Gupta, A. (2003) Role of seed mycoﬂora in deterioration of soybean seed during storage under ambient conditions. In: Singh, D.P. (ed.) Implications of Plant Diseases on Produce Quality. Kalyani Publishers, Ludhiana, India, pp. 85–96. Gupta, A. (2007) Integrated management of seed health for prolonging seed storability in parental lines of pearlmillet (Pennisetum glaucum). Indian Journal of Agricultural Science 77(4), 212–219. Gupta, A. and Aneja, K.R. (2004) Seed deterioration in soybean [Glycine max (L.) Merrill] cultivars during storage – physiological attributes. Seed Research 32(1), 26–32. Gupta, A. and Singh, D. (1990) Viability of fungicide treated seeds of mungbean and cowpea in storage. Seed Research 18, 70–76. Gupta, A. and Singh, D. (1993) Effect of fungicidal treatment on seed viability and mycoﬂora of stored vegetable seeds. Seed Research Special Volume 1, 270–276. Gupta, A., Singh, D. and Maheshwari, V.K. (1992) Effect of containers on the viability of fungicide treated chilli seeds. Seed Research 20, 160–161. Gupta, A., Singh, D. and Maheshwari, V.K. (1996) Effect of copper oxychloride treatment on sorghum in different containers. Agricultural Science Digest 16, 39–41. Gupta, J.P. and Chatrath, M.S. (1983) Persistence of Thiram on soybean seed in storage. Indian Phytopathology 36, 263–266. Gupta, R.P., Mehra, U. and Pandey, U.B. (1989) Effect of various chemicals on viability of onion seed in storage. Seed Research 17, 99–101. Harman, G.E. and Drury, R.E. (1973) Respiration of pea seeds (Pisum sativum) infected with Aspergillus ruber. Phytopathology 63, 1040–1044. Harman, G.E. and Nash, G. (1972) Deterioration of stored pea seed by Aspergillus ruber: evidence for involvement of a toxin. Phytopathology 62, 209–212. Harman, G.E. and Pﬂeger, F.L. (1974) Pathogenicity and infection sites of Aspergillus species in stored seeds. Phytopathology 64, 1339–1344. Harrington, J.F. and Douglas, J.E. (1970) Seed Storage and Packing. Applications for India. NSC Ltd., New Delhi, 222 pp. Indira, K. and Rao, J.G. (1968) Storage fungi in rice in India. Kavaka 14, 67–76. Joshi, S., Williamson, D., Sharma, M. and Iyer, S.R. (1988) Combined effect of fungi on hexoses, pentoses and phenolic contents of Pennisetum typhoides (Bajra) grain during storage: a biochemical estimation. Proceedings of the Indian National Science Academy 58 B, 149–153. Kushwaha, L.S. and. Raut, N.D. (1994) Effect of seed treatment, containers and storage period on seed health of soybean (Glycine max). Seed Tech News 24, 69–70. Lakshmi Shantha, B. and Gupta, J.P. (1997) Persistence of thiophanate-methyl on stored soybean seed. Indian Phytopathology 50, 358–361. Lal, S.P. (1975) Studies on storage fungi of wheat and maize. PhD thesis, IARI, New Delhi. Mahajan, P.D., Padule, D.N., Perane, P.R and. Mate, S.N (2001) Improvement of storage life of seeds of onion (Allium cepa l.,) var Phule Safed. In: Proceedings of 2001 National Seminar on Seed Science and Technology in New Millennium – Vistas and Vision. Department of Studies in Applied Botany, Seed Pathology and Biotechnology, Manasagangotri, Mysore, India, 86 pp. Mandal, A.K., De, B.K., Saha, R. and Basu, R.N. (2000) Seed invigoration treatment for improved storability, ﬁeld emergence and productivity of soybean (Glycine max L.) Seed Science and Technology 28(2), 349–355. Mishra, A.K. and Dharam Vir (1991) Assessment of losses due to discoloration of paddy grains. I. Loss during milling. Indian Journal of Mycology and Plant Pathology 21, 277–278. Misra, A.K. and Dharam Vir (1992) Effect of different agronomic practices in incidence of seed discolouration of paddy. Indian Journal of Plant Pathology 22, 44–48. Misra, R.R. and Kanaujia, R.S. (1973) Studies on certain aspects of seed borne fungi II. Seed borne fungi of certain oilseeds. Indian Phytopathology 26, 284–294. 344 A. Gupta Moreno, M.E. and Ramirez, J. (1983) Protective effect of fungicides on corn seed stored with low and high moisture contents. Seed Science and Technology 13, 285–290. Moreno, M.E., Mandogaro, L., Mandoze, M. and Valevcin, G. (1985) Use of fungicides for corn seed viability preservation. Seed Science and Technology 13, 235–241. Nair, L.N. (1982) Studies on mycoﬂora of seeds of some cucurbitaceous vegetables. Journal of the Indian Botanical Society 61, 343–345. Nkang, A. and Umoh, E.O. (1996) Six month storability of ﬁve soybean cultivars as inﬂuenced by stage of harvest, storage temperature and relative humidity. Seed Science and Technology 25, 93–99. Prasad, B.K., Rao, R.N., Narayan, N., Singh, R.N., Rahman, A., Singh, S.P., Daya, S. and Shankar, U. (1990) Changes in sugar and aminoacid contents in stored radish seeds due to Aspergillus ﬂavus. Indian Phytopathology 42, 457–460. Raju, T.N. and Chatrath, M.S. (1978) Persistence of thiram on stored wheat seed. Indian Phytopathology 31, 323–326. Ramamoorthy, K. and Karivaratharaju, T.V. (1986) Storability and biochemical composition of groundnut seed as inﬂuenced by packaging and seed treatment. Indian Agriculturist 30, 101–106. Rana, K.S. and. Sharma, B.K. (1995) Management of ginger yellows in storage and ﬁeld. In: Gupta, V.K. and Sharma, R.C. (eds) Integrated Disease Management and Plant Health. Scientiﬁc Publishers, Jodhpur, India, pp. 203–206. Rawat, A. and Kumar, J. (2003) Improving biological seed treatment through solid matrix priming. In: Sovenir and Abstracts of 5th National Symposium on Biocontrol Agents for Sustainable Management of Pests. GBPUAT, Pantnagar, Uttaranchal, India, 18–20 December 2003, 169 pp. Sandhu, K.S. (1989) Effect of different seed and soil treatments on the viability of pea seed. Plant Disease Research 4, 43–47. Sastry, R.K. and Chatrath, M.S. (1984) Persistence of carbendazin on stored wheat seeds. Indian Phytopathology 37, 637–640. Sheeba, T. and Ahmed, R. (1994) Variation in seed borne mycoﬂora of paddy in Mangalore Taluk of Karnataka. In: Singh, T. and Trivedi, P.C. (eds) Vistas in Seed Biology. Printwell, Jaipur, India, pp. 220–228. Vaidehi, B.K. (1997) Seed mycoﬂora in maize – an appraisal. In: Tewari, J.P., Saxena, G., Mittal, N., Tewari, I. and Chamola, B.P. (eds) New Approaches in Microbial Ecology. Aditya Books Pvt. Ltd., New Delhi, pp. 337–357. Vaidya, A. and Dharam Vir (1986) Efﬁcacy of fungicides XXXIV. Evaluation of antifungal chemicals for control of post-harvest spoilage of groundnut caused by Aspergillus niger and A. ﬂavus. International Journal of Tropical Plant Diseases 4, 173–176. Vaidya, A. and Dharam Vir (1987) Efﬁcacy of fungicides XLVII. Evaluation of antifungal chemicals for control of post-harvest spoilage of groundnut caused by Penicillium spp. Indian Journal of Mycology and Plant Pathology 17, 241–244. Van Toai, T.T., McDonald, M.B. Jr and Staby, G.L. (1986) Cultivar, fungicides seed treatment and storage environment interactions on carry over soybean seed quality. Seed Science and Technology 14, 310–312. Vyas, S.C. and Nene, Y.L. (1971) Degradation of thiram on treated seed in storage. JNKVV Research 7, 121. Zagrebenyer, D. and Bern, C.J. (1998) Variety Effect on Carbon Dioxide Production and Free Fatty Acid Development in Soybeans. American Society of Agricultural Engineers, No. 98182, Department of Agricultural and Biosystems Engineering, Davidson Hall, Iowa State University, Ames, Iowa, 17 pp. 26 Controlling Root and Butt Rot Diseases in Alpine European Forests Paolo Gonthier Department of Exploitation and Protection of Agricultural and Forestry Resources (DIVAPRA), Plant and Forest Pathology, University of Torino, Grugliasco, Italy Abstract Alpine European forests comprising of conifers and broadleaf trees at lower altitudes are facing major problems of poor regeneration and occurrence of numerous fungal diseases. Fungal organisms like Armillaria mellea and Heterobasidion annosum are taking their toll on a large number of conifers. These two pathogens are responsible for most of the root and butt rot diseases in natural forest stands. Diagnosis of disease can be done by macro/micromorphology of basidiomata. The wood-inhabiting fungi can also be identiﬁed by taxon-speciﬁc primers using PCR. This chapter deals with various biological and cultural control strategies and the promotion of disease-tolerant plants, which can reduce the occurrence of diseases. Integrated disease management plans are suggested for different species as found suitable in the Aosta Valley of the western Italian Alps. Introduction Mountains and uplands cover approximately one-ﬁfth of the earth’s surface and about one-tenth of the world’s population lives in mountain regions (Ives et al., 1997). Mountain forests have drawn growing attention in the past few decades in both North America and Europe. It has been hypothesized that if no forests existed in the Alps, humans would not inhabit most of the valleys (Motta and Haudemand, 2000). Forests protect cities and villages against avalanches, landslides, debris ﬂows and rockfalls. They ﬁx surface soil, prevent erosion and play an essential role in water resource management. They inﬂuence climate and air quality. At present, the main functions afforded by alpine forests are protection, tourism and recreation, wood production, landscape and nature conservation. Substantial economic and social changes in mountain areas over the past few decades have modiﬁed forest use drastically. Traditional forest functions (i.e. wood production) have been abandoned, while the importance of other functions has grown. Human activity has transformed mountain forests radically in various ways: large forest areas have been destroyed and the natural composition of forests has been modiﬁed through logging and thinning (Motta and Haudemand, 2000). European alpine forests comprise mostly of conifers, while broadleaves are generally widespread at lower altitudes, where they may form signiﬁcant stands. Only eight native coniferous tree species are present in the alpine region (Ozenda, 1985): Abies alba Miller (silver ﬁr), Picea abies (L.)  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) 345 346 P. Gonthier Karsten (Norway spruce), Larix decidua Miller (European larch), Pinus cembra L. (Swiss stone pine), P. sylvestris L. (Scots pine), P. uncinata Miller (mountain pine), P. mugo Turra (dwarf mountain pine) and P. nigra Arnold (Austrian pine). These conifers can grow in pure or mixed stands, depending on the site. Most forests in the Alps are naturally regenerated and current guidelines of forest management are aimed at maintaining adequate levels of natural regeneration. Another trait differentiating alpine forests from other forests located in ﬂat or even in mountain areas is that in alpine forests, given their prominent protective function, clear cut followed by artiﬁcial planting is generally forbidden. Furthermore, mixed, irregular or uneven-aged stands are supported locally and maintained through appropriate silvicultural practices (e.g. selective cutting, ‘forêt jardinée’) where such features fulﬁl particular functions better (i.e. protection, landscape). Several forests in the Alps, most of which are protected forests, are affected by major problems (Mayer, 1982), including a lack of regeneration, a scarcity of mediumaged trees, insufﬁcient stability and increasing vulnerability to natural disturbances. Plant pathogens, including those causing root diseases or butt rots, may behave as natural disturbances. In addition to causing severe economic losses, they are reported to inﬂuence patterns and processes in forest ecosystems and to be affected by forest development and landscape characteristics, as well as by human activities (Castello et al., 1995; Hansen and Goheen, 2000). This chapter reviews the signiﬁcance and the epidemiology of the most important and widespread root and butt rot diseases of alpine forests, as well as the most effective and promising control strategies to ﬁght them. Root and Butt Rot Pathogens, Their Signiﬁcance, Ecology and Infection Biology Root and butt rot fungi are important components of forest ecosystems worldwide. Although a large number of lignicolous fungal species are reported on conifers in European mountain areas, including the Alps (Breitenbach and Kränzlin, 1986, 1991, 1995; Bernicchia, 2005), only a few of them are aggressive organisms having a signiﬁcant impact on forests (Table 26.1). All these fungi belong to the Basidiomycota and are necrotrophic tree pathogens causing wood decay. They are facultative pathogens, being able to survive saprotrophically on dead wood. They may be classiﬁed either as white rot or brown rot agents depending on the component of the plant cell wall they are able to utilize, i.e. lignin or cellulose, respectively. The infection biology of wood decay fungi in living trees has been reviewed previously (Rayner and Boddy, 1986). In general, primary infections occur by means of airborne meiospores, which allow for the infestation of new forest areas. Some of these fungi may also operate a secondary, vegetative spread, allowing for the expansion of individuals established through primary infection. Depending on the tree pathogen, this expansion may occur vegetatively through root grafts or contacts, leading to a tree-to-tree contagion, or by free growth of the fungus in the soil through rhizomorphs or mycelial cords. In some pathosystems, insect vectors are essential for the transmission of wood decay fungi (Slippers et al., 2002). The relative importance of primary and secondary infection is signiﬁcant not only for our understanding of the epidemiology and population biology of these fungi, but also for control and management purposes. Pathogens like Armillaria spp. and Heterobasidion spp. are able to spread secondarily. When this happens, there is a carry-over of the pathogen into new generations; in this case, novel attacks are not necessarily caused by new primary infections, but by the inoculum established at that site previously. Most root and butt rot agents are wound pathogens able to gain entry into the trees through wounds or lesions. Some of them are obligate wound pathogens (e.g. Stereum sanguinolentum), while others are facultative wound pathogens (e.g. Heterobasidion spp.). Table 26.1. Summary of characteristics of the most important root and butt rot fungi present in alpine forests. Main hosts1 Rot type Disease/symptoms Infections Reference2 Armillaria mellea (Vahl:Fries) P. Kummer sensu lato Climacocystis borealis Kotl. & Pouzar Fomitopsis pinicola (Sw.) P. Karst. Heterobasidion annosum (Fr.) Bref. sensu lato Several conifer and broadleaf species Root rot and mortality; occasionally heart decay: butt rot Heart decay: butt rot Brown, cubic Decay: butt and stem rot White, pocket rot Root rot and mortality; heart decay in roots, butt and stem Laetiporus sulphureus (Bull.) Murrill sensu lato Larch, Norway spruce; oaks and chestnut among broadleaves Brown, cubic Root contacts; active pathogenesis through rhizomorphs Through wounds3 on roots and bole By spores through stem wounds By spores through fresh stump surfaces or wounds; root contacts and grafts By spores through wounds Guillaumin and Legrand, 2005 Norway spruce, silver ﬁr, larch, pines Silver ﬁr, Norway spruce, pines Conifers; mostly pines, Norway spruce, silver ﬁr White, ﬁbrous, wet, often with black zone lines White, mottled rot Onnia tomentosa (Fr.) P. Karst (= Inonotus tomentosus) Norway spruce, Scots pine White, pocket rot Phaeolus schweinitzii (Fr.) Pat. Stereum sanguinolentum (Alb. & Schwein.) Fr. Norway spruce, silver ﬁr, pines Norway spruce Brown, cubic Heart decay: butt and stem rot; Bernicchia, 2005; root rot and mortality when Butin, 2005 decay reaches the root system involving sapwood By spores through deep Root rot and mortality Tainter and root wounds; vegetatively Baker, 1996 through root contacts and grafts Heart decay: butt and stem rot Barrett and Greig, Vegetatively in the roots 1985; Butin, 1995 Butt and stem rot By spores through wounds Vasiliauskas et al., 1996; Solheim, 2006 Pale brown, stringy rot Solheim, 2006 Butin, 1995; Bernicchia, 2005 Asiegbu et al., 2005 Controlling Root and Butt Rot Diseases Fungi Note: 1Species are listed based on their in-ﬁeld susceptibility, the most susceptible listed ﬁrst; 2reference for the infection biology; 3wounds are considered of mechanical origin. 347 348 P. Gonthier Only a few root and butt rot disease agents do not need wounds to gain entry into the tree (e.g. Armillaria spp.). At the same time, most root and butt rot fungi are weak, secondary pathogens, being unable to attack vigorous trees. However, some of them are not (i.e. Heterobasidion spp., Onnia tomentosa) and behave as primary pathogens, which may cause signiﬁcant disease with or without pre-existing tree stresses. Weakened physiological conditions caused either by primary or by secondary pathogens may then trigger off attacks by other, secondary parasites (i.e. bark beetles) (Tainter and Baker, 1996; Jakuš, 2001). Wood decay fungi may rot standing trees in two ways, either starting from the cambium and then proceeding inward (sapwood decay), or by decaying the central portion of roots, bole and stem (heart decay) (Rayner and Boddy, 1986). When the cambium, functional xylem or outer sapwood are involved, several physiological functions in trees may be altered. This is particularly true for decays affecting the root system or the collar, which generally lead to a relatively rapid death of the host. In the second type of decay, only the smaller woody roots are killed, whereas the larger ones, the bole or the stem may remain physiologically functional for a long time (Rayner and Boddy, 1986; Gonthier et al., 2003). Very few root and butt rot diseases have been studied in detail, for instance those caused by A. mellea or H. annosum species complexes (reviewed in Shaw and Kile, 1991; Woodward et al., 1998a; Fox, 2000; Asiegbu et al., 2005; Guillaumin, 2005). Current knowledge on the other pathosystems here described is still very limited. For instance, only scanty information is available on Climacocystis borealis. This fungus is reported as a saprophyte and secondary pathogen (Bernicchia, 2005), being able to cause a typical heartwood rot in the roots and the bole, which seldom reaches more than 2–3 m in height (Solheim, 2006). Sometimes, the sapwood is also colonized. The borealis rot is a characteristic white mottle rot (Bernicchia, 2005) which, on a closer look, is cubic with white mycelium in between (Solheim, 2006). Cubes are easily distinguishable from those of typical cubical brown rots in that they are much ﬁner (1–2 mm). The fungus is rarely lethal, but has been reported to cause signiﬁcant economic losses locally and to amplify the mechanical instability of trees during storms, especially in mountain, mature Norway spruce stands (Rigling et al., 2005). Fomitopsis pinicola and Laetiporus sulphureus sensu lato are two powerful wooddestroying fungi, responsible for brown, cubic rots. In the alpine region, the former is associated mostly with severely damaged silver ﬁr and Norway spruce trees. The second species, which recently was investigated phylogenetically (Vasaitis et al., 2009), attacks mostly larch trees in alpine forests (Butin, 1995). They are wound pathogens, the second being able to progress towards the root system and here producing root rot and sapwood decay. Phaeolus schweinitzii is another widespread brown rot agent. It is reported on all coniferous tree species growing in the Alps and it causes a heart decay of the roots and the bole, spreading up to 1–2 m into the stem (Bernicchia, 2005). The infection biology of this fungus is still largely unknown. The pathogen is believed to infect the roots through the mycelium (Barrett and Greig, 1985; Bernicchia, 2005), which is unable to extend freely in the soil over long distances (Barrett and Greig, 1985). Thus, spores do not play any primary role in the infection. However, they are an important source of soil infestation (Barrett, 1985). It should be noted that other ways of infection, such as mechanical butt wounds or root contacts with diseased trees, have also been suggested for this pathogen (Tainter and Baker, 1996). There is very scanty information on the signiﬁcance of O. tomentosa in alpine forests. Its presence is probably overlooked. In fact, despite differences in the rot type, the fungus can be confused easily with P. schweinitzii since the basidiomata of the two species display similar macroscopic traits (Butin, 1995). O. tomentosa was found on Norway spruce and Scots pine trees. This fungus infects trees by spores through deep root wounds and is also capable of secondary spreading through root contacts and grafts (Tainter and Baker, 1996). Controlling Root and Butt Rot Diseases S. sanguinolentum is incapable of secondary spreading but is a very strong wound colonizer, especially on Norway spruce. Every wound, from root to top, is vulnerable to infection, even the oldest ones (Vasiliauskas et al., 1996). Very important factors for infection are wound size and depth (Solheim, 2006). Wound rot is initiated by injuries caused by bark-stripping red deer, as well as by harvest-induced injuries (Cermák et al., 2004). In Norway spruce, the potential economic losses caused by cut-off waste wood or low-quality logs are of considerable magnitude and wound rot affects the trees’ stability negatively (Cermák et al., 2004). Several taxa in the A. mellea and H. annosum species complexes are responsible for most of the root and butt rot diseases of conifers in natural forest stands and plantations throughout the northern temperate regions of the world (Kile et al., 1991; Asiegbu et al., 2005). A. mellea sensu lato encompasses about 40 biological species of varying geographic distributions, host ranges and virulence (Pegler, 2000), seven of which are present in Europe (Marxmüller and Guillaumin, 2005): A. mellea (Vahl: Fries) P. Kummer sensu stricto, A. ostoyae (Romagnesi) Herink, A. borealis Marxmüller and Korhonen, A. gallica Marxmüller and Romagnesi, A. cepistipes Velenovský, A. tabescens (Scopoli) Emel and A. ectypa (Fries) Lamoure. This last species is only marginally important since it is a non-lignicolous, nonparasitic species. All the European species may be found in the alpine area, although some of them (i.e. A. tabescens, A. mellea), being thermophilic, are more common at low elevations and in the Mediterranean region (Marxmüller and Guillaumin, 2005). There is general agreement on the fact that basidiospores play a marginal role in wood colonization and infection (Guillaumin and Legrand, 2005). Armillaria root disease may spread either through root contacts or rhizomorphs, depending on the Armillaria species. Root contacts are essential for the spread of A. tabescens and A. mellea, which is characterized by fragile and short-lived rhizomorphs, while the less pathogenic A. gallica and A. cepistipes generally infect through rhizomorphs. The ability of A. ostoyae and 349 A. borealis to produce rhizomorphs and to infect trees in this way is variable (Guillaumin and Legrand, 2005). In Europe, H. annosum sensu lato comprises three species, responsible for losses estimated at more than 800m/year (Woodward et al., 1998b). H. parviporum Niemelä & Korhonen primarily causes butt rots in Norway spruce, but it has also been reported to kill Scots pine saplings and attack exotics. H. abietinum Niemelä & Korhonen is commonly associated with root or butt rots in trees of the genus Abies, while H. annosum sensu stricto is associated typically with root rot and mortality of trees in the genus Pinus, but it can also be found on Picea, Juniperus and even on deciduous trees (Korhonen et al., 1998a). All the three species of the fungus are widespread in alpine coniferous forests (Korhonen et al., 1998a; Gonthier et al., 2001) and they are extremely pervasive locally. For instance, levels of disease incidence of up to 95% were reported in some subalpine Norway spruce stands in the western Alps (Gonthier et al., 2003). In a recent study, it was discovered that a large majority of gaps and mortality centres in mountain pine forests of the Swiss Alps was caused by a Heterobasidion species rather than by pathogenic Armillaria species (i.e. A. ostoyae) or other factors (Bendel et al., 2006). Heterobasidion primarily infects its hosts by means of airborne meiospores, normally through freshly cut stumps or wounds, and is capable of secondarily spreading from tree to tree through root grafts and contacts (Asiegbu et al., 2005). Airborne infection through thinning stumps not only may result in a rapid and heavy infection of a healthy stand in areas where Heterobasidion is common (Pratt and Greig, 1988; Swedjemark and Stenlid, 1993), but also may aid the spread of the fungus into new areas (Berry and Dooling, 1962). Thus, stumps play a crucial role in the epidemiology of this forest pathogen, as conﬁrmed indirectly by the positive relationship between the incidence of disease in residual trees and the intensity of earlier thinnings, as well as the proportion of thinning stumps infected (Rishbeth 1957; Vollbrecht and Agestam, 1995). 350 P. Gonthier Effects of Silviculture and Land Management With very few exceptions, root and butt rot fungi are opportunistic pathogens being able to take advantage of habitat modiﬁcations for their establishment and spread. Thinning and logging, as well as other forest management activities, appear to increase the damage caused by root and butt rots (Garbelotto, 2004). It has been suggested that the current high incidence of H. annosum in unmanaged mountain pine forests of the central Alps is due to intense logging in the past (Bendel et al., 2006). Large amounts of timber and fuel were needed to support mining activities, which were thriving in the area between the 14th and 17th centuries. Similarly, the high disease severity recorded in spruce forest stands of the western Alps (Gonthier et al., 2003) could also be associated with mining activities in the past. In some areas of the western Alps, intensive cutting occurred during the 17th and 18th centuries and this led to the creation of stumps over large surfaces (Nicco, 1997). At the same time, the current high level of infestation of subalpine forests (Gonthier et al., 2003) might also have a human origin. Cuttings have been performed regularly at the upper edge of forests to conserve alpine grasslands for summer grazing. As previously stated, the creation of stumps is particularly important for H. annosum, as stumps behave as main infection courts for primary infections. It has been reported that thinnings also promote the tree-to-tree vegetative spread of the pathogen in infected Norway spruce stands (BendzHellgren et al., 1999; Piri and Korhonen, 2008). The growth rate of the fungus in roots increases after the felling of infected trees (Bendz-Hellgren et al., 1999). In living spruce roots, Heterobasidion is conﬁned typically to dead heartwood. Hence, the transfer of the fungus between living trees may be limited to functional root grafts, which enable the fungus to grow from the xylem of infected roots into the xylem of healthy roots. After the tree is cut, Heterobasidion begins to expand outwards from the centre of the root and it may be able to spread into the surrounding trees more easily through nongrafted root contacts. Such an increase in spreading ability after cuttings could also occur with other heart rot fungi. Logging operations are likely to increase the probability of attack by most root and butt rot fungi, since new infection courts, i.e. wounds, are created. As an example, the wound rot caused by S. sanguinolentum, which is a relatively recent problem, seems to be the result of increasing mechanization of forestry (Butin, 1995). With the use of heavy machinery for the extraction of thinnings, large bark wounds occur much more frequently. This may be crucial for the establishment of fungi, like S. sanguinolentum, that need wounds larger than 10 × 10 cm to infect trees (Butin, 1995). In general, wounds play an important or fundamental role in the infection biology of fungi that are able to produce infective airborne inoculum. Nevertheless, it has been proposed that wounds could also trigger attacks by fungi unable to infect by spores, i.e. Armillaria spp., because they can induce a lowering of tree defences (Popoola and Fox, 1996). Diagnosis and General Control Strategies Management options are based on our knowledge of the ecology, epidemiology and infection biology of the causal agent. Hence, before planning control, there is good reason to perform an accurate diagnosis and identiﬁcation of the causal agent. The type of rot may aid in the diagnosis but in general it is not an exhaustive trait for the identiﬁcation of wood decay fungi (Bernicchia, 2005; Solheim, 2006). Traditionally, diagnosis is based on the macro- and/ or micromorphology of basidiomata and it may be achieved through the use of mycological keys (Eriksson et al., 1984; Breitenbach and Kränzlin, 1986, 1991, 1995; Bernicchia, 2005; Gonthier and Nicolotti, 2007). When performing ﬁeld diagnosis, a pathologist should consider that basidiomata of wood decay fungi usually emerge at advanced Controlling Root and Butt Rot Diseases stages of the fungal infection and they may be rarely or sporadically visible. Furthermore, basidiomata of some species (e.g. P. schweinitzii) are short-lived (Butin, 1995) or may be found only during particular periods of the year (i.e. Armillaria spp.); thus, the timing of diagnosis is also important. Most root and butt rot fungi can be cultured easily from decayed wood. A few of them (i.e. Heterobasidion spp., L. sulphureus) develop a fast-growing conidial stage in culture or when colonized wood is incubated in a damp room. Usually, asexual mitospores of these fungi do not play any signiﬁcant role in the infection biology. Nevertheless, they may have a diagnostic value. Identiﬁcation of wood-inhabiting fungi, non-sporulating in pure culture is also possible by using appropriate keys (Nobles, 1965; Stalpers, 1978). Pure culture analysis, however, is difﬁcult and time-consuming. A number of molecular techniques have been developed and are now available for 351 the identiﬁcation of the most important root and butt rot fungi. A summary of them is given in Table 26.2. Some techniques allow for the identiﬁcation of rots directly from wood. For instance, polymerase chain reaction (PCR) with taxon-speciﬁc primers provides reliable fungal diagnostics from both pure culture and environmental samples. Theoretically, a pathogen can be controlled during all stages of its life cycle, starting from primary infection and early establishment, through spreading inside the host, to formation, spread and survival of its propagules (Holdenrieder and Greig, 1998). Unfortunately, root and butt rot diseases are virtually impossible to eradicate once they are established. They may be controlled successfully only when pathogens have a small biomass and are therefore weakly competitive. In general, when dealing with root and butt rot diseases characterized by abundant primary infection events (i.e. H. annosum, S. sanguinolentum, etc.), forest management Table 26.2. Taxon-speciﬁc primers developed for the identiﬁcation of some of the most important root and butt rot fungi present in alpine forests. Fungi Forward primer Reverse primer Amplicon size Reference A. mellea sensu lato1 ARM-1 (agggta tgtgcacgttcgac) ITS3 (gcatcgat gaagaacgcagc) HET-7 (cttctcac aaactcttcg) MJ-F (ggtcctgtc tggctttgc) KJ-F (ccattaac ggaaccgacgtg) MLF (taaaaatttaa attagccataa) ARM-2 (ggaaagctaa gctcgcgcta) Armi2R (aaacccccat aatccaatcc) HET-8 (caggtccccca caatcg) MJ-R (ctgaagcacac cttgcca) KJ-R (gtgcggctcattc tacgctatc) Mito7 (gccaatttatttt gctacc) Mito5 (taagaccgctata waccagac) 660 bp Schulze et al., 1997 184 bp Guglielmo et al., 2007 400 bp Bahnweg et al., 2002 100 bp Hantula and Vainio, 2003 H. annosum sensu lato H. annosum sensu lato H. parviporum H. annosum sensu stricto H. abietinum H. parviporum L. sulphureus sensu lato O. tomentosa Stereum spp. MLS (aaattagcca tattttaaaag) 25sF (tggcgaga LaetR (ccgagcaaac gaccgatagc) gaatgcaa) It-ITS-209-f (gcta It-ITS-700-rc (agga aatccactcttaacac) gccgaccacaaaagat) ITS3 (gcatcgatg Ste2R (gtcgcaacaa aagaacgcagc) gacgcactaa) 350 bp 230 bp 195 bp 185 bp Garbelotto et al., 1998; Gonthier et al., 2001, 2003 146 bp Guglielmo et al., 2007 491 bp Germain et al., 2002 234–240 bp Guglielmo et al., 2007 Note: 1Some Armillaria species may be distinguished through PCR-restriction fragment length polymorphism (RFLP) (Harrington and Wingﬁeld, 1995; Schulze et al., 1997; Sierra et al., 1999). 352 P. Gonthier should focus on minimizing those activities likely to create good primary infection courts, e.g. wounds on roots, stems and stumps. Care should be taken in order to avoid harvestinduced injuries. Containment of wildlife populations, especially of bark-stripping deers, may also have some effects on wound rot severity (Cermák et al., 2004). Lowering stand density may regulate secondary infection events, especially for pathogens spreading through root grafts and contacts. In addition, a regulation of stand density aimed at reducing tree competition may prevent or reduce infections by a wide range of weakness pathogens, regardless of their mode of transmission (i.e. Armillaria spp., F. pinicola, etc.). The management of mature alpine forests that are diseased or under the risk of root and butt rots could be achieved through several other and more speciﬁc methods, which are reviewed below. Some of them are hardly applicable in mountain areas or may be justiﬁed only locally. Some others are currently used over large areas, including the alpine region. An integrated disease management approach could be more efﬁcient than single methods in controlling these diseases. Trenching Digging isolation trenches around diseased trees to prevent the secondary, vegetative spread of root and butt rot fungi is one of the most traditional control methods recommended against several pathogens including H. annosum sensu lato and A. mellea sensu lato (Korhonen et al., 1998b; Kliejunas et al., 2005; Legrand et al., 2005; Eyles et al., 2008). The trench should be at least 70–100 cm deep. Instead of an open trench, it could be more practical to bury a plastic sheet in a vertical position into the soil (Korhonen et al., 1998b; Legrand et al., 2005). As suggested by Eyles et al. (2008), the effectiveness of this method depends on the regular maintenance of trenches, to prevent the reestablishment of root contacts, and on the proper siting of the trenches to ensure all infected trees are isolated. It is usually quite difﬁcult to determine whether or not trees are infected; colonization of the root systems may go undetected for long periods of time. Trenching is a very impractical control method and its use is advisable only when pathogen inoculum is very localized. It should be noted that in the case of pathogens that are also able to spread aerially, trenching would offer limited protection. In the case of H. annosum, for instance, observational data suggest that instead of controlling the disease, trenching could actually promote the spread of the fungus by breaking and injuring roots (Korhonen et al., 1998b). Tree and stump removal Secondary mycelia of most root and butt rot fungi will survive and produce basidiomata for a long period of time on colonized wood. Thus, infected trees should be removed from the stand promptly in order to reduce the airborne inoculum of fungi spreading through spores. As most of these fungi can attack timber, asymptomatic felled trees should also be removed. In general, timber is unselective or less selective than standing trees to infection by wood decay fungi (Rayner and Boddy, 1986). As an example, S. sanguinolentum, which commonly attacks Norway spruce trees, is reported to colonize felled wood of spruce but also of pine and silver ﬁr, in which it causes a red streaking (Butin, 1995). Thus, attention should be given not only to preferential hosts, but also to other tree species that can become saprophytically colonized. Removing stumps and roots from the soil has been recommended for controlling both H. annosum sensu lato and A. mellea sensu lato (Korhonen et al., 1998b; Legrand et al., 2005). Beneﬁts of this method have been reviewed recently by Vasaitis et al. (2008). It should be noted that de-stumping can show effectiveness not only against diseases spreading from tree to tree through root grafts and contacts, but rather it may be effective against a wide range of wood decay Controlling Root and Butt Rot Diseases agents. In fact, for instance, basidiomata of P. schweinitzii growing on stumps are reported as sources of soil infestation for long periods of time (Barrett, 1985). Stump removal is an expensive, time-consuming control method (Korhonen et al., 1998b; Legrand et al., 2005) that requires the use of machines (Omdal et al., 2001). This method can be adopted optionally in certain artiﬁcial plantations after clear-felling. It is rarely used in mountain forests or after selective cuttings. Furthermore, de-stumping contrasts with current trends in forest management since it may have negative effects on biodiversity. Promoting tolerant species If a forest is heavily infested by a root or butt rot agent with restricted or deﬁned host range, control may be achieved by increasing the proportion of trees more resistant to the pathogen. In principle, a rotation of a resistant tree species can clean the site of the pathogen inoculum (Korhonen et al., 1998b). The concept of forest rotation has been widely advocated for the management of root rots of forest trees, with contrasting results (Korhonen et al., 1998b; Lygis et al., 2004). The pathogen inoculum usually persists in stumps and roots for decades after felling, and this was reported not only for H. annosum or A. mellea species complexes (Korhonen and Stenlid, 1998; Guillaumin and Legrand, 2005) but also for other root and butt rot fungus, i.e. P. schweinitzii (Barrett, 1985). Changes in tree species composition, turning a susceptible forest into a more tolerant one, can be achieved in a relatively short period of time through clear-felling only, followed by artiﬁcial plantation. In naturally regenerated, uneven-aged or irregular alpine forests, shifts in tree species composition are more difﬁcult to obtain and they should be driven by appropriate silvicultural practices. Obviously, in such conditions, for a reduction of the pathogen inoculum to occur, several decades, or even centuries, may be necessary. As for most plant diseases, it could be advisable to avoid the complete removal of the susceptible species, since the resulting selection pressure 353 on the pathogen might result in the infection of plants normally considered as nonhost (Garbelotto, 2004). This method of control might show some effectiveness in controlling Heterobasidion root and butt rots. In a recent unpublished study conducted in the western Italian Alps, and based on the analysis of about 2300 recently felled trees, it was found that disease incidence was signiﬁcantly higher on Norway spruce (43% of average incidence) than on other native tree species (Table 26.3). Silver ﬁr was also rather susceptible, while larch and, especially, Scots pine trees were more tolerant. Moreover, with the exception of H. annosum sensu stricto, a strict host preference of H. annosum species has been reported in the Alps (Gonthier et al., 2001). Thus, based on these data and on other observational data (Table 26.3), it is likely, for instance, that the establishment of deciduous tree species would have beneﬁcial effects in most Heterobasidion-infested forests, but not necessarily in H. annosum sensu stricto infected stands (Table 26.3). Also, the control of H. abietinum in heavily infected silver ﬁr forests could be achieved either by promoting Scots pine trees or Norway spruce trees. The disease in severely damaged Norway spruce stands can be lowered by favouring the more resistant larch. A. mellea sensu lato species display a lower degree of host preference with respect to H. annosum sensu lato species. Nevertheless, variation of tree species composition could have some beneﬁcial effects for this pathosystem also. For instance, Swiss stone pine trees have been reported as very susceptible to A. ostoyae in subalpine forest (Anselmi and Lanata, 1989). Cuttings promoting the regeneration and establishment of the most tolerant larch could be effective in the management of Armillaria root rots in these high elevation forests. Timing of thinning and cutting The timing of logging and harvesting may have a strong impact on the incidence of 354 P. Gonthier Table 26.3. Susceptibility of native alpine forest trees to H. annosum species based on the author’s experience and on previously published (Gonthier et al., 2002, 2003) and unpublished data. The list does not include susceptibility and symptoms of seedlings. Tree species Abies alba Picea abies Larix decidua Pinus cembra P. sylvestris P. uncinata Broadleaves Average Heterobasidion incidence1 17% 43% 12% 15% 4% ? ? H. parviporum H. abietinum H H H H ++++ ++ ++ H. annosum sensu stricto +++ H H H M M – ++ + ++ ++ ++?2 + Note: 1Based on the analysis of about 2300 recently felled trees from 22 forest stands in the western Italian Alps; 2symptoms and tentative susceptibility according to Bendel et al., 2006. Symbols: +, recorded; ++, occasionally diseased; +++, susceptible; ++++, very susceptible; M, root rot and mortality; H, heart rot; –, saprophyte. airborne infectious diseases. As a rule, operations should be done preferably when environmental conditions are unfavourable to the pathogen. For H. annosum sensu lato, winter thinning and logging operations have been used in Fennoscandia to take advantage of the low inoculum pressure during the cold season (Brandtberg et al., 1996; Piri and Korhonen, 2008). In that area, infections follow a bell-shaped curve with very low spore deposition rates in winter; an average of only 2% of Norway spruce stumps was infected following thinning in November–February compared with 34% in June–July (Brandtberg et al., 1996). Seasonal patterns of spore deposition of Heterobasidion species have been studied in the alpine region recently with the aid of woody traps (Gonthier et al., 2005). Here, the airborne inoculum of this pathogen, although present starting in February at most sites, is higher in August–October, reaching a peak in September. A relative peak, lower than the late summer one, appears in late spring (Fig. 26.1). Thus, despite the development of perennial basidiomata, in the Alps the inoculum production of Heterobasidion spp. is concentrated largely in a period of 2–3 months. Spore inoculum in winter, spring and early summer is generally low (Gonthier et al., 2005). Although the hazard of stump infection is not always described accurately by spore loads on woody traps (Driver and Ginns, 1969), recent unpublished data conﬁrm the above seasonal patterns of spore deposition and indicates that the highest risk of stump infection occurs in autumn (Gonthier and Nicolotti, unpublished). Winter operations may not be feasible at all sites in the Alps. However, Heterobasidion primary infections would be controlled successfully by planning logging and thinning in most of spring and in early summer. Although the above timing may be somewhat impractical, its advantage in Norway spruce includes limiting infection through wounds not only by H. annosum sensu lato but also by S. sanguinolentum, whose annual basidiomata are produced in autumn (Solheim, 2006). Biological and Chemical Control Several biological and chemical methods have been tested for the control of root and butt rot fungi, especially of H. annosum sensu lato and A. mellea sensu lato (reviewed in Holdenrieder and Greig, 1998; Pratt et al., 1998; Guillaumin et al., 2005a,b). Most Controlling Root and Butt Rot Diseases 355 100 90 Infected traps (%) 80 70 60 50 40 30 20 10 0 JAN FEB MAR APR MAY JUN JUL Months AUG SEP OCT NOV DEC Fig. 26.1. Average percentage of woody traps infected monthly by Heterobasidion spores in four forests of the western Alps from 1998 to 2000. Re-elaborated from data published by Gonthier et al. (2005). Bars show standard errors. experiments were conducted in vitro. Currently, only a very few control approaches are recommended in practical forestry and they are all devoted to H. annosum sensu lato. Stump treatment with appropriate biological or chemical products immediately after felling may prevent H. annosum primary infections, therefore reducing timber losses. A number of fungi have been tested on stumps as competitors or antagonists throughout North America and Europe (Holdenrieder and Greig, 1998), including in the alpine forests (Nicolotti et al., 1999). Only Phlebiopsis gigantea (Fr.) Jül is used currently, with good results over large areas (Holdenrieder and Greig, 1998; Thor, 2003; Berglund and Rönnberg, 2004; Thor and Stenlid, 2005). Three distinct products based on this saprotrophic fungus have been developed: PG Suspension® in the UK, PG IBL® in Poland and Rotstop® in Fennoscandia. Rotstop® showed a very good effectiveness in alpine Norway spruce stands heavily infected by Heterobasidion (Nicolotti et al., 1999; Nicolotti and Gonthier, 2005). In a recent comparative study performed in Austrian alpine protection forests, strains of Phlebiopsis gigantea from Poland resulted in a higher colonization frequency of spruce stumps with respect to the Rotstop® strain (Cech et al., 2008). Several chemicals proved to be effective as stump protectants against Heterobasidion airborne infections (Pratt et al., 1998), the most known of which are urea in Europe and borax in North America. Both compounds, as well as other chemicals, were tested in the western Alps on spruce stumps and they showed very good results, comparable to those obtained with the Rotstop® treatment (Nicolotti et al., 1999; Nicolotti and Gonthier, 2005). The effectiveness of urea was dependent on the concentration of the water solution: the best results were obtained with a 30% concentration. The rise of pH of stump surfaces, which occurs commonly during hydrolysis after treatment, rather than a toxicity of urea or ureaderivate compounds per se (e.g. ammonia and ammonium ions), is responsible for the inhibition of Heterobasidion germination and growth (Johansson et al., 2002). Such a high urea concentration allows high pH values on stumps to be maintained for at least the length of time these remain susceptible to infection, i.e. approximately 1 month. 356 P. Gonthier Because the costs of registration are very high, it is unlikely that any biological or chemical product will be registered in the near future for stump treatments in the alpine area. However, urea and borax are currently classiﬁed as fertilizers and their use is mostly unregulated for forestry purposes (Nicolotti and Gonthier, 2005). Urea could be preferred for its long history in stump treatment in Europe (Nicolotti and Gonthier, 2005; Oliva et al., 2008) and for its moderate effects on non-target organisms inhabiting stumps (Table 26.4). Furthermore, urea is effective on stumps of several native alpine tree species (Gonthier and Nicolotti, unpublished). Integrated Disease Management and Forest Protection: A Concluding Example It is generally agreed that systems combining cultural, biological, chemical or other methodologies to reduce parasites are more effective and even cheaper than single control methods. In the ﬁeld of forest trees, integrated pest management (IPM) systems have been developed especially for the protection of forests against insects or nurseries against diseases (Volney and Mallett, 1998; South and Enebak, 2006). Appropriate IPM systems may be developed only with a good understanding of the pathogen biology and disease epidemiology. Except for a few pathosystems (i.e. H. annosum sensu lato, A. mellea sensu lato), our current understanding of the epidemiology of root and butt rot diseases is still limited to allow the development of efﬁcient IPM systems. However, the biology and epidemiology of H. annosum sensu lato are well known and some weak points exist in its life cycle (i.e. stage of infection by spores). An integrated management system was designed to control H. annosum root and butt rots in the Aosta Valley, western Italian Alps. The system is based on stump treatment Table 26.4. A summary of the effectiveness and impact on non-target fungi of biological and chemical treatments against Heterobasidion airborne infections on Norway spruce stumps in the western Alps. Antagonist/competitor or active ingredient Biological Hypholoma fasciculare Phanerochaete velutina Phlebiopsis gigantea Vuilleminia comedens Verticillium bulbillosum V. bulbillosum Trichoderma harzianum Chemical Copper oxychloride Propiconazole Sodium tetraborate decahydrate Urea Urea Urea Effectiveness against Heterobasidion1 Impact on non-target fungi2 Wheat mash Wheat mash Rotstop® Wheat mash Culture ﬁltrate Conidial and mycelial suspension Conidial and mycelial suspension Low High High Low Medium Medium Low (after 2 years) Low (after 2 years) High Low (after 2 years) Very low Very low Low Very high Azuram® TILT (25% emulsion) Borax powder High High High Low (after 2 years) Low (after 2 years) Very high Water solution 10% conc. Water solution 20% conc. Water solution 30% conc. Low High High Very low Low (after 2 years) Low (after 2 years) Application method or commercial product Note: 1Categories (low, medium, high) were designed based on previously reported results (Nicolotti et al., 1999; Nicolotti and Gonthier, 2005); 2categories (very low, low, high, very high) were designed based on previously reported results (Varese et al., 1999; 2003a,b). Controlling Root and Butt Rot Diseases with urea at 30% concentration, combined with an appropriate timing of thinning and logging operations, and with practices aimed at promoting tolerant species (Fig. 26.2). In the Aosta Valley, forest management activities are planned yearly by the Regional Forest Administration, who decides the stands that need to be thinned each year. Every forest harvesting team is in charge of thinning a variable number of stands (3–8). While planning the timing of thinnings, priority is given to the most susceptible (see Table 26.3) and heavily infected stands, which are thinned preferably in spring and early summer, when the risk of stump infection is still limited. The average minimum air temperature of a 4-week period has been identiﬁed as a suitable predictor for modelling Heterobasidion primary infections in the Alps (Gonthier et al., 2005) and may be used for an accurate estimation of the seasonal risk of stump infection for each forest stand. Remaining, less susceptible and uninfected stands are thinned in summer or autumn. Stump treatment is necessary during summer and autumn thinnings and is strongly recommended whenever dealing with uninfected or susceptible stands, regardless of their location and distance from an infection source. In fact, despite a general limited Winter ? Highly susceptible stands Spring potential dispersal range of Heterobasidion spores (Gonthier et al., 2001), with spore densities undergoing huge dilution after the ﬁrst metres (Stenlid, 1994), the migration of even a few spores may be signiﬁcant for areas still not colonized by the pathogen (Garbelotto, 2004). Winter operations may be possible locally, in low elevation stands. Sanitation fellings are advisable in Scots pine forests to reduce bark beetle attacks. Depending on the forest function and on the economic injury level, practices such as de-stumping and, especially, the transformation of heavily infected susceptible forests into more tolerant ones can be arranged locally and they may be suited. For instance, increasing the larch component in subalpine spruce forests would have positive effects not only in reducing Heterobasidion incidence, but also in improving general forest stability (Motta and Haudemand, 2000). At lower elevations, the regeneration of diseased spruce forests with silver ﬁr or Scots pine could be advisable since H. parviporum very seldom attacks mature ﬁrs or pines (Korhonen et al., 1998b). Obviously, any integrated disease management system to ﬁght root and butt rots of forest trees should ﬁt and meet the requirements of the general forest management Summer Promote tolerant tree species Autumn Stump treatment ST Non-susceptible stands 357 Heavily infected stands Uninfected stands Sanitation cuttings (DT, WC) de-stumping (WC) Fig. 26.2. Diagram of the integrated disease management system developed to ﬁght Heterobasidion root and butt rots in the Aosta Valley, western Italian Alps. Arrows indicate the appropriate timing of thinning. Stump treatment is performed with urea at 30% concentration. Symbols: ?, where possible; ST, stump treatment; DT, dead trees; WC, where convenient. 358 P. Gonthier system of the area (Tainter and Baker, 1996). 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Forestry Chronicle 74, 597–605. Woodward, S., Stenlid, J., Karjalainen, R. and Hüttermann, A. (1998a) Heterobasidion annosum, Biology, Ecology, Impact and Control. CAB International, New York, 589 pp. Woodward, S., Stenlid, J., Karjalainen, R. and Hüttermann, A. (1998b) Preface. In: Woodward, S., Stenlid, J., Karjalainen, R. and Hüttermann, A. (eds) Heterobasidion annosum, Biology, Ecology, Impact and Control. CAB International, New York, pp. xi–xii. 27 Some Important Fungal Diseases and Their Impact on Wheat Production Aakash Goyal and Rajib Prasad Agriculture and Agri-Food Canada, Lethbridge Research Center, Lethbridge, Canada Abstract Wheat, an important cereal crop, is cultivated worldwide and is second highest in production, just after maize. Due to the increasing world population, there is need for a 40% increase in wheat production to meet global food requirements. Wheat production is diminished mainly by biotic and abiotic stresses all over the world. Of these, pathological diseases are the most important limiting factor of wheat production as different pathogens infect wheat plants, causing severe losses in yield and quality. Wheat can be infected by biotrophic fungi, necrophytic species and nematodes, as well as viruses and bacteria. Among these, different fungal diseases are the most prominent and pose a great challenge to wheat production. Development of resistant varieties is the only solution to overcome this problem and to attain the required wheat production. The development of resistant varieties has beneﬁted immensely from the use of molecular markers, genetic maps, physical maps, QTL analysis and markerassisted selection (MAS). However, we have to develop multidisease-resistant varieties to fulﬁl the demand for wheat globally. This review highlights some major fungal diseases of wheat in different parts of the world and the associated problems. Introduction Common wheat (Triticum aestivum L. em. Thell) is an important staple food crop and ranks ﬁrst among the three major crops (wheat, maize and rice), which together constitute about half of the total world food production. Wheat feeds about 40% of the world population and provides 20% of the total food calories and protein in human nutrition (Varshney et al., 2006). It is not only used for bread making but is also used for making biscuits, cakes, breakfast cereals, pasta and fermented products like beer, alcohol, vodka, etc. It is also becoming popular as a forage crop. Wheat straw has been 362 used as a fodder material and for ethanol production for the past few years. Unlike rice and maize, which prefer tropical environments, wheat is best adapted to temperate regions, occupying 17% (one-sixth) of the total crop acreage worldwide (Gupta et al., 2008). According to the FAO (2007), wheat occupies 20% of the cultivated crop area (in 2007, 213m ha versus 150m for rice and 143m for maize) and its annual production is 619 Mt of grain. Over the past 20 years, there has only been a small increase in the area of land on which wheat is cultivated worldwide, but the tonnage of wheat grain produced on this land has tripled as a result of improved farming practices and  CAB International 2010. Management of Fungal Plant Pathogens (eds A. Arya and A.E. Perelló) Some Important Fungal Diseases and Wheat Production the development of better wheat varieties (Marshall et al., 2001). A signiﬁcant increase in wheat production has been observed in the past four decades; however, a slowing down has been witnessed during the past few years (Gupta et al., 2008). Due to a consistent increase in world population, there is need for a 40% increase in wheat production to meet this requirement. Despite the enormous progress that has taken place around the world, there is less hope in achieving this goal. Resistance to both biotic and abiotic stresses will be critical for reaching this target. Abiotic stresses include drought, untimely or excess heat, untimely or excess rain, water logging of soils, wind, extreme cold, frost, acid soils and salinity, nutrient imbalances and/or shortages, as well as micronutrient deﬁciencies. The impact of biotic stress on wheat production and quality is highly devastating. Diseases in wheat, most caused by fungal pathogens and a few by viruses and bacteria, are important production constraints in almost all wheat-growing environments (Rajaram and van Ginkel, 1996). Wiese (1987) identiﬁed over 40 fungal, 32 viral and 81 bacterial diseases that attack wheat plants at different growth stages. Although it is difﬁcult to obtain accurate estimates of crop losses to different fungal diseases, the British Agrochemicals Association (1993) suggests that, under farm conditions where crop rotations, good husbandry and the application of pesticides are practised, losses to diseases can still be around 13%, while under conditions where crop protection measures are not taken, losses can be as high as 50%. It is the goal of wheat breeders to introduce genetic resistance into their varieties to minimize chemical protection measures and losses due to diseases. Under different environments, breeders face problems of different spectra of locally prevalent diseases caused by speciﬁc biotypes, serotypes and strains. For many diseases, genes for resistance segregating in a simple ‘Mendelian’ fashion have been identiﬁed; while for other diseases, resistance genes still remain to be detected, due to either a complex mode of inheritance or imprecise disease-screening procedures (Gowda et al., 363 2006). Recently, available molecular markers and functional genomics tools have helped the breeder to manipulate the wheat genome to develop disease-resistant cultivars and achieve the target of wheat production. Fungal Diseases of Wheat Most of the important diseases of wheat are caused by fungal pathogens, while only a few are caused by viruses and bacteria (McIntosh et al., 1995; Rajaram and van Ginkel, 1996). Infection of fungal diseases in wheat depends on the availability of free water on the host plant surface, susceptibility of the host, the density of inoculum, temperature and other environmental factors. Moreover, host–parasite interaction plays a signiﬁcant role in the development of disease and subsequent symptoms on the wheat plant. In this chapter, some of the commonly reported fungal diseases of wheat are described. Table 27.1 lists major fungal diseases of wheat reported by different pathologists around the world. Fusarium head blight (FHB) Several species of Fusarium can cause Fusarium head blight (FHB), also known as scab of cereal crops. Among these, F. graminearum, found mainly in the USA, Canada, China and the EU, is accountable for severe losses in yield and quality of wheat production (Parry et al., 1995). An epidemic of FHB in the USA and Canada in 1993 was a result of changes in crop management practices (minimum or reduced tillage), changes in rainfall patterns and a low resistance in the cultivars against FHB (Dill-Macky and Jones, 1997). In the case of wheat, Fusarium spp. attacks different plant organs but mainly targets the ear, which leads to great loss in seed quality. On the ear, Fusarium enters through the stomata to the palea and lemma and destroys these tissues completely. The ﬁrst symptom of FHB is a tan or brown discoloration at the base of a ﬂoret within the spikelets of the head. The infection may be 364 A. Goyal and R. Prasad Table 27.1. The major fungal diseases of wheat reported across the world. Name of the disease Pathogenic fungal species Tolerant varieties/ genotypes References Black point/kernel smudge Common bunt Common root rot Ergot Fusarium head blight (scab) Leaf rust Alternaria alternata Sunco, Cascades Lehmensiek et al., 2004 Tilletia caries, T. foetida Cochliobolus sativus Claviceps purpurea Fusarium graminearum AC Domain ND 652 Carleton, Kenya farmer Bizel, Sumai 3 Fofana et al., 2008 Mergoum et al., 2005 Platford and Bernier, 1970 Bourdoncle and Ohm, 2003 Puccinia recondita (P. triticina) Tangmai 4, ND 652 Loose smut Powdery mildew Speckled leaf blotch Glume blotch Spot blotch Stem rust Ustilago tritici Erysiphe graminis Septoria avenae f. sp. tritici Stagonospora nodurum Cochliobolus sativus P. graminis f. sp. tritici DT676 Tangmai 4 Arina and Riband; Courtot and Tonic Red Chief Ning 8201, K8027 ND 652, Tangmai 4 Li et al., 2004; Mergoum et al., 2005; Kolmer et al., 2007 Knox et al., 2008 Li et al., 2004 Chartrain et al., 2009 Stripe rust Take-all P. striiformis Gaeumannomyces graminis var. tritici Tangmai 4 Xinong 1376, Xinong 918, R859 limited to one spikelet, but if the fungus invades the rachis, the entire head may develop symptoms of the disease. Discoloration of the head starts due to the production of mycotoxins [zearalenones and deoxynivalenol (DON)] by the Fusarium. The mycotoxins affect seed quality adversely, producing toxic dust and thus making the seeds unsuitable for human and livestock consumption (Eudes and Laroche, 2003). The mycotoxin DON, even in low doses of 1–3 ppm, can cause reduced feed intake and less weight gain in animals, while a high dose up to 10 ppm can cause vomiting and refusal to feed. DON is also very harmful to humans; therefore, different countries have established laws to protect consumers. For example, the EU Member States allow a maximum of 1.25 ppm DON in unprocessed bread, 0.5 ppm in bread and bakery products and only below 0.2 ppm in baby foods (Buerstmayr et al., 2009). The USA Food and Drug Administration recommend only 1 ppm DON in ﬁnished wheat products, while Health Canada have established guidelines Laubscger et al., 2008 Sharma et al., 2007 Li et al., 2004; Mergoum et al., 2005 Li et al., 2004 Xiaoning et al., 2004 of 2 ppm and 1 ppm DON in soft wheat in non-stable and baby foods, respectively. Control of this disease has been difﬁcult, because of the complex nature of the host/pathogen interaction. Cultural practices, such as rotation with non-host crops and management of crop residues, in combination reduce primary infection. A mixed fungicide composed of carbendazim and triadimefon was reported to have a signiﬁcant synergistic action (Wang, 1997). Under high disease pressure, Bravo or Folicur were reported to reduce levels of FHB, though these are not cost-effective under low disease pressure (Agrios, 1997). Host resistance is a promising and effective management solution, but resistance has not been easy to achieve in the adapted cultivars. Wheat rust Wheat rust pathogens belong to genus Puccinia, family Pucciniaceae, order Uredinales and class Basidiomycetes. Rust disease Some Important Fungal Diseases and Wheat Production is capable of causing considerable economic loss throughout the world (FAO, 2008). Rust in cereals, found back in the late 17th century, was caused by a fungal parasite which was named later as Persoon’s P. graminis (Chester, 1946). In the beginning of the 20th century, different fungal species were identiﬁed for different rusts with contrasting host ranges. In wheat, rust diseases are so important that in 2007, the CSIRO, Australia, published a special issue on wheat rust in the Australian Journal of Agricultural Research. Stem rust Stem or black rust of wheat is a major disease problem, caused by the fungus, P. graminis Pers. f. sp. tritici. It has been a major disease on wheat since the rise of agriculture and the Romans even prayed to a stem rust god, ‘Robigus’. The Italians, Fontana and Tozzetti, independently provided the ﬁrst report on stem rust in wheat in 1767. In the early to mid 1950s, stem rust epidemics caused approximately 50% yield losses of wheat in North America (Leonard, 2001). During the 1950s, Norman Borlaug and other scientists started developing high-yielding wheat varieties that were resistant to stem rust and other diseases in North America and throughout the world. The rust-resistant, highyielding wheat variety banished chronic hunger in much of the world, ended stem rust outbreaks and won Borlaug the Nobel peace prize in 1970 (Singh et al., 2006). In most areas of the world, the life cycle of P. graminis consists of continual uredinial generations. The disease spreads either via airborne spores or occasionally locally from wild susceptible barberry (Berberis sp.) plants (Eversmeyer, 2000). Ug99, so called as it was ﬁrst seen in Uganda in 1999, is a new devastating race of ‘stem rust’ which has already travelled from Africa to Iran and can proceed to India, Pakistan and Bangladesh (Pretorius et al., 2000). It is particularly dismaying because of its ability to infect crops in just a few hours and its vast cloud of invisible spores can be carried by the wind for hundreds of miles (Singh et al., 2006). 365 Cultural control provides at least partial control of wheat rust epidemics. Planting early maturing varieties is an efﬁcient way to avoid losses due to stem rust infection. Propiconazole (Tilt) and triadimefon (Bayleton) are found to be effective against stem rust (Agrios, 1997), though these chemicals are cost-prohibitive. To save the world from the wheat epidemic, CIMMYT and ICARDA started the Global Rust Initiative (GRI) to coordinate efforts to track and study Ug99 and develop resistant varieties of wheat (Stokstad, 2007). Later in 2008, it was taken over by the Borlaug Global Rust Initiative (BGRI), chaired by Dr N.E. Borlaug, who said he was optimistic that the fungus would be beaten again (Stokstad, 2007). Efforts were also taken to understand the rust’s epidemiology and evolution, which led to the barberry eradication programme in North America and Europe (Singh et al., 2006). Leaf rust Wheat leaf rust, also known as brown rust, is caused by the rust fungus, P. triticina Rob. Ex Desm. f. sp. tritici Eriks (syn. P. recondita). De Candole (1815) reported for the ﬁrst time that leaf rust was caused by fungus and named it Uredo rubigovera. Later in the 19th century, the name was changed to P. recondita (Cummins and Caldwell, 1956). However, the present name, P. triticina, was suggested by Savile (1984) and Anikster et al. (1997). Up to 2007, more than 50 races of leaf rust were detected all over the world (Kolmer et al., 2007; Mebrate et al., 2008). Leaf rust is the most prevalent of all the wheat rust diseases, occurring in nearly all areas where wheat is grown. Depending on the severity and duration of infection, losses in wheat can vary by up to 50% (Nagarajan and Joshi, 1975; McIntosh et al., 1995). The disease has caused serious epidemics in North America, Mexico, South America and some other countries. This fungus can infect wheat plants with a 3 h dew period at temperatures near 20°C. However, more infections occur with longer dew periods. The fungus initially starts covering leaves with 366 A. Goyal and R. Prasad orange pustules of urediniospores (uredinia). The urediniospores are reddish-brown, elliptical to egg-shaped, echinulate structures. In the later stage, the postules eventually darken due to the formation of black teliospores (Roberson and Luttrell, 1987). Infections can result in a 1–20% yield loss since infected leaves die earlier and all the nutrients are directed to the growing fungi. Infection can also cause grains to shrivel. The loss in yield depends on several factors that include time of initial infection, crop development stages, relative resistance or susceptibility of the wheat cultivars. Higher yield losses result when the initial infection occurs early in the growing season before tillering. Infection occurring after heading when grain ﬁlling is in progress will cause lesser crop loss (Agrios, 1997). Chemical control with trizole fungicides has been reported as useful in controlling infections up to ear emergence, but is difﬁcult to justify economically in attacks after this stage. Varietal control is again the best control for leaf rust. Resistant varieties possess one or more special leaf rust resistance genes called Lr genes. Currently, there are more than 58 different Lr genes available in wheat (Bansal et al., 2008; Chhuneja et al., 2008; McIntosh et al., 2008), but most varieties have only a few Lr genes. So, there is a need to develop multi Lr gene-carrying varieties to defeat leaf rust disease. Yellow rust or stripe rust Another rust of wheat, stripe or yellow rust which is caused by P. striiformis f. sp. tritici, can be as damaging as other rusts. Due to a requirement for a very low optimum temperature for its development, stripe rust is not found in many areas of the world. However, a total area of 9.4m ha (> 35%) under wheat cultivation is affected by stripe rust (Singh et al., 2004). On the world level, stripe rust is found predominantly in northern Europe, the Middle East, East Africa, China, India and the continents of South America, Australia and New Zealand (Saari and Prescott, 1985). In the USA, it was ﬁrst reported in 1915 (Carleton, 1915) and serious outbreaks were reported in the western states in the 1960s (Line, 2002; Boyd, 2005). For this disease, generally no cultural control measures are applicable, but in the USA, where the disease occurs commonly, the removal of the alternate host is an established method of cultural control. Identiﬁcation and use of the resistant gene in resistant varities is the only way to reduce the impact of the disease on wheat production. Many yellow rust resistance genes have been identiﬁed in wheat by different wheat workers and to date, 41 of these (Yr1 to Yr41) have been designated (McIntosh et al., 2008). Most of the identiﬁed yellow rust resistance genes have proven to be race-speciﬁc, with resistance being effective only against isolates of P. striiformis f. sp. tritici carrying the corresponding avirulence gene. Different wild wheat varieties were also used to transfer the resistance gene to hexaploid wheat for stripe rust resistance (Kuraparthy et al., 2007a,b; Singh et al., 2007; Chhuneja et al., 2008). More recently, a highly resistant gene with broad spectrum on strip rust races, namely Yr36, from wild emmer wheat was used for positional cloning (Fu et al., 2009). Karnal bunt Karnal bunt (partial bunt) of wheat has become a disease of serious concern in some parts of the world as it causes direct yield losses and also has signiﬁcance as an export problem because many believe the pathogen to be a quarantine pest. Consequently, stringent quarantine measures have been adopted in several countries, which may affect not only the wheat grain trade but also germplasm exchange (Royer and Rytter, 1988). Karnal bunt caused by the smut fungus Tilletia indica Mitra Neovossia indica (Mitra, 1931), a Basidiomycetes fungus, is a serious ﬂoral-infecting disease of wheat in the major wheat-growing areas of India (Gill, 1990) and some other wheat-growing countries of the world (Nath et al., 1981). The pathogen is known to infect bread wheat, durum wheat and triticale (Agarwal Some Important Fungal Diseases and Wheat Production et al., 1977). The disease was ﬁrst reported in 1931 in experimental wheat crop at the Botanical Station at Karnal, India (Mitra, 1931), and was for many years known only in the plains of India and Pakistan (Ahmad and Attaudin, 1991). Currently, it occurs in Afghanistan, India, Iran, Iraq, Mexico, Nepal and Pakistan and in limited areas of the USA (Durán, 1972; Munjal, 1975; Singh et al., 1989; Ykema et al., 1996). Recognition of fungal structures (teliospores) on grain samples from Lebanon and Syria suggest that the disease is established in these countries as well (Locke and Watson, 1955). Karnal bunt requires free water in the soil for teliospores, the overwintering life stage of Karnal bunt, to germinate. Teliospores are brown to dark brown, spherical or subspherical, or oval, 22–42 × 25–40 µm in diameter, occasionally having an apiculus (Roberson and Luttrell, 1987), papilla (Mitra, 1931) or a vestige of attached mycelium (Durán and Fischer, 1961). The disease cycle (Fig. 27.1) starts with the introduction of teliospores on to a ﬁeld. Contaminated seeds are considered to be the major source of teliospores, while other sources include wind, animals, contaminated equipment or contaminated vehicles. Teliospores may remain dormant but viable for several years (Ottman, 2002). Although planting infected seed is the primary means of getting the spores into the soil, this may or may not produce infected plants directly in the ﬁrst year. The greater threat of disease occurs the following year as the soil is turned over, bringing these teliospores back to the surface. At the ﬂowering stage of host plants, the teliospores produce sporidia that infect the plant ﬂorets and fungal hyphae enter the ovary (Aujla et al., 1977; Singh and Prasad, 1978; Khetarpal et al., 1980; Krishna and Infected grains Primary infection (partial systematic spread) Combining and threshing Germination of soilborne teliospores Subsequent spread to late tillers Multiplication on wheat and other plant leaves 367 Germination of primary spordia on whorl Allantoid secondary sporidia Fig. 27.1. The life cycle of Karnal bunt caused by Tilletia indica Mitra. Filliform secondary sporidia 368 A. Goyal and R. Prasad Singh, 1982). Subsequent disease development in the embryo end of the kernel results in the formation of new teliospores, which are deposited back in the soil at harvest, adding further to soil inoculum. Cool, cloudy and very humid conditions or rainfall between awn emergence and the end of ﬂowering are required for sporidia production, infection and for the disease to ﬂourish (Dhaliwal et al., 1983; Goates, 1988). The incidence of Karnal bunt is usually very low and rarely seen if the environmental requirements are not met. Karnal bunt affects the heads of wheat plants. The disease is not easily detected in the ﬁeld because few ﬂorets are typically infected and the area of the kernel affected might be small and facing inwards. A mass of black teliospores is found at the embryo end of the kernel and, at higher levels of infection, along the crease or in the entire kernel (Goel et al., 1977; Dhaliwal et al., 1983). A ﬁshy odour is emitted from infected seeds due to the presence of trimethylamine (Mehdi et al., 1973). Conventional approaches to control this disease consist of the adoption of various cultural practices such as crop rotation for longer periods, sowing of disease-free seeds, adjustment of the nitrogen balance in the soil and adjustment of the time of irrigation to minimize disease incidence (Mitra, 1937; Munjal, 1974; Goel et al., 1977; Singh and Prasad, 1978; Aujla et al., 1981, Singh and Singh, 1985; Gill et al., 1993). Control through fungicides is not completely effective as the disease is seed- and soilborne (Singh et al., 1985). However, application of Tilt at heading and 1 week later can reduce disease incidence by 90% when environmental conditions are conducive to disease development (Ottman, 2002). Hence, the most economical, eco-friendly and effective approach to control the disease is the cultivation of resistant varieties. The main sources of resistance against Karnal bunt have been the Indian, Chinese and Brazilian wheats (Fuentes-Davila and Rajaram, 1994). A new range of genetic variability for resistance to Karnal bunt has been observed in synthetic hexaploid wheat derived from T. turgidum × T. tauschii crosses (Villareal et al., 1996). Powdery mildew Powdery mildew of wheat, a wind-dispersed disease, is an important and most common disease worldwide, particularly in humid regions (Oerke et al., 1994). It is of special interest in epidemiology because it results in reduced kernel size and seed weight, and ultimately lower yield. The fungal pathogen, Blumeria graminis f. sp. tritici (an Ascomycete), causing powdery mildew on wheat, is a biotrophic obligate parasite (Cooke et al., 2006), which is highly sensitive to the environment and its presence can vary from season to season (Jenkyn and Bainbridge, 1978; Jorgensen, 1988; Wolfe and McDermott, 1994). The powdery mildew fungus is made up of different races and forms that are highly specialized. Wheat cultivars might be resistant to a certain race of the mildew fungus, but susceptible to another race. Some of the special features of powdery mildew, such as wide distribution, rapid development within or on host tissue, massive production of spores, the ability to remain viable after long-distance dispersal and a high capacity to become virulent on previously resistant cultivars, make it a devastating disease of wheat (Boshoff et al., 2002). Powdery mildew oversummers on volunteer crops in the asexual stage, infects the autumn-sown crop and, eventually, overwinters on the volunteers to infect the crops in spring (Zadoks, 1961). In mild areas, volunteer wheat plants are abundant because of the relatively frequent rainfall in summer, while in dry regions, oversummering can depend on grass species (Boshoff et al., 2002). For mildew, the asexual cycle is the production of haploid conidia, while occasionally the ascospores, which are the result of sexual cycle, can initiate epidemics (Cooke et al., 2006). Mildew also differentiates a sexual stage, which contributes to oversummering. In early summer, B. graminis f. sp. tritici initiates the formation of generative mycelium and cleistothecia starting on the lower leaves. In the cleistothecia, 15–20 asci develop, each containing eight haploid Some Important Fungal Diseases and Wheat Production ascospores which are dispersed by wind, even under high humidity after rain (Gotz et al., 1996). Ascospores can develop at any time during the last half of the year; therefore, sexual reproduction is more important for powdery mildew on wheat. Apart from ascospores, conidia from the summer crop can also infect volunteer plants; thus, a mixture of ascospores and conidia forms the inoculum for the winter crop. However, the mildew population grown during autumn on the winter crop can survive the cold period in vegetative stage on overwintering green plants (Cooke et al., 2006). Mildew is more severe in dense stands of heavily fertilized wheat. Plants are most susceptible during periods of rapid growth, especially from stem elongation through heading growth stages. Powdery mildew on wheat is recognized by small, effuse patches (colonies) of cottony mycelia on the upper and lower surfaces of the leaves. As these patches sporulate and age, they become a mass of dull tan colour. Chlorotic (yellow) patches may later surround the mildew colonies (Purdy, 1967; Kingsland, 1982). Powdery mildew attacks the leaves, but stems and heads are also affected. The fungus grows primarily on the surface of the host and feeds on the living green cells of the plant. Damage occurs from reduced photosynthetic ability when green surfaces are shaded and the host is robbed of moisture and food by fungal growth. Yields may be reduced by 20% or more. Spring wheat, other than soft white wheat, are seldom affected at economic levels on the prairies, while winter wheat is affected to a greater degree. The disease will reduce yields seriously if the ﬂag and second leaves are affected (Gotz et al., 1996; Boshoff et al., 2002). Incorporating wheat residues into the soil, destroying volunteer wheat and crop rotation can lessen the amount of overwintering inoculum in the ﬁeld. Powdery mildew thrives where high rates of nitrogen have been used. Therefore, use of a correct and balanced fertilization programme with proper levels of N, P and K is advised. It is important to keep the top two leaves of the plant as disease free as possible so that the 369 plant can use its full potential to ﬁll the grain. Fungicides can be applied based on the level of disease in the ﬁeld, the known susceptibility of the variety and the selling price of the grain (Agrios, 1997). Growing mildew-resistant cultivars is the most economical way to control powdery mildew, though wheat varieties vary in their resistance to powdery mildew and new races of the fungus can attack previously resistant varieties. Conclusions Wheat is a most important cereal crop and is becoming more in demand due to the signiﬁcant increase in the world population. To protect the world from the upcoming threat of hunger, food and nutrition, wheat production must be doubled in time. Major concern about wheat quality and production is related to biotic stress. Different methods, such as chemical control, cultural methods and eradication of alternate hosts, are used to prevent the disease, but the most important and effective one is the development of resistant varieties. For durability of resistance against fungal diseases, breeders should focus on new sources of race-speciﬁc resistance genes from either adapted cultivars or wild varieties. 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This page intentionally left blank Index Note: Page numbers in italic refer to tables and ﬁgures in the text Abies spp., root and butt rot 347, 349, 353, 354 abiotic stresses endophyte protection of plants 189–190 fenugreek 246–247 wheat 363 Acaulosporaceae 166 accelerated ageing germination (AAG) 332–333 acetaldehyde 8 acetic acid 43, 341 Achyranthes japonica 40 acibenzolar-s-methyle (ASM) 9 Acremonium terricola 284 Actinomycetes, antibiotics 123 Adgen Phytodiagnostic Septoria ELISA kit 299 aﬂatoxins 15, 29, 31, 39 production inhibited by plant extracts 39, 60, 61 recommended limits 34 agglutinins 127 Agrobacterium radiobacter 125, 125 agronomic characters, and disease resistance 73, 81–82 ajoene 20, 22 aldehydes 42 alfalfa 173, 207 alimentary toxic aleukia (ATA) 31 alkaloids 33, 150, 190 allicin 20, 22 Allium sativum extract 40 allyl-isothiocyanate (AITC) 43 Aloe vera gel 9 alpine European forests 345–346 ecological functions 345 management 350 root and butt rot biological and chemical control 122–123, 354–356 diagnosis 350–351, 351 effects of forest management 350 general control strategies 351–354 infection biology 346–349, 347 integrated management 356–358 wood decay 348 tree species 345–346 Alternaria spp. 232 fruit crops 5 inhibition by plant extracts 40–41 leaf blight castor 271–272 safﬂower 267–268 wheat 232–233, 241 leaf blight/black point 234–236 leaf spot sesame 270 sunﬂower 265–266 Alternaria alternata 364 Alternaria carthami 271 Alternaria helianthi 266 Alternaria infectoria 234–236 Alternaria padwickii 40–41, 55 Alternaria sesame 270 Alternaria solani 173 Alternaria triticina 232–233 aluminium tolerance 189 375 376 Index Amaranthus spp. 311–312 smut incidence in cultivars 315, 316 wild species 313, 315–316 Amaranthus hybridus 315–316 Amaranthus retroﬂexus 315–316 AMF, see arbuscular mycorrhizal fungi amino acids 177 anise 56, 60 antagonists 122 criteria for commercial production 111 endophytes 185–186 fruit storage pathogens 6–7, 110–111 intergration with other control measures 115–116 mechanisms of action 111–113 root and butt rot fungi 355 tan spot 283–284 antibiosis 9, 111, 123 KB-8A 270 Trichoderma 123, 127 antiseptics, stored produce 34 Aosta Valley forests 357, 357 Aphanoderma album 212 apoplast, protease activity 304 apples 6, 43, 174 hot air treatment 116 potassium iodide wraps 6 arbuscular mycorrhizal fungi (AMF) 124 effects of agricultural practices 163–165 importance in agriculture 162–163 interaction with fungal pathogens 122–123, 172–175 mechanisms of disease control 175–177 role in plant nutrition and growth 172 signalling pathway 175 soil propagule bank 164 effects of tillage 164–167 taxonomy 172 Argentina fungicide use 292 wheat pathogens 231 Alternaria leaf blight 232–233 Alternaria leaf blight/black point 234–236 Ascochyta hordei leaf spot 236–237 Cephalosporium gramineum stripe 238–239 Cladosporium herbarum leaf spot 239–241 monitoring changes 241–242 Phoma soghina leaf spots 237–238 Pyricularia grisea spot blight 241 tan spot 276–284 wheat production 276, 291–292 arginine 177 Armillaria mellea sensu lato 347, 348, 349 control 133, 351–358 diagnosis 351, 351 aroeira extract 61 aromatic compounds, fruit and vegetables 7–8, 42–43 asarone 20, 22 Ascochyta hordei var. europaea 236–237 Ascochyta leaf spot 252 Aspergillus spp. plant extract treatment 59–61 seed spoilage 330–331 Aspergillus ﬂavus 29, 31, 32, 330 Aspergillus niger 265, 330 Aspergillus ruber 330–331 AspireTM 110, 114 Avr gene products 129 AVR-Pita avirulence gen family 98 Bacillus spp. 124–125, 252 bacteria biocontrol of soil diseases 124–125, 125 endophytes 150 bacterial disease endophyte plant protection 186–187 fenugreek 247, 248 bags, seed storage 336, 338, 340, 341 bajra, rusts 209 banana, mycorrhizae 176 bark beetle 357 bark extracts 40 barley, Fusarium head blight 79, 81–83, 87–88 barley yellow dwarf virus 187 basil extracts 40, 60–61 bavistin 332, 332, 333 bayletan 332 bean, rust fungi 209–210, 214, 216 beet, leaf endophytes 151–152, 153 benlate 332 benodanyl 214 benomyl 226 benomyl thiabendazole 34 benzaldehyde 42 benzanilide 214 benzimidazole 214 Beta vulgaris var. esculenta, see beet biological control airborne disease 124 commercial products and systems 125, 125 components 131–134 constraints in development 113–115 Trichoderma spp. 131, 133 deﬁnition 121–122 efﬁcacy, consistency of 114 endophytes 150–151, 185–190 mechanisms 122–123 Phytophthora sojae 323 Index postharvest diseases 17–18, 110 rusts 212 Septoria tritici blotch 303–305 soilborne diseases 124–125, 125 tan spot of wheat 159, 283–284 see also botanicals; essential oils; Trichoderma spp.; yeasts bioprotection, AM fungi 171, 172–175 BioSaveTM 7, 110 Bipolaris spp., seed pathogens 55–56 Bipolaris sacchari 223–224 Bipolaris sorokiniana 55 bitter leaf 40 ‘black mars’ 29 black pepper 174, 176 black point, wheat (Alternaria infectoria) 234 black stem rust 203, 214 bleaching powder, seed treatment 333 blue mould decay 44, 116 Blumeria graminis f.sp. tritici 368–369 borax 355–356 bordeaux mixture 214 botanicals 7–9, 17–18 chemical structures 21–22 effective against toxin producing fungi 39 effects on seed fungi 55–61 efﬁcacy in fungal control 19–20 essential oils 8–9, 18 seed treatments 38–39, 52–53 fruit crop disease control 7–9 potential advantages 45 potential risks 45–46 seed treatments 41 seedborne fungi Alternaria 55 Aspergillus 59–61 Bipolaris 55–56 Colletotrichum 56 Curvularia 56–57 Fusarium 57–59 Macrophomina 59 Penicillium 59–60 Botryosphaeria spp. 133 Botrytis spp. botanical control 42, 43–44 grey mould of fruit 4 grey rot of castor 272 Trichoderma control products 133 Botrytis cinerea 4, 42, 43–44 Botrytis ricini 272 brassicol 332 brinjal, seed treatments 334, 335, 335 Bromus spp., head smut 139–140, 140, 141 brown rot 4 brown rust, wheat 364, 365–366 Bt genes 342 Burkholderia ambifaria 188 α-cadinol 20, 22 calcium chloride 115 camphor 20, 21 captafol 214 captan, seed dressings 332, 332, 333 caraway oil 38 carbendazim 99, 226 carboxin, seed treatment 334 Carthamus oxycantha (Pohli weed) 269 Carthamus tinctorius, see safﬂower carvacrol 20, 22 carvone 20, 21, 40, 53, 57 caryophyllene 20, 21 cash crops, mycorrhizae 174 castor 271–273 cedarwood oil 38 Cephalosporium gramineum stripe 238–239 Ceratocystis paradoxa 225 Cercospora arachidicola 263–264 Cercospora leaf spot fenugreek 249–252, 250 sesame 271 Cercospora sesami 271 Cercospora traversiana 250–252, 250 cereal crops mycorrhizae 172–173 rust diseases 206 seedborne fungi, treatment with plant extracts 57 see also individual cereal crops cerebrosides 187 charcoal rot, fenugreek 250 charcoal stump rot 174, 177 chemical fungicides 36, 183 drawbacks of 36, 62 Phytophthora sojae control 323 regulation 110 resistance 99–100 rice blast disease 99–100 root and butt rot of alpine forests 355–356 rust diseases 213–214 seed treatments 331–333, 332 storage diseases 16–17, 34 sugarcane diseases pineapple disease 225–226 rusts 221 smut 219 tan spot 283 wheat 283, 292 Chenopodium procerum 40 chestnut blight fungus 123, 186 chickpea mycorrhizae and fungal diseases 176 seed treatments 333, 334 chilli seeds 335, 335 chitinases 125, 127, 177 377 378 Index chitosan 9 Cicer arietinum 209 1,8 cineole 20, 22 cinnamaldehyde 20, 21, 53 cinnamate derivatives 95 cinnamon oil 56, 58 citral 39, 53 citronellol 20, 21 citrus fruit 115 brown rot 4 essential oil treatments 44 green mould 115–116 root rot 174 Cladosporium spp. 212 Cladosporium herbarum, wheat leaves 239–241 Claviceps 54 Clavicipitaceae 150 Climacocystis borealis 347, 348 control 351–358 diagnosis 351, 351 climate India 15, 37 and postharvest diseases 15 and powdery mildew of wheat 368–369 Septoria tritici blotch development 298–299, 298 climatic factors, seed storage fungi 340 clove oil 38, 56, 60 clover, rusts 207 cocoa beans, postharvest damage 29 coffee rust diseases 204–205, 206, 208 fungicides 214 cold storage 15 collar rot castor 272 fenugreek 250, 252 groundnut 265 Colletotrichum spp. 56, 186 Colletotrichum falcatum 221–222 Colletotrichum gloeosporioides 7, 154 common bunt (Tilletia laevis) 143–145 competition 112, 122–123 Trichoderma 127–128 ‘compound interest diseases’ 205 containers, seed storage 336, 338, 340, 341 copper-based fungicides 214, 335, 340 corn smut 140–142, 143 cotton mycorrhizae, effects on fungal diseases 176 rusts 207, 210 coumestrol 177 cowpea mycorrhizae 173, 176 seed treatments 333, 334 Crambus spp. 188 crop residues, wheat 283 crop rotation forests 353 Phytophthora sojae control 323 tan spot control 283 cross-protection 123 Cryphonectria (Endothia) parasitica 123 cryptocin 186 cucumber, mycorrhizae, effects on fungal diseases 176 cultural practices alpine forests 350 effects on mycorrhizae 163–165 fruit production 5 wheat diseases 365, 368, 369 Septoria leaf blotch resistance 74 tan spot 283 see also no-tillage systems cumin 60 Cuminum cyminum 174 curcumene 20, 22 Curvularia spp. 56 Curvularia protuberata 190 cyanide (HCN) precursors 177 ρ-cymene 20, 21 Cyprus rotundus extracts 40 cytomegalovirus, human (hCMV) 187 Dalbergia rust 206 damping-off diseases fenugreek 249, 252 sunﬂower 267 Trichoderma control products 133 defences, see plant defence responses deformed plants 30 deoxynivalenol (DON) 80, 81, 83, 86, 364 inhibition by essential oils 58 recommended levels 364 Dichanthelium lanuginosum 190 diene antifungal compounds 4 discoloration of crops 29, 54 disease resistance agronomic characters 73, 81–82 FHB 80–81 induced 9, 123–124, 128–129 morphological 80, 81–82 non-speciﬁc (partial) 95–96, 322 physiological, types of 80–81 race-speciﬁc 321–322 RBD 95–99 Septoria leaf blotch 70–73 see also endophytes, plant protection; plant breeding; resistance genes DON, see deoxynivalenol downy mildew, sunﬂower 266–267 Drechslera tritici-repentis, see tan spot Index drought tolerance, and endophytes 189 drying of produce 33, 341 dryland crops 246 Dutch elm disease 188 early leaf spot, groundnut 263–264 egusi melon 61 endophytes bacteria 150 beet leaves 151–152, 153 deﬁnitions 149–150, 183–184 ecological role and strategy 150–151 as gene vectors 151 groups 184 non-grass plants 150 plant protection 151, 159 abiotic stress 189–190 bacterial disease 186–187 fungal disease 159, 185–186 insects 188–189 nematodes 188 viral disease 187 potential of 190 research 151, 184–185, 190–192, 191 soybean leaves 154–155, 154 tomato leaves 152–154, 153 wheat 155–156, 157–158, 159 environmental conditions and powdery mildew of wheat 368–369 seed storage 337–340, 341 Septoria tritici development 298–299 stored crops 31–33 enzyme-linked immunosorbent assays (ELISA) 299 enzymes AM fungi 177 biological control agents 112, 125, 127 fungal pathogens 29–30, 32 epidemiology, deﬁnition 292 ergot 31, 54, 364 ergovaline 150 Erysiphe cichoracearum 270 Erysiphe polygoni 254–256, 257, 258 Escherichia coli, recombinant 125 essential oils 8–9, 18, 37–38, 43–44 active components 37, 53 biocide formulation 42 chemical structures 21–22 efﬁcacy 19, 20 phytopathogenic fungi 38 seedborne fungi 38–39, 52–53, 55–61 toxin producing fungi 39 mode of action 44–45, 53 toxicity 45–46 estrobirulinas 283 379 ethanol 115–116 eugenol 20, 53 Eutypa dieback 9 expressed sequence tags (EST) 87 eye spot disease, sugarcane 223–224 fenchone 20, 21 fennel 60 fenugreek abiotic disease 246–247 bacterial disease 247, 248 biology 245–246 crop potential 246 disease-resistant cultivars 257 fungal disease 249–256, 257 collar rot 250, 252 Fusarium wilt 252–253 leaf spot 250, 252 pod spot 250, 253 powdery mildew 254–256, 257, 258 spring black stem/leaf spot 253–254 insect pests 247, 248, 249 nematodes 247, 248 seed extracts, antifungal activity 249 seed treatments 334–335, 335 viral diseases 247 ferbam 213 fertilization 74, 369 fescue, tall 150, 187, 188 fescue toxicosis 150 FHB, see Fusarium head blight ﬁbre crops, rusts 207 ﬁg (Ficus spp.), rust fungi 210–211, 214 ﬁr, silver, root and butt rot 347 ﬂavour, damage by fungi 29 ﬂavour compounds, fruit 7–8, 42–43 ﬂuzilazol propiconazol 283 Fomes annosum, see Heterobasidion annosum Fomitopsis pinicola 347, 348 control 351–358 diagnosis 351, 351 food grains, loss in storage 329–330 food security 14 foot rot, black pepper 174 forage crops, rusts 207, 216 forest rotation 353 forests role of mycorrhizae 174, 175 see also alpine European forests free fatty acids (FFAs) 29–30, 331 fruit antifungal compounds in unripe 4 aromatic and ﬂavour compounds 7–8, 42–43 cultural disease control 5 disease-resistant transgenic plants 10 380 fruit continued nutritional value 3 postharvest diseases 3, 4 integrated management 7, 115–116 plant extract treatments 7–9, 42–44 prevention 5–7 production in India 4 rust fungi 213 frutiafol 283 fumigation, botanicals 5, 42, 43, 44, 60 fumonisins 39, 80 fumonism 31 fungicides, see chemical fungicides fusarenon-X (FUS-X) 80 Fusarium spp. mycotoxins 80 plant extract treatments 57–59 postharvest pathogens 79 toxin production 31, 364 Trichoderma control products 133 Fusarium avenaceum 79, 80 Fusarium culmorum 79, 80 Fusarium graminearum 31, 32, 79, 80 Fusarium head blight (scab/FHB) 78–79, 363–364, 364 epidemics and crop losses 79 host resistance 80–81 barley 81–83, 87–88 wheat 83–87 mycotoxins 364 species isolated 79 symptoms and effects 79–80, 363–364 Fusarium moniliformae 224–225 Fusarium oxysporum 252–253 Fusarium oxysporum f.sp. ricini 272–273 Fusarium oxysporum f.sp. sesame 270 Fusarium poae 79, 80 Fusarium wilt and AM fungi 173 castor 272–273 endophyte-induced resistance 185 fenugreek 250, 252–253 safﬂower 268 sesame 270 galactomannans, fenugreek 246 garlic extracts 56, 59, 60, 61 gene silencing 9 geothermal soils 190 geraniol 53 germination and seed mycoﬂora 331, 331 and seed treatments 333–336, 333, 337 Gibberella moniliformis 224 Gigasporaceae 166 ginger 44, 56 Index Gliocladium spp. 130, 270 Gloeosporium rot 5 Glomeromycota spp. 172 in no-tillage systems 163–164, 166–167 Glomosporium amaranthi, see Thecaphora amaranthi Glomus coronatum 188 beta-1,3-glucanases 125, 177 glucosinolates 8, 43 glume blotch 364 Glycine max, see soybean gram (Cicer arietinum), rusts 209 grapes, postharvest technology 6 grapevine, Eutypa dieback 10 grass species endophytes 150, 187, 188, 189 head smut 139–140, 140, 141 tan spot hosts 279 green mould, citrus fruit 115–116 green revolution, India 205–206 grey mould, strawberry 4 grey rot, Botrytis 272 groundnut 263 collar rot 265 early leaf spot 263–264 late leaf spot 264 postharvest damage 29, 30, 33 role of mycorrhizae 173 rust 203, 206, 208 fungicides 213, 214, 264 resistant varieties 264 seed and seedling diseases 264–265 growth abnormalities, damaged seed 30 guava 4 hairpin-encoding genes 99 halogenation, seeds 342 head blight, see Fusarium head blight (scab) head rot, Rhizopus 267 head smut, Bromus spp. 139–140, 140 heading date 73, 81–82 heat tolerance, and endophytes 190 heat treatments 15–16 Alternaria leaf blight 268 fenugreek seed 253 fruit crops 5, 116 red rot of sugarcane 223 heating of crops (deleterious) 30 Helianthus annuus var. macrocarpus, see sunﬂower Helicobacter pylori 187 Helminthosporium carbonum 10 Helminthosporium oryzae 56 Heterobasidion annosum sensu lato 347, 348, 349, 350 control 122–123, 351–358 Index diagnosis 351, 351 species susceptbility 353–354, 354 Heterosporium medicaginis 253 hevien 9 hexanal 42–43 hexenal 42–43 hinosan, seed dressings 332, 332, 333 honeybees 45 horticultural crops mycorrhizae 173–174, 176 rust diseases 207 ‘host shifts’ 94 host-speciﬁc toxins (HST) 223 hrf1 gene 99 hydration-dehydration treatments 342 hyperparasitism 123 hypersensitive response (HR), soybean 320 hypovirulence 123 immunoassays, Septoria tritici 299 India castor production 271 climate 15, 37 endophyte research 190–192, 191 fruit production 4 green revolution 205–206 indole derivatives 186 induced resistance 9, 123–124, 128–129 insect pests fenugreek 247, 248, 249 protective effects of endophytes 188–189 stored products 28–29, 31 integrated disease management 62, 124 fruit crop diseases 7 Phytophthora sojae 323 postharvest fruit disease 7, 115–116 postharvest fungi 115–116 root and butt rot fungi of trees 356–358 rusts 215 Septoria leaf blotch 73–74 ionizing radiation treatments 5–6, 16 iron 130 isoleucine 246 jasmonates 8, 43 javanicin 187 jowar, rusts 206, 208–209, 216 jute bags, seed storage 336, 338 karnal bunt (partial bunt) 366–368, 367 Laetiporus sulphureus 347, 348 Laetisaria arvalis 284 381 larch (Larix), root and butt rot 347, 353, 354 late leaf spot, groundnut 264 latex 9 leaf blight Alternaria carthami 267–268 Alternaria infectoria 234, 241 Alternaria triticina 232–233, 241 leaf blotch (Septoria) 70–74 leaf rust, wheat 364, 365–366 leaf spot Alternaria 265–266, 270 Ascochyta 236–237, 252 Cercospora, sesame 271 groundnut 263–264 Phoma sorghina 237–238 Pyricularia grisea 241 lectins 127 legume crops mycorrhizae 173 rust diseases 209–210 fungicide treatment 214 seed treatments 333, 334 lemongrass oil 39, 58 leucine-rich repeats (LRRS) 212 Leveillula taurica 270 Lewia infectoria 235–236 lime-sulphur 213 limonene 20, 21, 53 Limonomyces roseipellis 284 linalool 20, 21, 53 lineage exclusion hypothesis 98 linseed 207, 209 lipases 29–30, 127 logging, impact on root and butt rot fungi 350 lolitrem B 150 Lr genes 366 Luffa acutangula 340 Lycopersicon esculentum, see tomato lyso-phosphatidylcholine 175 Macrophomina phaseoli 29 Macrophomina phaseolina 272 Magnaporthe graminicola 293 Magnaporthe grisea 92–93 pathotypes 93–95, 97 maize postharvest damage 30 rusts 206, 209, 216 seed spoilage in storage 330 seedborne fungi, plant extract treatments 58 smut 140–142, 143 mancozeb 213, 333, 333 maneb 213 manganese 130 mango 4 382 marker-assisted selection (MAS) soybean 323 wheat 86–87, 88, 369 medicinal plants fenugreek 246 northern India 185, 190 rusts 207 Meloidogyne incognita 188, 247 melon seeds, plant extract treatments 41, 61 menthol 20, 22 menthone 20, 21 mercury fungicides 214 methyl bromide 183 methyl jasmonate 43 methyl salicylate 46 MGR586 DNA repeat element 94 microtubules 177 minerals, solubilization and sequestration by Trichoderma 128–129, 130 moisture, stored crops 31–32 molecular diagnostics, root and butt rot fungi 351, 351 molecular markers Phytophthora sojae 321 soybean 323 wheat 86–87, 369 moringa 41 morphological disease resistance 80, 81–82 mulberry 206 mung bean 173, 333, 334 muskmelon seed 334, 335 mustard 334 mycoparasitism 123, 127 mycoparasitism related genes (MRGs) 126 mycorrhizae 124 soil infectivity 164 see also arbuscular mycorrhizal fungi (AMF) Mycosphaerella arachidis 263–264 Mycosphaerella graminicola 69, 70, 231 mycotoxins aﬂatoxins 15, 29, 31, 39 control of production 39 DON 58, 80, 81, 83, 86, 364 Fusarium head blight 80, 364 mould species producing 32 safe limits 34 myrcene 20, 21 Myrothecium roridum 284 nabam, rust diseases 213 neem extracts 6, 8, 34, 40, 41 neem oil 42, 56, 60 neem seedlings, AMF 174, 175 neembicidine 333 nematodes Index and endophytes 188 fenugreek 247, 248 and mycorrhizae 176–177 nitrogen fertilization 74, 369 nivalenol (NIV) 80 no-tillage systems and arbuscular mycorrhizae 163–164, 166–167 problems of 163 soil property changes 163 and tan spot control 283 wheat crops 74, 276 Norway spruce, root and butt rot 347, 352, 353, 354, 354 notchi powder 41 nucleotide-binding site plus leucine-rich repeat (NBS-LRR) genes 98 nutrients competition for 112 see also plant nutrients nutritional requirements, fungi 32 oat, rust 206 oats 283 ochratoxin 31 odour changes 29 Oidium erysiphoides 270 oils fruit skin coatings 6 see also essential oils oilseed crops mycorrhizae 173 rust diseases 206 seed treatments 333, 334 see also individual oilseed crops olive oil 38 onion, mycorrhizae 176 onion-pink rot 173 Onnia tomentosa 347, 348 oocydin 186 oranges 44 orchard hygiene 5 oregano oil 38, 60 ornamental plants, rusts 207 orthodihydroxy (O-D) phenols 177 oxanthiin-carboxin, rusts 214 oxycarboxin 214 oxygen 32 ozone treatments 5 paddy, seed treatments 334, 335, 335, 336 palm oil 30, 33 palmarosa oil 58 papaya 5, 9, 41 pawpaw 40 Index PCR-based diagnosis, root and butt rot fungi 351, 351 pea, rust 209, 213 pearl millet rust diseases 206, 209 seed spoilage/treatments 330, 335–336, 336, 337, 338 Penicillium, plant extract treatments 59–61 pepper, mycorrhizae 176 peppermint oil 38 Peronospora trifoliorum 249 peroxidase 129, 177 Pestalotiopsis microspora 186 Phaeoisariopsis personata 264 Phaeolus schweinitzii 347, 348 phalsa 174, 213 phenylalanine 177 Phlebia gigantea 122–123 Phoma spp., Trichoderma control products 133 Phoma pinodella 249, 253–254 Phoma sorghina 237–238 Phomopsis oblonga 184 Phomopsis psidii 4 physiological specialization Phytophthora sojae 320–321 powdery mildew of wheat 368 rice blast fungi 93–95, 97 tan spot on wheat 281–282 Tilletia laevis 143–145 Ustilago bullata 139–140, 141, 142 physiology of resistance, rice blast 95 phytoalexins 95, 129 in AMF-containing plants 177 induction by yeast antagonists 112 Phytophothora spp., Trichoderma control products 133 Phytophthora drechsleri 268 Phytophthora infestans 185 Phytophthora nicotianae var. parasitica 173–174 Phytophthora parasitica var. sesame 269 Phytophthora sojae 319 life cycle 319–320, 319 physiologic races 320–321 Phytophthora spp., blight of sesame 269 Pi-ta gene 98 Picea spp., root and butt rot 347, 349, 353, 354 pigeon pea blight 173 pine oils 53 pine, Scots, root and butt rot 347 pineapple disease, sugarcane 225–226 α-pinene 20, 21 Pinus spp., root and butt rot 347, 349, 353, 354 plant breeding Fusarium head blight (FHB) resistance 80 powdery mildew resistance 257 rust disease resistance 212 383 Septoria leaf blotch resistance 71–73 wheat 86–87, 363, 369 plant defence responses 95, 123–124 effects of mycorrhizae 175–176 provocation by Trichoderma spp. 304–305 plant extracts 39–40 effective against phytopathogenic fungi 39–40 effective against seed fungi 40–41 fruit crop treatments 44 total number 37 see also botanicals; essential oils plant growth and mycorrhizal associations 172 and Trichoderma spp. 129–130 plant height, and disease resistance 73 plant nutrients and fruit storage rot 5 solubilization/sequestration by Trichoderma 128–129, 130 uptake and mycorrhizal associations 176–177 plantation crops rust diseases 206–207 see also alpine European forests; forests plantavax w.p. 214 plantibodies 9 Plasmopara halstedii 266 Plasmopara patens 266 Plasmophara perennis 266 Plebiopsis gigantea 355 pod spot, fenugreek 253 Pohli weed 269 pokkah boeng disease 224–225 polyphenol oxidase 177 polythene bags, seed storage 336, 338 population genetics 292–293 postharvest diseases 14–15, 28, 29–30, 54–55 biochemical effects 29–30 conditions favouring 31–33 crop losses 109 crop weight loss 30 discoloration of crops 29, 54 ﬂavour and odour changes 29 fruit crops 4–5 botanical as antifungal agents 7–9 integrated control 115–116 management 5–7 Fusarium spp. 79 growth abnormalities 30 insects 28–29, 31 management 33–34 biocontrol products 110 botanicals 17–18, 19–22 preparation for attack by other agents 30–31 rotting and caking 30 384 postharvest diseases continued see also seed storage; storage diseases potassium metabisulphite 341 powdery mildew fenugreek 249, 250, 254–256, 257, 258 plant extracts 38 sesame 270–271 wheat 368–369 predation 124 preservatives, chemical 34 prochloraz 283 propagative materials, prevention of storage damage 34 propionic acid 341 proteases, apoplast 304 Pseudonmonads 124 Puccinia arachidis 264 Puccinia graminis f.sp. tritici 364, 365 Puccinia helianthi 266 Puccinia kuehnii 220–221 Puccinia melanocephala 220–221 Puccinia striiformis f.sp. tritici 366 Puccinia triticina f.sp. tritici 365–366 pulegone 20, 21 pulses mycorrhizae 173 rust diseases 207, 209–210, 216 pungam 41, 42 Pyrenophora tritici-repentis, see tan spot Pyricularia grisea 241 Pyricularia oryzae 186 Pythium spp. damping-off 249 Trichoderma control products 133 Qfhs.ifa-5A QTL 84 QFhs.ndsu-3BS QTL 84–85 Qrgz-2H-8 gene 83 QTL, see quantitative trait loci quantitative trait loci (QTL) Fusarium head blight resistance 78, 82, 84–87 Phytophthora sojae resistance 323 Septoria leaf blotch resistance 71, 72 ‘quelling’ 10 race typing, Ustilago scitaminea 218 radiation, stored food commodities 5–6, 16 Radopholus similis 188 rainfall and Septoria tritici blotch development 298–299, 298 and storage fungi 340 rainforests, mycorrhizae 175 random ampliﬁcation of polymorphic DNA (RAPD) 95–96, 321 raspberries 8 Index RBD, see rice blast disease reactive oxygen intermediates (ROI) 95 reactive oxygen species (ROS) 190 recombinant inbred lines (RILs) 85, 323 red rot, sugarcare 221–223 red smudge 278 relative humidity (RH) seed storage 337–339, 339 Septoria tritici blotch development 298, 298 stored crops 31–32 repeat induced mutation (RIP) 10 repeated DNA sequences, Magnaporthe grisea 93–95 resistance gene analogues (RGA) 99 resistance (R) genes 10 FHB 82–83 Hm1 10 race-speciﬁc 321–322 rice blast disease 96–99, 97 rusts 212 soybean 322–323 wheat 366 resorcinols 4 Reynoutria spp. 38 RFLP techniques 301 rhizobacteria-induced systemic resistance (RISR) 128 Rhizoctonia solani 133, 249, 250, 252 rhizome rot, ginger 174 Rhizopus arrhizus 267 Rhizopus nigricans 267 Rhizopus oryzae 267 Rhizopus stolonifer 5 Rht-D1 locus 86 rice postharvest damage 30 seed fungi 55, 57 sheath rot 42 rice blast disease (RBD) control 99–100, 100, 186 crop losses 92 epidemiology 93 fungal agent (Magnaporthe grisea) 92–93 races 93–95, 97 fungicide resistant 99–100 host resistance genetics 95–99 non-speciﬁc 95–96 physiology 95 symptoms 93 Ricinus communis, see castor RISR, see rhizobacteria-induced systemic resistance ROI, see reactive oxygen intermediates root and butt rot fungi, biological and chemical control 122–123, 354–356 root knot nematodes 176–177 Index root rot Macrophomina 250, 272 Phytophthora 268–269 safﬂower 268–269 wheat (S. rolfsi) 172, 177 ROS, see reactive oxygen species roses, rusts 210 rubber plant 174, 177 Rumex crispus 40 rust fungi 204 bean 209–210 coffee 208 cotton 210 epidemics and losses from 204–205 fenugreek 249, 250 ﬁg 210–211, 214 grain crops 206 gram 209 groundnut 203, 206, 208, 209, 264 key to species/varieties 216 linseed (ﬂax) 209 maize 206, 209, 216 management strategies 211–215 orange 220 pea 209 pearl millet 206, 209 rose 210 safﬂower 269 Sorghum spp. 206, 208–209, 216 soybean 210 sugarcane 220–221 sunﬂower 266 systematics 201–204 teleutospores 204 urediniospores 203–204 wheat 207–208, 364–366, 364 rye, rusts 206 ryegrass, endophytes 187, 188, 189 ‘ryegrass staggers’ 150 safﬂower 267–269 Alternaria leaf blight 267–268 Fusarium wilt 268 Phytophthora root rot 268–269 rust 206, 213, 214 Salvia ofﬁcinalis 38 sambangi 41 savoury oil 58 sclerotia 33, 54 Sclerotinia spp., Trichoderma control products 133 Sclerotinia sclerotiorum, endophyte protection 185 Sclerotium rolfsii 172, 177, 265 stem rot 265 Trichoderma control products 133 385 Scots pine, root and butt rot 347, 353, 354 seed abortion 54 seed extracts, disease suppression 249 seed health 329 seed necrosis 54 seed piece infection, pineapple disease 226 seed storage containers 336, 338, 341, 342 postharvest strategies 341 seed treatments chemical fungicides 331–333, 332 fenugreek 251–252 persistence during storage 339–340 plant extracts 39, 40–41 essential oils 38–39, 52–53 Trichoderma spp. 129, 134 and viability 333–336, 333, 337 seed viability loss in storage 30, 330, 331–336, 331 and seed treatments 333–336, 333, 337 and storage environment 337–340, 341 seedborne fungi Fusarium spp. 79 management, plant extracts 40–41, 55–61 symptoms of disease 54–55 seeds artiﬁcial 342 drying 341 free fatty acid content 29–30, 331 invigorating treatments 342 pelleting 342 Septoria leaf blotch causal agent 70 crop yield losses 70 integrated management 73–74 resistance 70–73 Septoria tritici blotch (STB) 293 ascendant movement of disease 299–301 biological control 303–305 early detection 299 epidemiological studies 293–298 impact of climate 298–299 population genetic studies 301–303 serine 177 Serratia marcescens 186 sesame 269–271 Fusarium wilt 270 Phytophthora blight 269–270 powdery mildew 270–271 Sesamum indicum, see sesame sheath rot, rice 42 siderophores 130 skin coatings, fruit 6 smut 138–139 amaranth 311–312 characterization of pathogen 313–315, 314 386 Index smut continued amaranth continued incidence in amaranth cultivars 315, 316 wild hosts 313, 315–316 head smut, Bromus spp. 139–140, 140, 141 maize 140–142, 143 sugarcare 218–219 ‘smut whip’ 219 sod webworms 188 sodium bicarbonate 115–116 sodium bisulphate 5 sodium metabisulphite 341 soils acidity 189 diseases control 124–125, 125 mycorrhizae propagule banks 163, 164–167 no-tillage systems 74, 163–164, 166–167, 283 sorghum, seed treatments 335 Sorghum spp., rusts 208–209, 213 sowing time 249 soybean endophytic fungi 154–155, 154 mycorrhizae 173, 177 Phytophthora root/stem rot 318 crop losses 318 disease cycle 319–321 management 321–323 pathogen 319, 320–321 symptoms 319 rust fungi 206, 210 seed treatments 333, 333 Sphaerellopsis ﬁlum 212 Sphaerotheca fuliginea 270 spike morphology 81–82 spinach, seed treatments 334, 335 sponge-gourd 41 spring black stem, fenugreek 253–254 spruce, Norway, root and butt rot 347, 352, 353, 354, 354 star anise 56, 60 stem rot, S. rolfsi 265 stem rust (black rust), wheat 364, 365 Stereum sanguinolentum 347, 349, 350 control 351–358 diagnosis 351, 351 steroidal sapogenins 246 storage diseases common fungal species 330 conditions favouring 31–33 management 15–17, 33–34, 340–341 botanicals and plant extracts 40–41 postharvest strategies 341 preharvest conditions 340 storage environment fruit crops 33, 34 seeds 337–340, 341 strawberries 4, 5, 8 Streptomyces 124–125 stripe, Cephalosporium gramineum 238–239 stromatization 54 stylar end rot 4 sugarcane economic importance 217 eye spot disease 223–224 pineapple disease 225–226 pokkah boeng disease 224–225 red rot disease 221–223 rust diseases 220–221 smut diseases 218–219 sulphur 213 sulphur dioxide 5 sunﬂower 265–267 Alternaria leaf spot/blight 265–266 downy mildew 266–267 head rot 267 rusts 206, 213, 214, 266 Suryanarayanan, Prof. T.S. 190, 191 sustainable disease management rice blast 100, 100 see also biological control; integrated management T-muurolol 20, 22 take-all 172, 364 tamarind 41 tan spot 231–232, 241, 276 crop losses 276–277 disease cycle 278–280 management 159, 282–284 pathogen 277–278 physiological specialization 281–282 prevalence and range in South America 276 symptoms of infection 278 tannins 40 tea tree oil 53 tebuconazol 283 teliospores 219 temperatures, seed and crop storage 32, 337–339, 339 Terminalia ivorensis 175 terpenoids 44 terpine-4-ol 20, 21 Thecaphora amaranthi 311–312 Thecaphora amaranthicola 312 characterization 313–315, 314 incidence in amaranth cultivars 315, 316 in wild amaranth species 313, 315–316 thermotherapy see heat treatments red rot of sugarcane 223 Index thiram rust diseases 213 seed dressings 332, 332, 333 thujone 20, 21 thyme oil 9, 19, 38, 44–45, 56 thymol 20, 21, 44–45, 53 tillage effects on mycorrhizae 163, 164–166 see also no-tillage systems Tilletia indica 366–368, 367 Tilletia laevis (common bunt) 143–145 time of sowing 249 tobacco 174, 177 tomato endophytes 152–154, 153, 185 mycorrhizae 173–174, 176 toxic metabolites 15, 29, 31, 39 Alternaria 232 Fusarium head blight 80 mould species producing 32 toxicosis, endophytes 150 transgenic plants, disease-resistant 10 trenches, isolation 352 triazoles, systemic 283 Trichoderma spp. 123, 125–126 chemicals produced 129 commercial use 131 compatibility testing 132–134 delivery methods 134 mass production and formulation 130–131, 132, 134 mechanisms of action 126–129 pesticide susceptibility 130 plant growth promotion 129–130 range of biocontrol uses 126, 126 red rot of sugarcane 223 safﬂower wilt 268 Septoria tritici blotch 303–305 solubilization/sequestration of plant nutrients 128–129, 130 tan spot control 284 Trichoderma harzianum 126, 223, 265, 270, 273, 284, 303–304 mass production and delivery 134 Trichoderma viride 126, 265, 270, 273 trichothecenes 80 Type A 80 Type B 80 Triticum spp., see wheat Triticum dicoccoides 87 Triticum macha 87 γ tubulin 177 ‘tulsi’ 8 UG99 (stem rust) 365 urea treatments 355–356 387 urediniospores, Puccinia 221, 269 Ustilago bullata 139–140, 140, 141 Ustilago maydis 140–142, 143 Ustilago scitaminea 218–219 Ustilago tritici 364 UV illumination 5–6 vegetable crops endophytes 185–186 mycorrhizae 173–174, 176 rust diseases 207, 209–210, 216 seed treatments 334–335, 335 verbenol 20, 22 verbenone 20, 22 Verticillium spp. antagonistic effects of endophytes 185–186 parasitic on rusts 212 protective role of AMF 173 viral diseases endophyte plant protection 187–188 fenugreek 247 vitavax 214 vomitoxin, see deoxynivalenol (DON) Vrs1 locus 82 warehouse conditions 33, 34, 337–340, 341 water requirements, fungi 32 weed hosts, rice blast disease 94 wheat abiotic stresses 363 Alternaria leaf blight 232–233, 241 Ascochyta leaf spot 236–237 black point/leaf blight (A. infectoria) 234–236 breeding for disease resistance 363, 369 MAS 86–87, 369 Cephalosporium gramineum 238–239 Cladosporium herbarum 239–241 common bunt (T. laevis) 143–145 endophytic fungi 155–159, 156–158 fungal diseases 69–70, 277, 363–369, 364 pathogen-speciﬁc thresholds 292 Fusarium head blight 363–364, 364 crop losses 79 resistance 83–87 global demand and production 69, 275, 291–292, 362–363 karnal bunt (T. indica) 366–368, 367 Phoma sorghina leaf spots 237–238 powdery mildew 368–369 Pyricularia grisea 241 root rot 133, 172, 177, 265 rust diseases 204, 207–208, 364–366, 364 fungicides 213, 214 leaf rust 364, 365–366 388 Index wheat continued rust diseases continued stem rust 364, 365 yellow (stripe) rust 366 seed disease, plant extract treatments 57–58 seed dressings 333–334 Septoria leaf blotch 70, 71–73 Septoria tritici blotch (STB) 293–305 ascendant movement 299–301 biocontrol 303–305 early detection 299 epidemiological studies 293–298 impact of climate 298–299 population genetic studies 301–303 Sumai 3 cultivar 85–86, 87 tan spot 231, 241, 276 crop losses 276–277 disease cycle 278–280 management strategies 159, 282–284 pathogen 277–278 physiological specialization 281–282 prevalence and range in South America 276 symptoms of infection 278 wilt, Fusarium and AM fungi 173 castor 272–273 endophyte-induced resistance 185 fenugreek 250, 252–253 safﬂower 268 sesame 270 wintergreen, oil of 46 wood decay fungi 348 wrappings, fruit 6 xanthan gum 114 Xanthomonas alfalfa 247, 248 Xylaria sp. 187 yams 30, 34, 40 yeasts (biocontrol agents) 111 constraints in commercial development 113–115 integration with other control measures 115–116 mechanisms of action 111–113 yellow rice disease 31 yellow rust, wheat 364, 366 Yield-PlusTM 110, 114 Zea, see maize zearalenone (ZEN/F2-toxin) 80 zero-tillage, see no-tillage systems zineb, rust diseases 213 zingiberene 20, 22 ziram 213

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