Plant Pathology 0070473994, 9780070473997

Table of contents :
Title
Contents
1 Introduction
2 History of Plant Pathology
3 Pathogenesis
4 Enzymes and Toxins in Plant Diseases
5 Alteration in Plant Physiological Function due to Plant-Pathogen Interaction
6 How Plants Defend Themselves Against Infection
7 Genetics of Plant Pathogen Interaction
8 Effect of Environmental Factors and Nutrition on Disease Development
9 Dispersal of Plant Pathogens
10 Plant Disease Epidemiology and Plant Disease Forecasting
11 Management of Plant Diseases
12 Plant Diseases Caused By Fungi—Characteristics of Plant Pathogenic Fungi, Classification of Plant Pathogenic Fungi
13 Rots, Damping Offs, Downy Mildews and White Rusts
14 Powdery Mildews
15 Smuts and Bunts
16 Rusts
17 Wilts and Root Rots
18 Leaf Spots, Leaf Blights and Anthracnoses
19 Galls and Abnormal Growths
20 Post-Harvest Diseases
21 Root Diseases
22 Seed-Borne Diseases
23 Mycoplasmas and Plant Diseases
24 Bacteria and Bacterial Diseases
25 Viruses, Viroids and their Diseases
26 Diseases Caused by Nematodes and Flagellates
27 Diseases Due to Angiospermic Parasites
28 Non-Parasitic Diseases
29 Application of Bio-Technology and Plant Pathology
30 Phytopathological Techniques in Plant Pathology
Index

Citation preview

PLANT PATHOLOGY Second Edition

ABOUT THE AUTHORS

Dr. R.S. Mehrotra A Ph.D. from the university of Saugor (now Dr. Hari Singh University). Dr. Mehrotra retired as Professor of Botany from Kurukshetra University in the year 1997. He has vast teaching experience of around ten years at the Univesity of Saugor and of more than twenty five years at Kuruksehtra University. He had been a Post Doctoral Fellow at the University of Western Ontario, London, Ontario, Canada and a Fulbright Fellow at the University of California, Riverside, California, U.S.A. He has published around 200 research papers in Indian and foreign journals of international repute. Dr. Mehrotra is a Fellow of the National Academy of Sciences, Fellow of the Indian Botanical Society, Fellow of Indian Phytopathological Society. He was the president of the Indian Phytopathological Society in the year 1990, President of the Indian Society of Plant Pathologists in the year 1992, President of the Indian Mycological Society in the year 1996, Sectional President of the Botany Section of the Indian Science Congress in the year 1997 and was the recipient of the Birbal Sahni Gold Medal of the Indian Botanical Society in the year 1998. Dr. Ashok Aggarwal Dr. Ashok Aggarwal is currently Reader in the Department of Botany, Kurukshetra University, Kuruksehtra. He joined the Department of Botany, Kurukshetra University in the year 1987. He is a member of several academic societies and has published more than 60 research papers in national and international journals. Recently he has written a monograph in collaboration with Dr. R.S. Mehrotra entitled “Phytophthora Diseases in India” in 2001. Dr. Aggarwal has considerable experience in the taxonomy, biology and control of Phytophthora species. He has also edited another book entitled “Glimpses of Plant Sciences” in 2002.

PLANT PATHOLOGY Second Edition

R.S. Mehrotra Ex Professor and Dean Faculty of Science Kurukshetra University Kurukshetra Ashok Aggarwal Reader Department of Botany Kurukshetra University Kurukshetra

Tata McGraw-Hill Publishing Company Limited NEW DELHI McGraw-Hill Offices New Delhi New York St Louis San Francisco Auckland Bogota Caracas Kaula Lumpur Lisbon London Madrid Mexico City Milan Montreal San Juan Santiago Singapore Sydney Tokyo Toronto

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© 2003, 1980, Tata McGraw-Hill Publishing Company Limited No part of this publication can be reproduced in any form or by any means without the prior written permission of the publishers This edition can be exported from India only by the publishers, Tata McGraw-Hill Publishing Company Limited ISBN 0-07-047399-4 Published by Tata McGraw-Hill Publishing Company Limited, 7 West Patel Nagar, New Delhi 110 008, Typeset at Script Makers, 19, A1-B, DDA Market, Paschim Vihar, New Delhi 110 063 and printed at Gopaljee Enterprises 190/5, Main Road, Maujpur, Delhi - 53 Cover: Pushp Print Services RZXCRDLDDDZDX

PREFACE TO THE SECOND EDITION

Ever since the publication of the first edition of this book in the year 1980, a lot of changes have taken place in terms of new information and advancements in the area of plant pathology. The changes are particularly in the field of plant molecular biology, molecular approaches to manipulate disease resistance, changes in the nomenclature of plant pathogens, our understanding of induced resistance, physiological aspects of plant pathology interaction, plant disease management like some newer fungicides for plant disease control, environmentally safe approaches to disease control and the concept of integrated disease management. During the last few decades, the application of biotechnology to plant pathology has revolutionized our understanding of genetics for building resistance in plants against diseases, by way of producing transgenic plants. And hence, a new chapter “Application of Biotechnology to Plant Pathology” has been added along with the chapter “Phytopathological Techniques in Plant Pathology”. This would familiarize the student of plant pathology to some of the basic techniques. Also, to know more about Phytopathological literature, web sites of several important journals have been provided in this chapter. The present edition gives the latest information available on plant diseases and their management. Several chapters have been thoroughly revised to bring in new information. Chapters like “Alteration in Plant Physiological Functions Due to Plant Pathogens”, “ Plant Disease Epidemiology and Plant Disease Forecasting”, “Post Harvest Diseases”, “Plant Disease Caused by Fungi—Characteristics of Plant Pathogenic Fungi, Classification of Plant Pathogenic Fungi” have been revised according to the latest classification of Hawksworth et al, (1995); so is the classification of Bacteria and Viruses. This edition also contains discussion on a number of diseases that were not included in the earlier edition. Diseases like Powdery mildew of mango, Rust of groundnut, Ascochyta blight of chickpea, Mango malformation, Stem rot of paddy, Common scab of potato, Ratoon stunting disease of sugarcane, Citrus greening (earlier thought to be mycoplasmal; but now, bacterial), Angular leaf spot and wild fire of tobacco, Coconut root wilt, Tristeza of citrus, Citrus exocortis, Cadang-cadang disease of coconut palms, Soybean cyst nematode, Phloem necrosis of coffee and Hartrot of coconut are discussed at length. Most of the chapters relating to disease control in plants are exhaustive enough and would permit interested students and researchers to delve more deeply into some of the diseases of plants. The intention behind including so many references is to help the student have access to wide-ranging literature on plant pathology. The various diagrams, tables would be useful to students at the postgraduate levels. A number of figures in this text have been retained from the previous edition, while new ones have been included and their sources gratefully acknowledged at appropriate places. It is hoped that the second edition of the book will provide the latest information on plant pathology and give a better insight into the subject to undergraduate students and postgraduate students of agriculture and plant sciences. This book would be equally handy for teachers and researchers. R.S. MEHROTRA ASHOK AGGARWAL

PREFACE TO THE FIRST EDITION

Plant pathology is intimately related to the health of plant and the production of crop yields. It has special relevance in agricultural countries like India which are fast developing and where much still needs to be done in the modernization of control measures against plant diseases. Rough estimates have suggested that in India, on an average, there is a loss of about 10 percent in crop yield due to plant diseases. This loss would certainly amount to hundreds of crores of rupees annually, taking into account the output of all the cultivated crops. Hence, a book on plant pathology is always welcome. During the last quarter of the century there have been enormous developments in the science of plant pathology. Many new techniques and chemicals have been evolved to fight plant diseases. Efforts have been made to understand the nature of diseases at molecular levels and the relationship between the host and parasite has been put, in many cases, on a sound biochemical footing. It is a stupendous task to present this knowledge in a volume of suitable size to a young post graduate or research student. The book has several important features, one of which is the “all-in-one” approach which will be useful to postgraduate students. To the best of my knowledge there is no book with both generalized and specialized topics discussed in parallel which caters to the needs of undergraduate, postgraduate and research students at the same time. Most of the important aspects of plant pathology, such as physiology of diseased plants, root diseases, seed-borne diseases, post harvest diseases of fruits and vegetables, diseases caused by fungi, bacteria, viruses, mycoplasmas, nematodes and non-parasitic diseases have all been dealt with suitable detail for the benefit of students. Besides, the book should also serve as a good reference for research workers. It is hoped that this work will enrich the background of those who wish to study plant pathology intensively. Suggestions for improving the book will be gratefully received and acknowledged. R.S. MEHROTRA

ACKNOWLEDGEMENTS

The authors thank Dr. B.B. Nagaich, former Director, Central Potato Research Institute, Shimla and Dr. S.M. Paul Khurana, present Director C.P.R.I., Shimla, for providing photographs of diseases of potato; Dr. J.C. Majumdar of BASF India Limited, for permission to reproduce some photographs; Dr. M.M. Payak, for providing photographs of diseases of maize; Dr. R.K. Grover, former Professor and Head, Department of Plant Pathology, CCS Haryana Agriculture University, Hisar, for providing photographs of downy mildew of bajra and angular leaf spot of cotton; Dr. R.N. Gupta, for providing photographs of the germinating chlamydospore of Protomyces macrosporous, and Dr. D.K. Chakravarti, Department of Horticulture, N.D. University of Agriculture and Technology, Faizabad, U.P. for providing the photographs of mango malformation and providing relevant information on this disease. Acknowledgements are also due to the following: · Agrios, G.N. (1969) Plant Pathology, Academic Press, New York, for reproducing Figures 6.7, 22, 26, 45, 47, 49, 52, 118 and 121. · Bateman, D.F. and H.G. Basham (1976), Physiological Plant Pathology, edited by R. Heitefuss and P.H. Williams, Springer Verlag, Berlin, Heidelberg, New York, for giving permission to reproduce Figure 1 from their article. · Dickinson, C.H. and J.A. Lucas (1977), Plant Pathology and Plant Pathogens, Blackwell Scientific Publications, Oxford, London, Edinburg, Melbourne, for giving permission to reproduce seven figures. · Raychaudhuri, S.P. and T.K. Nariani (1977), Virus and Mycoplasmal Diseases of Plants in India, Oxford and IBH Publication Co., New Delhi, Bombay, Calcutta, for granting permission to reproduce the photographs of papaya leaf curl. It must be emphasized that it would not have been possible to complete the second edition of this book without the willing help rendered to the authors by their colleagues and research associates. Finally, the authors express their appreciation and thanks to their family members for their unwavering support and encouragement. R.S. MEHROTRA ASHOK AGGARWAL

CONTENTS

Preface to the Second Edition Preface to the First Edition Acknowledgements

vii ix xi

1. INTRODUCTION

Nature and concept of plant diseases 1 Cause of disease 3 Various levels of parasitism 6 Importance of plant diseases 10 Effect of plant diseases on human affairs 11 Classification of plant diseases 16 Trends in teaching and research in plant pathology worldwide References 18 Questions 20

1

18

2. HISTORY OF PLANT PATHOLOGY

The herbalists 22 The systematists 22 The beginning of modern period 23 Confirmation of Prevost's work 24 Doctrine of spontaneous generation 24 Forest pathology 26 Discovery of Bordeaux mixture 26 Plant pathology in the twentieth century 27 Physiological plant pathology 27 Genetics of the host and the pathogen 28 Fungicidal research 28 Environment in relation to plant disease 29 Nature of disease resistance 29 Biochemistry and physiology of the diseased host plant 29 Molecular biology of pathogenesis and induced systemic resistance Tissue culture in plant pathology 30 Biotechnological approach to disease control 30 Ecological studies of soil-borne fungal plant pathogens 31 Bacteriology 31 Virology 31 Mycoplasma 32

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Contents

History of the development of plant pathology in India Plant disease clinics 37 References 39 Questions 43

34

3. PATHOGENESIS

Penetration and entry by plant pathogens Prepenetration 47 Entry through natural openings 50 Direct penetration 52 Entry through wounds 57 Wounds caused by other fungi 58 Wounds caused by nematodes 58 Entry through root hairs 59 Entry through buds 59 Development inside host tissue 59 References 62 Questions 63

45

45

4. ENZYMES AND TOXINS IN PLANT DISEASES

64

Enzymes in plant diseases 64 Composition of cell wall materials and middle lamella 65 Enzymes for waxes and cutins 70 Pectic enzymes 72 Macerating enzymes 73 Cellulolytic enzymes 75 Hemicellulases 76 Lignolytic enzymes 77 Proteolytic enzymes 77 Lipolytic enzymes 78 Inactivation of enzymes 78 Toxins and plant diseases 80 Classification of toxins 81 References 94 Questions 100 5. ALTERATION IN PLANT PHYSIOLOGICAL FUNCTION DUE TO PLANT-PATHOGEN INTERACTION

Permeability changes in diseased plants 101 Effect of plant pathogens on translocation of water and nutrients in the host plant 103 Photosynthesis 104 Photosynthesis as influenced by viral infections 107 Respiration 112

101

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xv

Changes in nitrogen metabolism of infected plants 118 Changes in protein metabolism 120 Phenols 121 Growth regulators in plant diseases 127 Transcription and translation in diseased plants 133 References 134 Questions 141 6. HOW PLANTS DEFEND THEMSELVES AGAINST INFECTION

142

Morphological or structural defence mechanisms 142 Biochemical defence 148 Defence through induced synthesis of proteins and enzymes 159 Defence through formation of substrates resisting the enzymes of the pathogen 162 Defence through detoxification of pathogen toxin 163 Defence through altered respiration 163 Defence through hypersensitive reaction 164 The concept of phytoncides 164 References 165 Questions 169 7. GENETICS OF PLANT PATHOGEN INTERACTION

170

Genetics of host-parasite interaction 170 Resistance and susceptibility 170 The common antigen hypothesis 173 The phytoalexin-induced hypothesis 173 Vertical and horizontal resistance 174 Mutation 174 Heterokaryosis 175 Parasexual recombination 177 Adaptation 177 Saltation 178 Cytoplasmic variation 178 Sexual recombination 178 Transformation 179 Transduction 179 Physiological specialization 180 Adaptation of fungi to different hosts 183 References 185 Questions 188 8. EFFECT OF ENVIRONMENTAL FACTORS AND NUTRITION ON DISEASE DEVELOPMENT

Effect of temperature on the development of a disease

189

189

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Contents

Humidity and moisture 191 Soil pH 193 Soil texture 193 Effect of light 194 Effect of oxygen and carbon dioxide concentration 195 Effect of nutrients 195 Role of biotic environment 196 Role of environmental factors in epiphytotics 197 References 197 Questions 198 9. DISPERSAL OF PLANT PATHOGENS

199

Direct transmission 199 Indirect transmission 200 References 209 Questions 211 10. PLANT DISEASE EPIDEMIOLOGY AND PLANT DISEASE FORECASTING

212

Introduction 212 Some important epiphytotics of the past and present 214 Epiphytotic growth and analysis 215 Computer simulation of epidemics 215 Systems approach in epidemiology 216 Methods used in plant-disease forecasting 219 Examples of plant disease forecasting systems 222 References 224 Questions 227 11. MANAGEMENT OF PLANT DISEASES

Cultural methods 228 Chemical methods 242 Breeding for disease resistance 266 Innovative methods of plant disease control References 284 Questions 290

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12. PLANT DISEASES CAUSED BY FUNGI—CHARACTERISTICS OF PLANT PATHOGENIC FUNGI, CLASSIFICATION OF PLANT PATHOGENIC FUNGI

General features of fungi 291 Classification of fungi 293 References 311 Questions 311

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Contents

13. ROTS, DAMPING OFFS, DOWNY MILDEWS AND WHITE RUSTS

Damping off fungal pathogens, downy mildews and white rusts 312 Key to the families of the order peronosporales 312 Damping off disease of seedlings 314 Fruit rot of cucurbits 317 Stem or foot rot of papaya (Carica papaya ) 318 Rhizome rot of ginger (Zingiber officinale) 319 Late blight of potato (Solanum tuberosum) 320 Blight of colocasia (Colocasia esculenta) 327 ‘Koleroga’ or ‘mahali’ of areca palms (Areca catechu) 330 Bud rot of palms 332 Gummosis or gum disease of citrus 333 Leaf rot and foot rot of ‘pan’ (Piper betle) 335 Downy mildew of peas (Pisum sativum) 339 Downy mildew of crucifers 341 Downy mildew of cucurbits 342 Downy mildew of grapes (Vitis vinifera) 344 Downy mildew of bajra (Pennisetum typhoides) 345 Downy mildew of maize 349 Brown stripe downy mildew of maize 351 Sugarcane downy mildew of maize (corn) caused by S. sacchari 352 Philippine downy mildew of maize caused by sclerospora philippinensis Downy mildew or leaf-shredding disease of sorghum 355 White rust of crucifers 356 References 360 Questions 368 14. POWDERY MILDEWS

Powdery mildews 369 Powdery mildew of wheat (Triticum spp.) and barley Powdery mildew of peas (Pisum sativum) 375 Powdery mildew of grapevine 378 Powdery mildew of cucurbits 379 Powdery mildew of apple 383 Powdery mildew of mango 385 References 386 Questions 389

xvii 312

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372

15. SMUTS AND BUNTS

Modes of infection 392 Loose smut of wheat 394 Flag smut of wheat 397 Hill bunt (or European bunts or stinking smuts) of wheat

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Karnal bunt of wheat 403 False smut of paddy 406 Bunt of rice (syn. Black smut, kernel smut, rice smut) 408 Leaf smut of rice 410 Covered smut, kernel smut, short smut or grain smut of jowar (Sorghum vulgare) 411 Loose smut of sorghum 414 Head smut of sorghum and maize 416 Long smut of sorghum 417 Smut of bajra (Pennisetum typhoides) or pearl millet 420 Common smut of maize 422 Whip smut of sugarcane 423 Covered smut of barley (Hordeum vulgare ) 427 Loose smut of barley 429 References 431 Questions 434 16. RUSTS

Plant rusts 436 Life cycles 437 Heteroecism and autoecism 438 Rusts of wheat 443 Black stem rust of wheat 444 Brown rust of wheat 449 Yellow rust of wheat 452 rust of linseed (Linum usitatissimum) 456 Rust of beans (Phaseolus vulgaris ) 458 Rust of pea 459 Rust of gram (Cicer arietinum) 462 Rust of coffee 464 Rust of pearl millet or bajra (Pennisetum typhoides) Rust of jowar (Sorghum vulgare) 467 Rust of groundnut 470 References 471 Questions 474

436

466

17. WILTS AND ROOT ROTS

Vascular wilts 475 Wilt of pigeon pea (Cajanus cajan) 481 Wilt of cotton (Gossypium arboreum and G. herbaceum) Wilt of linseed (Linum usitatissimum) 487 Panama disease of banana 488 Dutch elm disease 490 Root rots 493

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xix

Rhizoctonia root rot of cotton 493 Root and stem rot of jute 495 Black scurf of potato 496 Stem rot of rice 498 References 500 Questions 504 18. LEAF SPOTS, LEAF BLIGHTS AND ANTHRACNOSES

505

Leaf spots and leaf blights 505 Leaf spot of turmeric (Curcuma longa) 506 Brown spot of maize 507 Early blight of potato 509 Leaf blight of wheat 512 Stripe disease of barley 514 Brown spot (helminthosporiosis) disease of rice 516 Blast disease of rice 520 Symptoms 520 Tikka disease of groundnuts 526 Anthracnoses 529 Red-rot of sugarcane 530 Ripe fruit-rot and die-back of chillies 533 Mango anthracnose 536 Anthracnose of grapes 537 Ascochyta blight of chickpea (Cicer arietinum) 539 References 542 Questions 548 19. GALLS AND ABNORMAL GROWTHS

Club root disease of cabbage (Brassica oleracea var. capitata) Wart disease of potato (Solanum tuberosum) 552 Leaf curl of peach (Prunus persica) 556 Ergot of bajra (Pearl millet) 558 Malformation of mango 561 References 565 Questions 568

549

549

20. POST-HARVEST DISEASES

Bacterial rots 570 Fungi 571 Non-pathogenic diseases 573 Mode of infection and factors influencing post-harvest diseases Control of post harvest diseases 575 References 587 Questions 592

569

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Contents

21. ROOT DISEASES

Root exudates and soil-borne pathogens 594 Associative and antagonistic effects 595 Pre-emergence killing, damping off and seedling blight Root rots 596 Vascular wilt diseases 597 Diseases involving hypertrophy 597 Non-parasitic root pathogens 597 Predisposing factors 597 Survival 598 Dispersal of soil-borne pathogens 599 Control of root diseases 599 References 602 Questions 605

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596

22. SEED-BORNE DISEASES

606

Seed-borne bacterial diseases 606 Seed-borne viral diseases 607 Seed-borne fungal diseases 608 Seed-borne nematode diseases 613 Pathological effects of seed-borne diseases 613 Quarantine programmes for seed borne diseases and seed stocks Seed treatment 616 References 617 Quesitons 619 23. MYCOPLASMAS AND PLANT DISEASES

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620

Sandal spike 624 Disease cycle 625 Sesamum phyllody 626 Grassy shoot disease (gsd) of sugarcane 627 Little leaf of brinjal (Solanum melongena) 629 Coconut (root) wilt 630 References 633 Quesitons 639 24. BACTERIA AND BACTERIAL DISEASES

Structure, reproduction and classification of bacteria Growth and reproduction in bacteria 645 Classification of bacteria (prokaryotes) 651 Plant pathogenic bacteria 658 Symptoms of bacterial plant diseases 659 Bacterial plant diseases 661 References 693 Questions 702

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Contents

25. VIRUSES, VIROIDS AND THEIR DISEASES

xxi 703

Plant viruses and plant viral diseases 703 Symptoms due to viral infections in plants 706 Transmission of plant viruses 709 Control of viral diseases 713 Stuructre of plant viruses 716 Physical and chemical properties of plant viruses 722 Infection, multiplication and movement of viruses within plants 723 Tobacco and tomato mosaic 724 Viral diseases of potato 727 Leaf curl of tomato 732 Mosaic disease of tomato 733 Viral diseases of papaya 734 Yellow vein mosaic of bhindi (Abelmoschus esculentus) 735 Viral diseases of sugarcane 736 Bunchy disease of banana 737 Bean mosaic 738 Tristeza of citrus 739 Citrus exocortis 741 Cadang-cadang disease of coconut 742 Potato spindle tuber viroid 743 References 744 Questions 748 26. DISEASES CAUSED BY NEMATODES AND FLAGELLATES

Diseases caused by plant parasitic nematodes 750 Isolation of nematodes 753 Symptoms caused by nematodes 754 Mechanism of nematode injury to plants 755 Interrelationship between nematodes and other plant pathogens 758 Ecology and spread 759 Characteristics of plant pathogenic nematodes 759 General methods of control of nematode diseases 762 Root knot of vegetables 768 Molya disease of barley and wheat 770 Citrus nematode 771 Symptoms 771 Ear-cockle of wheat 772 Soybean cyst nematode 774 Plant diseases caused by flagellate protozoa 775 Phloem necrosis of coffee 776 Hartrot of coconut palms 777 References 778 Questions 780

750

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Contents

27. DISEASES DUE TO ANGIOSPERMIC PARASITES

781

Parasitic flowering plants 781 Dissemination of angiosperm parasites 783 Control of angiosperm parasites 784 Loranthus or ‘banda’ or giant mistletoe 784 Striga 786 Cuscuta 787 References 789 Questions 790 28. NON-PARASITIC DISEASES

Non-infectious diseases of plants 791 Nutritional deficiencies 792 Tip burn of paddy or pansukh of rice 795 Blossom end rot of tomato 796 Heat canker of linseed 796 Black heart of potato 797 Mango necrosis or black tip 798 Khaira disease of rice (zinc deficiency disease of rice) Zinc deficiency disease of citrus 800 References 800 Questions 801

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29. APPLICATION OF BIO-TECHNOLOGY AND PLANT PATHOLOGY

The use of tissue culture techniques of importance to plant pathology Selection for disease resistance 808 Recombinant-DNA technology 808 The use of monoclonal antibodies in plant pathology 809 Somaclonal variants as a source of disease resistance 811 Conclusion 812 References 812 Questions 814

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30. PHYTOPATHOLOGICAL TECHNIQUES IN PLANT PATHOLOGY

815

Isolation of fungi 815 Requirement for isolation of fungi 815 Plant disease assessment methods 822 References 826 Questions 828 INDEX

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Contents

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Introduction 1 History of Plant Pathology 21 Pathogenesis 45 Enzymes and Toxins in Plant Diseases 64 Alteration in Plant Physiological Function due to Plant-Pathogen Interaction 101 How Plants Defend Themselves Against Infection 142 Genetics of Plant Pathogen Interaction 170 Effect of Environmental Factors and Nutrition on Disease Development 189 Dispersal of Plant Pathogens 199 Plant Disease Epidemiology and Plant Disease Forecasting 212 Management of Plant Diseases 228 Plant Diseases Caused By Fungi-Characteristics of Plant Pathogenic Fungi, Classification of Plant Pathogenic Fungi 291 Rots, Damping Offs, Downy Mildews and White Rusts 312 Powdery Mildews 369 Smuts and Bunts 390 Rusts 435 Wilts and Root Rots 474 Leaf Spots, Leaf Blights and Anthracnoses 504 Galls and Abnormal Growths 548 Post-Harvest Diseases 568 Root Diseases 592 Seed-Borne Diseases 605 Mycoplasmas and Plant Diseases 619 Bacteria and Bacterial Diseases 639 Viruses and Viral Diseases 702 Diseases Caused by Nematodes and Flagellates 750 Diseases Due to Angiospermic Parasites 781 Non-Parasitic Diseases 791 Application of Bio-Technology and Plant Pathology 803 Phytopathological Techniques in Plant Pathology 816

1 Introduction

NATURE AND CONCEPT OF PLANT DISEASES

It is imperative to define and discuss the meanings of certain terms commonly used in phytopathology before we discuss the basic principles and concepts of plant pathology. It is essential to include these definitions and terms in an introductory chapter, since an understanding of these terms is required before the reader can be made aware of the details of the processes involved in plant diseases. Phytopathology (phyton—plant; pathos—suffering; logos—knowledge) is the study of the diseases of plants and covers the entire field of biological and scientific activity concerned with the understanding of this complex phenomenon. Phytopathology is thus the study of the nature, development, and control of plant diseases. Disease, being a complex phenomenon, is difficult to define in a few words. We have to keep in mind what Locke said in Human Understanding: “Though definitions will serve to explain the names of substances as they stand for our ideas, yet they leave them not without great imperfection as they stand for things.” According to modern conception, disease is an interaction among the host, parasite, and environment. A simple dictionary meaning of disease is: any departure from health, presenting marked symptoms, malady, illness, disorder. Diseased plants are distinguished by changes in their morphological structures or physiological processes, which are brought about by unfavourable environment or by parasitic agencies. Several definitions of diseases in plants have been proposed. Some examples are: “a series of harmful physiological processes caused by continuous irritation of the plant by a primary agent”; “a harmful deviation from the normal functioning of physiological processes”; “a continuous impairment of metabolism”. Stakman and Harrar (1957) defined plant disease as “a physiological disorder or structural abnormality that is harmful to the plant or to any of its part or products that reduces the economic value”. Agrios (1997) has defined disease in plants as a series of invisible and visible responses of plant cells and tissues to a pathogenic micro-organism or environmental factors that result in adverse changes in the form, function, or integrity of the plant and may lead to partial impairment or death of the plant or its parts. However, A Guide to the use of the terms in Plant Pathology prepared by the Federation of British Plant Pathologists recommends that the use of the terms should be restricted to malfunctions

2

Plant Pathology

caused by plant pathogenic organisms or viruses and those caused by other factors (the so called noninfectious diseases) should be termed as ‘disorders’. According to Horsfall and Cowling (1977a), disease is a malfunctioning process that is caused by continuous irritation. Of course this process may result in some suffering, and this produces symptoms. This conception of the disease is accepted by the Committee of Terminology of the American Phytopathological Society and by the counterpart Committee of the British Mycological Society. Simple Interest Disease

This denotes a disease, the increase of which is mathematically analogous to simple interest in money. There is only one generation of disease in the course of one epidemic. A simple interest disease develops from a common source of inoculum, that is, the capital is constant, and often there is one generation of infection in a season. In the increase of simple interest diseases, the loge [1/(l – x)] is plotted against time (where x = the proportion of diseased tissue), thus allowing for the fact that with increasing infection the number of plants susceptible to infection decreases (Fig. 1.1). Soil-borne diseases such as vascular wilts and soil-borne smuts, which infect seedlings and subsequently sporulate in the inflorescence of the mature plants, provided that the pathogen does not spread from the smutted heads, are examples of simple interest diseases.

x

0.50

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0.0

Loge

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Fig. 1.1

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14 Aug.

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3 Sept.

The progress of an epidemic of wilt cotton caused by Fusarium oxysporum f. vasinfectum. In the upper graph, x is plotted against time; the lower part plots Loge [1/(1 – x)] against time. (Redrawn from JE Van der Plank. Plant Diseases— Epidemics and Control, Academic Press, New York and London, 1963, p. 34.)

Compound Interest Disease

This denotes a disease, the increase of which is mathematically analogous to compound interest money. There are several or many generations of the pathogen in the life of the crop, that is, the capital is increased by the amount of interest. In plotting the amount of disease against time, the transformation loge(x/l – x) is used, where x represents the proportion of infected susceptible tissue or of infected plants

Introduction

3

if the pathogen develops in a systemic manner (Fig.1.2). Late blight of potato, stem rust of wheat or powdery mildew of wheat are examples of compound interest diseases. If we know that a particular disease is of the simple interest type, suitable control measures can be applied. For example, sanitation affects the simple interest disease more than the compound interest type. If the disease is of the former type, sanitation, including the eradication of the inoculum of the pathogen from planting material and sites, should be insisted upon. If it is of the compound interest type, and the epidemic spreads rapidly and lasts long, the infection rate should be reduced by a timely application of fungicides. The stress should shift from sanitation to reducing the infection rate in proportion to the increase in the rate of spread and duration of the epidemic.

Fig. 1.2

Progress of stem rust of wheat caused by Puccinia graminis tritici. (Redrawn from JE Van der Plank, Plant Diseases—Epidemics and Control, Academic Press, New York and London, 1963, p. 32.)

CAUSE OF DISEASE

When a parasitic micro-organism enters into the causal complex of a disease, it is commonly considered to be the cause of the disease. However, we know that sometimes a variation in the environment also makes a plant liable to infection. The micro-organism is thus not the sole causal factor. After a microorganism has successfully infected a susceptible plant, the subsequent interaction between the plant and the micro-organism (which results in a disease) is also subject to environmental influences. Thus, strictly speaking, it is not correct to refer to a micro-organism as the cause of a given disease since this implies that it is the sole cause. The term ‘causal organism’ is proper since it implies that the organism is a part of the causal complex. Most causal organisms are parasites. A parasite is not synonymous with the pathogen. In some cases, organisms are parasitic without becoming causal factors in the disease. Many parasitic establishments do not become pathogenic if the parasite compensates for the damage done by its presence. The mycorrhizal fungus is certainly parasitic on the roots of trees but it is not pathogenic. The root nodule bacterium (Rhizobium leguminosarum) is another example. In other cases, the byproducts of a strict saprophyte are an important part of the causal complex. Leaf tip injury to oats may be due to the absorption of Griseofulvin from soil. Milo root rot is caused by a powerful toxin produced by the fungus (Periconia circinata) growing in the root zone (Oswald, 1951; Leukel, 1948).

4

Plant Pathology

Thrower (1966) modified a definition offered by the British Mycological Society in 1950 and defined a parasite as “an organism or virus existing in an intimate association with another living organism from which it derives an essential part of the materials for its existence”. In a literal sense, a pathogen is any agent that causes damage. According to A Guide to the use of terms in Plant Pathology prepared by the Federation of British Plant Pathologists (1973), a pathogen is an organism or virus capable of causing disease in a particular host or range of hosts. Most, but not all pathogens, are also parasites in that they derive the materials they need for existence from a living plant (the host or the suscept) as distinct from saprophytes, which derive these materials from dead organic matter. Pathotype

This is a subdivision of a species distinguished by the common characters of pathogenicity, particularly in relation to the range of hosts. Serotype

A serotype is a population of a pathogen (usually a bacterium or virus) in which all individuals possess a given character of serology in common (Robinson, 1969). On the basis of serological tests, differences between apparently similar organisms or viruses may be found and the subdivisions thus formed are called serotypes or strains. For example, only one serotype was designated amongst 45 isolates of Xanthomonas oryzae (X. campestris pv. oryzae) on the basis of agglutination reactions and gel diffusion tests (Addy and Dhal, 1977). Physiotype

It is a population of pathogens in which all individuals have a particular character of physiology (but not pathogenicity) in common (Robinson, 1969). Pathogenicity

It is the quality or characteristic of a pathogen capable of causing disease. Pathogenesis

It is the sequence of progress in disease development from the initial contact between a pathogen and its host to the completion of the syndrome. Horsfall and Dimond (1960) liken the relationship between the micro-organisms and higher plants to a situation in which your mother-in-law lives with you. If she contributes to the harmonious running of the household, your relation is symbiotic; if you support her and she does not do her share of work she is a parasite and you are the host; if she stirs up trouble between you and your wife she is a pathogen. Her actions, your reaction, and her reactions constitute pathogenesis.

Introduction

5

Host

An organism that harbours or supports the activities of a parasite is known as the host. Whetzel (1929) termed the diseased plant suscept, but suscept may actually mean the plant that is susceptible or prone to disease. Inoculum

Inoculum is the infectious material that can cause disease, and it is that portion of individual pathogens that is brought into contact with the host. Inoculum Potential The term inoculum potential describes the degree of infectivity. Garrett (1956) defined inoculum potential as “the energy of a fungal parasite available for infection of a host at the surface of the host organ to be affected”. Primary Inoculum The overwintering or oversummering stage may be in or on the seed of the host, in the perennial wild host, in debris of infected plant of the previous season, in dormant spores, sclerotia or other organs of the soil, or in the form of an inhabitant of the soil flora. Whatever the source, it causes primary infection. It may serve as an infective agent and thus becomes the primary inoculum, or it may multiply as a saprophyte (or as a parasite on the perennial host) to produce spores which become the primary inoculum. The rust fungus oversummers in India in the uredial stage on summer wheat in the hills, from where the primary inoculum is carried by air currents to the next crop grown at the foot of the hills and then to the plains. When the host becomes diseased, the organism commonly produces another crop of spores or infective bodies which are known as the secondary inoculum and cause secondary infection. This last cycle may be repeated several times. The uredial pustules formed in the wheat crop as a result of primary infection become a source of secondary infection to other plants as the urediospores are blown down to these leaves and plants. Penetration This is the first step in the contact of the inoculum with the host. Penetration may take place in one of a number of ways characteristic of the organism and are sometimes influenced by the environment. The methods of penetration have been discussed in Chapter 3. There is a clear distinction between penetration and infection. Penetration refers to the initial invasion of the host by an organism. We may have many cases of penetration without infection, for example, in the case of apple scab fungus (Venturia inaequalis) conidia, the infection hyphae penetrate readily whether or not the host variety and the fungal strain are compatible (Nusbaum and Keitt, 1938), but in the resistant host variety, the fungus is not able to develop further and cause infection due to the influence of the vital activities of the cell. Infection It implies the establishment of the pathogen inside the host following penetration in which a parasitic relationship between the two organisms is established. Incubation Period There may be a long period between penetration and infection and the appearance of the disease, but ordinarily one follows the other. This interval of time between the penetration of the host by a pathogen and the first appearance of symptoms on the host is known as the incubation period. In the case of black stem rust of wheat, the pathogen Puccinia graminis tritici enters the host (wheat) a day or so after the urediospores have germinated, but the actual symptoms of the disease in the form of rust pustules develop only after a week or so at 20°C. In the case of Sphacelotheca sorghi

6

Plant Pathology

(covered smut of Sorghum), the very young seedling is infected from soil-borne or seed-borne spores, but no visible symptoms appear until the infected plant produces smutted flowers. In such cases, the incubation period is technically several months, although increased respiration of the diseased plant may occur much sooner. In case of the pathogen Mycosphaerella musicola, the incubation period is about three or four weeks. Disease Development It refers to a series of events which occur between the time of infection and complete development of the disease. Epidemiology Epidemiology of disease is a study of the factors affecting the outbreak of an infectious disease. Van der Plank (1963) defined epidemiology as the science of disease in a population. The founders of epidemiology have kept the identity that epidemiology deals with population of the host and pathogens under varying environment. Symptoms Disease development is a long drawn process, which involves the production of secondary inoculum and progression of external or internal evidences of the disturbance of morphological and physiological processes of the host plant. These external or internal reaction or alterations as a result of a disease are referred to as symptoms. Syndrome Diseased plants show several valuable symptoms by which a disease is recognized. These symptoms are collectively known as a syndrome. Disease Cycle A series of events involved in disease development, including the stages of development of the pathogen and the effect of the disease on the host, is called the disease cycle. When a pathogen is involved, the disease cycle is intimately associated with the organism. It is distinct from the life cycle of the organism. Infectious Organism An infectious organism is one that may be transmitted from a diseased host plant to a healthy plant and which is capable of inciting the disease in a favourable environment. Infectious Disease An infectious disease is one in which a transmissible causal organism is part of the causal complex. In other words, on infection the disease can spread from a diseased to a healthy plant. VARIOUS LEVELS OF PARASITISM Symbiosis

Two living organisms may live in association with one another and in a sense, each may be parasitic upon the other. However, they are not only non-pathogenic to one another but are also essential to each other’s development. This phenomenon is known as symbiosis. Lichens are classical examples of symbiosis in which a fungus and an alga live in close association. The alga through its photosynthetic activity, converts the kinetic energy of sun’s rays into the potential energy of carbohydrates which is useful for its own development and essential to the life of the fungus. The latter through its property of producing hydrolytic enzymes breaks down material in the substrate, and thus makes energy available

Introduction

7

and provides minerals and organic nutrients essential to itself and the alga. Another case of symbiosis is that of the Rhizobium species forming root nodules in leguminous plants. Obligate Parasites

There are various categories of parasitism. Parasites which are restricted to living tissues (also known as biotrophs) are called obligate parasites. Obligate is also used to denote parasites which have not been grown in axenic cultures, that is, free from any other organism. Facultative Saprophytes

The term facultative is both cumbersome and confusing. Comprehension of the phrase facultatively saprophytic requires a mental pause for translation to strongly parasitic. Facultative saprophytes ordinarily have a life cycle similar to that of obligate parasites in that they pass most of the cycle in association with the host and do not subsist as saprophytes. They may, however, subsist as saprophytes for part of their life cycle but do not complete their life cycle away from the host. Many smut fungi are good examples of this. Facultative Parasites

This term implies that they are ordinarily saprophytic but under certain conditions they may become parasitic. Many damping off fungi, such as Pythium and Rhizoctonia are good examples of facultative parasites. Saprophytes

Saprophytes are those organisms which have no relation with the living cell and secure their nutrients from dead organic tissues or from available inorganic materials. In fact, a number of fungi, once considered to be obligate parasites, have been cultured away from their hosts on artificial media. This makes the term obligate inappropriate. Luttrell (1974) recognizes three broad categories of parasitism which are as follows: 1. Biotrophs

These are organisms which, regardless of the ease with which they can be cultured in the laboratory or not, in nature (in vivo) obtain their food from the living tissues on which they complete their life cycles. Some typical examples are rusts, smuts and mildews. 2. Hemibiotrophs

These are organisms which attack living tissues in the same way as biotrophs but continue to develop and sporulate after the tissue is dead. Typical examples of these are leaf-spotting fungi.

8

Plant Pathology

3. Perthotrophs

These are organisms which kill host tissues in advance of penetration and then live saprophytically. Sclerotium rolfsii is a typical fungus included in this category. Virulence

Aggressiveness and virulence are ambiguous terms. Many pathologists equate virulence with pathogenicity; nevertheless, some shade of difference in usage is usually apparent. Thus, the statement that an organism is pathogenic merely implies that it can cause a disease. But a virulent organism is considered to have a high capacity to do so. Aggressiveness is equally ambiguous and has been used by Gaumann (1950) to describe the capacity of a parasite to invade and grow in its host plant and to reproduce on or in it. High aggressiveness can be combined with low pathogenicity as in the case of some obligate parasites which invade plants efficiently but cause minimal damage to it, at least in the early stages of attack. Aggressiveness is also used as a measure of pathogenicity. Predisposition

It may be defined as the effect of one or more environmental factors which makes a plant vulnerable to attack by pathogen. It is a process which antedates penetration and infection. Susceptibility

It is the inability of a plant to resist the effect of a pathogen or any other damaging factor. Susceptibility of a given individual may be increased or decreased by environmental factors. It may also vary between individuals, varieties or species because of differences in the inherited characteristics which affect susceptibility. Resistance

Resistance can be described as the inherent ability of a plant to prevent or restrict the establishment and subsequent activities of a potential pathogen. It may be regarded as the opposite of susceptibility and may also be influenced by environmental factors. It is the ability of the plant to withstand, oppose or overcome the attack of a pathogen completely or to some degree. The new concepts of vertical resistance (VR) and horizontal resistance (HR) were put forward by JE Van der Plank (1968). Resistance may be defined as horizontal (uniform) when it is evenly spread against all races of a pathogen, or as vertical (differential) when it is completely effective against some races of a pathogen but not against others. It may be polygenic (determined by many genes), oligogenic (several genes) or monogenic (one gene). The genes involved may be major or minor depending on their effectiveness in conferring resistance. Figure 1.3 shows the foliage resistance of two potato varieties to 16 races of Phytophthora infestans. Both varieties show vertical (complete) resistance to races (0), (2), (3), (4), (2,3), (2,4), (3,4), and (2,3,4), while resistance to other eight races is horizontal and greater in Maritta than in Kennebec.

Introduction

9

Vertical resistance is complete but impermanent, while horizontal resistance, though incomplete, is of a permanent nature.

Fig. 1.3

Vertical and horizontal resistance in two varieties of potato (Redrawn from JE Van der Plank. Plant Diseases—Epidemics and Control, Academic Press, New York and London 163, pp. 175.)

Hypersensitivity

This results in a violent local reaction to attack by a pathogen and the prompt death of tissue around the points of entry prevents further spread of infection. Hypersensitivity thus confers high resistance and is sometimes used in the sense of immunity. Symptomless Carriers

Symptomless carriers are those plants in which the agent inciting disease is present but shows no symptoms. This is an extreme case of tolerance in some plants. Active Resistance

Active (or dynamic) resistance is “due to reactions incited by the attack”, while passive (or static) resistance is “due to qualities innate in the host prior to the attack”. These terms describe resistance mechanisms and do not necessarily connote any epidemiological significance.

10

Plant Pathology

Pathodeme

Pathodeme is that population of a host in which all individuals have a particular character of resistance in common (Robinson, 1969). Biotype

A biotype is a population of individuals which are genetically identical (literally any species subdivision which is based on criteria other than morphological). Immunity

This implies exemption from infection by a pathogen and in this sense is an absolute quality. Aetiology

Aetiology is the science of the cause of disease. When used in a broader sense it includes the study of causal factors, their nature, and their relation with the host. Fungitoxic, Fungicidal, Fungistatic

Fungitoxic substances are harmful to fungi and include fungicides. The word fungicide has originated from two Latin words, fungus and caedo. The word caedo means ‘to kill’. Thus, literally speaking, a fungicide is any agency which has the ability to kill a fungus. According to this meaning, physical agencies like heat and ultraviolet light should also be called fungicides. However, this is not so. In common usage the meaning of the word fungicide is restricted to chemicals. Hence, fungicide means a chemical capable of killing fungi. Some chemicals do not kill fungi but inhibit fungus growth temporarily. Such a chemical is called fungistat and the phenomenon of temporarily inhibiting growth is called fungistasis. For further references to the definitions and terms, you are referred to the lists of definitions published by the British Mycological Society (1950 and 1953), the American Phytopathological Society (1940 and 1943) and A Guide to the use of terms in Plant Pathology prepared in 1973 by the Terminology Subcommittee of the Federation of British Plant Pathologists. IMPORTANCE OF PLANT DISEASES

Man is directly dependent upon plants for his survival because plants are his prime source of food, fibre and drugs. The late Professor James G Horsfall, formerly Director of the Connecticut Agricultural Experiment Station, said that 30 million mouths were added every year. When we cannot feed the present population of the world, which is around four thousand million, how can we expect to feed it in 2000, by which year the world population will have doubled? This cannot be brushed aside with the quip

Introduction 11

that man should not be as prolific as plant pathogens. The role of plant pathology is important for alleviating losses to our food, fodder, fibre, and other plant products. Plants are also important to man because they utilize CO2 in photosynthesis and release O2. EFFECT OF PLANT DISEASES ON HUMAN AFFAIRS

An example of the tremendous impact that plant disease can have on the course of human history is given by the late blight of potatoes, caused by the fungus Phytophthora infestans. Between 1840 and 1847 it resulted in a famine among the working class population of Ireland who had come to depend on potato as their major source of food. In 1845 the potato blight devastated millions of acres in Europe, the USA and Canada. So sudden and so complete was the catastrophe that in only a few days, fields which had promised abundant harvests, were transformed into blackened wastes of vegetation overlying foul and putrefying masses of rotten tubers. As a result of this total failure of potato crop in Ireland there was a great famine and about a million people died and almost the same number of people migrated to other continents. In the early 1870s another fungal disease, the coffee rust (Hemileia vastatrix), wiped out the coffee plantations of Ceylon (Sri Lanka). The eminent English plant pathologist, H Marshal Ward, made a thorough investigation of the disease but did not succeed in devising ways of checking its ravages. Coffee growing was therefore abandoned, at least for many decades, and much of the world’s coffee production was shifted to the western hemisphere where the disease did not occur. The tea and rubber industry replaced coffee in Sri Lanka and other eastern areas. In the 1930s the entire banana industry in Central and South America was threatened with extinction by Sigatoka (Mycosphaerella musicola) and the industry could be saved only by 1940 when Bordeaux mixture was used on a massive scale (Wardlaw, 1961). The eating habits of people are very difficult to change and plant diseases appear to be responsible for many deeply implanted eating habits. While in northern and southern Europe, wheat bread is more common, in central Europe people eat rye bread. In the southern parts of the USA people eat corn bread while in the north, wheat bread is more common. Horsfall (1956) had suggested that this situation resulted due to the ravages of wheat rust. In France, between 1878 and 1882, the wine industry was threatened due to the introduction of downy mildew (Plasmopara viticola) from the USA. In India, the 1942 Bengal famine was perhaps largely due to the Helminthosporium disease of rice caused by Helminthosporium oryzea Breda de Haan. About two million people died of starvation. According to Padmanabhan (1973), nothing as devastating as the Bengal epiphytotic of 1942 has been recorded in plant pathological literature. In 1946–47, the wheat rust epidemic was responsible for food shortage as well as seeds in Madhya Pradesh. The bacterial blight of rice, thought to be confined to Japan, was reported in 1951 in Bombay (Srinivasan et al., 1959) and in a short time became very serious and widely distributed, attacking the commonly cultivated varieties. The disease broke out in an epidemic form in the Shahbad district of Bihar in 1963. With the introduction of Taichung Native-1, the disease appeared in a severe form in 1966 throughout the country wherever the crop was grown. Thus, with the introduction of one susceptible variety, a disease which was unknown in all but two states earlier became pandemic within two years. At present bacterial blight is a major hurdle in stepping up rice yields (Srivastava et al., 1966; Srivastava, 1967). Recently, there has been an epidemic of ergot disease of bajra caused by Claviceps microcephala almost throughout the country. In the USA (1969–70), there occurred one of the most devastating epidemics in the recent history of plant pathology, the southern corn leaf blight caused by Helminthosporium maydis. In 1970, over 70 per cent of the North American

12

Plant Pathology

maize crop carried Texas male sterile cytoplasm because a single type of cytoplasmic male sterility (Tms) was used in the production of hybrid seed. A virulent race of Helminthosporium maydis, known as race T, was present and the weather conditions favoured the development of the epidemic. The epidemic led to a great crisis in North America at that time. Coffee rust (Hemileia vastatrix) was recently discovered in Brazil where the pathogen has spread rapidly over the entire area. Coffee rust epiphytotic caused colossal losses in Brazil during 1974-1975. Thus, the disease has crossed the Atlantic Ocean to Brazil and Columbia where it is spreading. Chestnut blight caused by the fungus Cryphonectria (Endothia) parasitica nearly wiped out the American chestnut. It is believed that the causal organism was introduced from eastern Asia at the beginning of this century. The first report of the disease came from the New York Zoological Park in 1904. A year later, the disease assumed serious proportions and the Forestry Division of the Department of Agriculture became alarmed. Although the causative agent of the disease was soon discovered, no suitable control measures could be found to control the disease. The result was that about 30 billion board feet of wood, used for furniture, flooring, poles, railroad tiers (sleepers) and coffins, was lost. The tannin industry, which was dependent on this tree, collapsed and this forced the leather makers of the USA to obtain this item from other sources. The loss of one species created far reaching effects that have not been completely overcome till now. The dramatic biological and human events of this epiphytotic and the attempts to stop it have been documented in an excellent paper by Hepting (1974). Another instance of a serious loss by a fungus disease is due to the red rot of sugarcane (Glomerella tucumanensis) which reached its peak in 1938-39 in the white sugar belt of northern India—Bihar, Punjab and Uttar Pradesh. In the badly affected areas, most of the mills could crush only 33 per cent of their normal quantity (Woodhead et al., 1945). The farmers suffered tremendously. Catastrophic plant diseases are ably summarized by Klinkowski (1970) in which he mentions diseases like peach yellow, phony peach, sugarcane mosaic, pear decline, swollen shoot of cacao, plum pox, sugarbeet yellow, tobacco veinal necrosis, late blight of potatoes, blue mould of tobacco, hop downy mildew, Dutch elm disease, St. Anthony’s fire, cereal rusts, white pine blister rust, brown spot of rice, red rot of sugarcane and certain catastrophic nematode diseases which had disastrous consequences for man and made a dramatic impact on his affairs. Information on internationally important plant diseases exists (Holliday, 1971; Klinkowski, 1970; Meredith, 1973; Padwick, 1956; Riker, 1964). Some of the plant diseases which can assume serious proportions have been ably discussed by Thurston (1973). Some common epidemics have been discussed by Horsfall and Cowling (1978). A large number of plant diseases and the destruction they caused can be quoted and the above few examples amply prove that diseases can entirely change the course of history and the economy of a country, and they have been and still are a limiting factor in crop production. The advances made in food production in developing countries due to the green revolution could be lost if proper attention is not given to plant diseases and other pests. The higher and increased yields of green revolution are based on practices which are generally favourable to attacks by plant pathogens. Today, there is an increasing awareness on the part of the scientific agricultural community that only through a deeper and more fundamental understanding of all the interacting components of agriculture can we expect to improve our capabilities of feeding the expanding world population. Therefore, the role of plant pathologists is assuming greater importance in the present day world. The role of plant pathology in the scientific and social development of the world had been discussed by Stakman (1958) who has also dissertated on the opportunities and social obligations in plant pathology (1964). The future and the sociology of plant

Introduction 13

pathology have been discussed by Walker (1963) and Horsfall and Cowling (1977b), respectively. The scope and contribution of plant pathology have been dealt with by Houten (1959). Zentmyer (1994) has given a 55-year retrospective of plant pathology. In our country it is believed that the following are the (Table 1.1) key diseases of national and international importance (Pathak et al., 1996). TABLE 1.1 Crop Rice

Disease Tungro Bacterial blight/wilt Blast Brown leaf spot Sheath blight False smut

Wheat

Nematodes pest Rusts Smut of wheat Karnal bunt Foliar blights Nematode pests Molya Ear cockle

Pearl Millet

Sorghum Corn or maize Pigeon pea

Chick pea Soybean Pea Potato

Downy mildew Ergot Smut Grain mould Stalk rot Sterility mosaic Phytophthora blight Fusarium wilt Ascochyta blight Yellow mosaic Bud blight Powdery mildew Viral diseases Bacterial wilt Late blight Nematode pests

Pathogen Viral Xanthomonas oryzae pv. oryzae Pyricularia oryzae Helminthosporium orzyae Rhizoctonia solani (Perfect state— Thanatephorus cucumeris) Ustilaginoidea virens (Perfect state—Claviceps oryzae sativae) Nematodes Puccina graminis f. sp. tritici, P. striiformis f. sp. tritici, P. recondita Ustilago tritici Neovossia indica Helminthosporium sativum and Alternaria triticina Heterodera avenae Corynebacterium michiganense var. tritici and Angunia tritici Sclerospora graminicola Claviceps fusiformis Tolyposporium penicillariae Fungal and bacterial Viral Phytophthora drechsleri f. sp. cajani Fusarium udum (perfect state Gibberella indica) Ascochyta rabie Viral Viral Erysiphe polygoni Viral Pseudomonas solanacearum Phytophthora infestans Globoderma rostochiensis (Heterodera rostochiensis) and several species of Meloidogyne. (Contd.)

14

Plant Pathology

Crop Ground nut

Disease Aspergillus root rot Rust Tikka disease

Mustard

White rust Alternaria blight Mango malformation

Mango

Banana

Guava Apple Tea Coconut

Areca-Nut Sugarcane Cotton Tobacoo

Tapioca Ginger Cardamom Coffee Rubber Jute

Black Pepper

Powdery mildew Diplodia stem and other rots of fruits Bunchy top Bacterial wilt Nematode pests Wilt Scab Blister blight Root wilt Phytophthora bud rot Ganoderma wilt Fruit drop Red rot Smut New cotton wilt Leaf curl Phytophthora black leg Bacterial wilt Mosaic Rhizome rot Katte disease Die back Rust Phytophthora leaf fall Pink disease Macrophomina stalk rot Bacterial wilt

Phytophthora fruit fall and Phytophthora wilt Nematode pests

Pathogen Aspergillus spp. Puccinia arachidis Cercospora arachidicola and Cercosporidium personatum (Perfect stage Mycospaerella berkeleyii) Albugo candida Alternaria spp. A complex caused by Fusarium moniliforme var. subglutinans and mites Oidium mangiferae Diplodia sp. Mycoplasmal Pseudomonas solanacearum Nematodes Fusarium oxysporum f. sp. psidi Venturia inaequalis Exobasidium vexans Mycoplasmal Phytophthora palmivora Ganoderma lucidum Colletotrichum sp. Colletotrichum falcatum Ustilago scitaminea Fusarium oxysporum f. sp. vasinfectum Viral Phytophthora nicotianae var. nicotianae Bacterial Viral Pythium aphanidermatum and Pythium myriotylum Viral Colletotrichum coffeanum Hemileia vastatrix Phytophthora palmivora Pellicularia salmonicolor Macrophomina phaseoli Pseudomonas solanacearum var. asiaticum along with Rhizoctonia bataticola and Fusarium solani. Phytophthora capsici

(Contd.)

Introduction 15

Crop Brinjal Okra Cauliflower and Cabbage Tomato

Disease Little leaf Yellow bein mosaic Sclerotinia rot Curd rot of cauliflower Leaf curl complex Bacterial wilt

Pathogen Mycoplasmal Viral Sclerotinia sclerotiorum Bacterial Viral Pseudomonas solanacearum

According to Mukhopadhyay and associates (1992), the following (Table 1.2) are the diseases of international importance. TABLE 1.2 Fungal Diseases Karnal bunt of wheat Blast of rice Wheat rusts Brown spot of rice Sheath blight of rice Downy mildew of maize Downy mildew of pearl millet Ergot of pearl millet Anthracnose of sorghum Eye spot of cereals Pigeon pea blight Wilt of pigeon pea Chick pea wilt Ascochyta blight of chick pea Grey mildew of lettuce Bean anthracnose Potato late blight Downy mildew of lettuce Phomopsis blight and fruit rot of egg plant Rust of groundnut Tikka disease of groundnut Apple scab Sunflower rust Powdery mildew of apple Downy mildew of grapes

Bacterial Diseases Bacterial blight of rice Fire blight of apples and pears Moko disease of bananas Bacterial canker of stone fruits Common bacterial blight of bean Black rot of crucifers Black leg of crucifers Bacterial blight of soybean Citrus canker Ratoon stunting of sugarcane

Viral Diseases/Mycoplasmal Diseases/Viroid Disease Barley yellow dwarf Rice tungro Maize dwarf mosaic, Maize chlorotic dwarf and maize streak Sterility mosaic of pea Bean mosaics Pea seed borne mosaic virus disease Potato mosaics Citrus tristeza Sandal spike Cadang-cadang of coconut palm Lethal yellowing of coconut palm (Contd.)

16

Plant Pathology

Fungal Diseases Phytophthora disease of citrus Fusarium wilt of banana Phytophthora root rot of papaya Mango malformation Phytophthora root rot of avocado Red rot of sugarcane Smut of sugarcane Leaf scald disease of sugarcane Cercospora leaf diseases of sugarbeet Blight and wilt of cotton Blue mould of tobacco Leaf rust of coffee Coffee berry disease Armillaria root disease of trees Phytophthora root rot and dieback of forest trees

Bacterial Diseases Bunchy top of banana

Curly top of sugarbeet Nematode diseases Cereal cyst nematodes Root knot of vegetables Soybean cyst nematode Red ring disease of coconut palm White tip disease of rice

CLASSIFICATION OF PLANT DISEASES

Diseases may be classified in various ways on the basis of: 1. Host plant affected, such as cereals, millets, fruits, trees, vegetables, etc. 2. Part of the plant affected, such as root, stem, leaf, flower, fruit, etc. 3. Symptoms produced in the host plants, such as wilt, blight, soft rot, anthracnose, rust, smut, mildew, damping off, etc. 4. The mode of spread and severity of infection, such as epiphytotic, endemic, sporadic and pandemic. 5. Causal organism/ factor. Classification of Plant Diseases on the Basis of Spread and Severity of Infection Endemic Diseases A disease is classified as endemic when it is constantly present in a moderate or severe form and is confined to a particular country or district, for example, the wart disease of potato caused by Synchytrium endobioticum is endemic in Darjeeling. In plant pathology, this term is generally applied to simple interest diseases which are either indigenous or of ancient introduction. Epidemic or Epiphytotic Diseases An epidemic or epiphytotic disease usually occurs widely, but periodically, in a destructive form. The pathogen may be present, as in endemic diseases, but the environmental factors responsible for the development of the disease occur only periodically, for example, cereal rusts, powdery mildews, etc.

Introduction 17

Sporadic Diseases Sporadic diseases, in reality, belong to the epidemic group. The term is applied to those diseases which occur at very irregular intervals and locations and in relatively fewer instances. Angular leaf spot and blotch disease of cucumber are sporadic diseases (Pathak, 1972). Pandemic Diseases Pandemic outbreak is an epidemic which occurs over vast and extensive areas causing severe losses. These occur all over the world and result in mass mortality, for example, late blight of potato. Diseases Classified According to Major Causal Factors

Walker (1969) has classified plant diseases into three main types: 1. Non parasitic diseases 2. Parasitic diseases 3. Mycoplasmal and viral diseases based on the major causal factors These are further subdivided on the basis of the causal agency or organism involved. These are as follows: Non-parasitic diseases

Parasitic diseases

(1) 1. Low temperature, i.e. freezing injury

(2) Bacteria, i.e. bacterial blight of paddy

2. High temperature, i.e. heat canker of flax

Plasmodiophoromycota, i.e. club root of crucifers

3. Unfavourable oxygen, i.e. black heart of potato 4. Unfavourable soil moisture, i.e. flooding injury 5. Injurious atmosphere, i.e. apple scald or black tip of mango. 6. Lightning injury, i.e. damage to potato plants due to lightning 7. Mineral excess, i.e. internal bark necrosis of delicious apple 8. Mineral deficiency, i.e. red leaf of cotton or khaira disease of rice

Chytridiomycota, i.e.wart of potato

Mycoplasmal, Spiroplasmal, Viral and Viroidal diseases (3) Such as little leaf of brinjal, citrus stubborn, tobacco mosaic, and potato spindle tuber.

Oomycota i.e. late blight of potato Ascomycota. i.e. ergot of pearl millet Basidiomycota, i.e. pink disease of citrus Deuteromycota, i.e. blast of rice Phanerogams, i.e. diseases due to giant mistletoe. Animal parasites (Nematodes, i.e. ear cockle of wheat; insect, i.e. insect galls of various trees; Protozoans, i.e. Hart rot of coconut palms, phloem necrosis of Coffee).

18

Plant Pathology

TRENDS IN TEACHING AND RESEARCH IN PLANT PATHOLOGY WORLDWIDE

Agrios (1997) has very briefly summarized the trends in teaching and training in plant pathology. Trends in plant pathology-significance for the future have been critically discussed by Weinhold (1988). Sequeira (1986) has critically evaluated the issues facing the development of the subject of plant pathology. Phytopathology has existed barely for the last 150 years when it was separated from the basic sciences, of botany and medicine. As the date of its origin the publication of the work of Julius Kuhn Die Krankheiten der Kulturgewachse (Diseases of the cultivated plants) in 1858 may be considered. To obtain a clear understanding of the modern trends in phytopathological research it may be necessary to briefly recapitulate the earlier developmental trends of the subject. From the time of Theophrastus or as he was called in his era “Father of Botany”, a transcendental origin was attributed to plant diseases and epidemics. This view dominated until the end of the middle ages. This era is called the predispositional era in plant pathology. The etiological view developed essentially in the 19th century and culminated in the activities of Prevost, Tillet and de Bary. The mycological trend of de Bary and Brefeld was followed by the bacteriological and virological directions of research. The requirement of industry for agricultural raw materials rose considerably in the 20th century. Efficient field-plant protection machines such as aircraft, helicopters, and new means of mechanical and thermo-mechanical aerosol treatment, use of potent fungicides and so on spread considerably in the modern control of plant diseases and epidemics. According to Ubrizsy (1964), phytopathology has been characterized in the 20th century by prophylaxis and breeding for resistance and more particularly pathophysiological trends of research started in the 1950s. The next 50 years saw the development of research in the areas of the physiological-biochemical processes of diseased plants, the nature of parasitism as well as disease resistance. The modern trends of research tend to disclose the true causes of cell proliferation, elucidate the nature of biosynthesis of viruses, possibilities of inhibition of virus multiplication, the mechanism of fungal toxicity, the manner of action of toxins, the role of hormone-like growth substances (regulators) in pathogenesis and the importance of extracellular enzymes in the etiology of the disease. Chemotherapy and chemically-induced resistance are areas where a lot of attention is being directed. Some of the frontline areas of research in plant pathology are the molecular biology of disease resistance, plant disease epidemic modelling, forecasting and modern methods of disease control including biocontrol of plant pathogens.

REFERENCES Addy, S.K. and N.K. Dhal (1977), “Serology of Xanthomonas oryzae”, Indian Phytopath., 30 (1): 65–69. Anonymous (1940), “Report of the committee on technical words”, Phytopath., 30: 361–68. Agrios, G.N. (1991), “Frontiers and Challenges in Plant Pathology Communication”, (1992), Phytopath, 82: 32–34. Agrios, G.N. (1997), Plant Pathology, Fourth Edition, Academic Press, San Diego; p. 635. Garrett, S.D. (1956), Biology of Root Infecting Fungi, Cambridge University Press, London. Gaumann, E. (1950), Principles of Plant Infection, (English Translation), WB, Briely (Ed.), Crosby Lockwood, London. Hepting, G.H. (1974), “Death of the American chestnut”, Forest History, 18: 60–67. Holliday, P. (1971), “Some tropical plant pathogens of limited distribution”, Rev. Pl. Pathol., 50: 337–348.

Introduction 19

Horsfall, J.G. (1956), “The fight with fungi on the rusts and the rots that rob us, the blasts, and the blights that beset us”, Am. J. Bot., 43: 522–536. Horsfall, J.G. and A.E. Dimond (1960), “The pathogen: The concept of causality”, in: Plant Pathology, JG Horsfall and A.E. Dimond (Eds.), Academic Press, New York, 2: 1–18. Horsfall, J.G. and E.B. Cowling (1977a), “Prologue: How disease is managed”, in: Plant Disease—An Advanced Treatise, J.G. Horsfall and E.B. Cowling, (Eds.) Academic Press, New York, 1: 1–9. Horsfall, J.G. and E.B. Cowling (1977b), “The sociology of plant pathology”, in: Plant Disease—An Advanced Treatise, J.G. Horsfall and E.B. Cowling, (Eds.) Academic Press, New York, 1: 12–33. Horsfall, J.G. and E.B. Cowling (1978), “Some epidemics man has known”, in: Plant Disease—An Advanced Treatise, J.G. Horsfall and E.B. Cowling, (Eds.) Academic Press, New York, 2: 17–31. Houten, JG (1959), “Scope and contributions of plant pathology”, in: Plant Pathology—An Advanced Treatise, J.G. Horsfall and A.E. Dimond (Eds.), Academic Press, New York and London, 1: 19–60. Johann, Helen, J.R. Holbert and J.G. Dickson (1931), “Further studies in Penicillium injury to corn”, J. Agr. Res., 43: 757–790. Klinkowsky, M. (1970), “Catastrophic plant diseases”, Ann. Rev. Phytopath., 8: 37–60. Leukel, R.W. (1948), “Periconia circinata and its relation to Milo disease”, J. Agr. Res., 77: 201–222. Luttrell, E.S. (1974), “Parasitism of fungi on vascular plants”, Mycologia, 66: 1–15. Meredith, D.S. (1973), “Epidemiological considerations of plant diseases in the tropical environment”, Phytopath., 63: 1446–1454. Mukhopadyay, A.N., J. Kumar, U.S. Singh and H.S. Chaube (1992), Plant Diseases of International Importance, Vols. 1-4, Prentice Hall, Englewood, Cliffs, New Jersey. Nene, Y.L. (1987), “Indian plant pathology—2000 AD Presidential address”, Indian Phylopath, 40(1): 1–13. Nussbaum, C.J. and G.W. Keitt (1938), “A cytological study of host–pathogen relation of Venturia inaequalis on apple trees”, J. Agr. Res., 56: 595. Oswald, J.W. (1951), “The relation of Periconia to Milo root-rot in California”, Phytopath., 41: 28–29. Padmanabhan, S.Y. (1973), “The great Bengal Famine”, Ann. Rev. Phytopath., 11: 11–26. Padwick, G.W. (1956), “Losses caused by plant diseases in the colonies”, Commonw. Mycol. Inst. Phytopath., Paper No. 1. Pathak, V.N. (1972), Essentials of Plant Pathology, Parkash Publishers, Jaipur, p. 448. Pathak, V.N., N.K. Khatri and M. Pathak (1996), Fundamentals of Plant Pathology, Agro Botanical Publishers (Indian), Bikaner, p. 356. Riker, R.J. (1964), “Internationally dangerous tree diseases and Latin America”, J. Forestry, 62: 229–232. Robinson, R.A. (1969), “Disease resistance terminology”, Rev. Appl. Mycol., 48: 593–606. Saksena, H.K. (1977), “Reminiscences and observations on the growth of plant pathology in India”, Indian Phytopath., 30(2): 165–175. Sequeria, L. (1986), “Facing the issues in Plant Pathology, (1987)”, Phytopathology, 77: 24–28. Srinivasan, M.C., M.J. Thirumalachar and M.K. Patel (1959), “Bacterial blight of rice”, Curr. Sci., 28: 469–470. Srivastava, D.N., Y.P. Rao and J.C. Durgapal (1966), “Can Taichung Native-1 stand up to bacterial blight?”, Indian Farming, 16: 15. Srivastava, D.N. (1967), “Epidemiology of bacterial blight of rice and its control in India”, Tropical Agr. Res. Series, Agriculture, Forestry and Fisheries Research Council, Ministry of Agriculture and Forestry, Japan, 1: 11–18. Stakman, E.C. (1958), “The role of plant pathology in the scientific and social development of the world in plant pathology”, in: Plant Pathology—Problems and Progress, C.S. Holton et al., (Eds.), University of Wisconsin Press, Madison, pp. 3-13. Stakman, E.C. (1964), “Opportunities and obligations in plant pathology”, Ann. Rev. Phytopath., 2: 1–12. Stakman, E.C. and J.G. Harrar (1957), Principles of Plant Pathology, Ronald Press, New York, p. 581. Thrower, L.B. (1966), “Terminology for plant parasites”, Phytopathogische Zeitschrift, 56: 258–259.

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Thurston, H.D. (1973), “Threatening plant disease”, Ann. Rev. Phytopath., 11: 27–52. Ubrizsy, G (1964), “Modern Trends in Plant Pathological Research”, in: Host Parasite Relations in Plant Pathology, Z Kiralay and G. Ubrizsy (Eds.), Reasarch Institute for Plant Protection, Budapest, Hungary, pp. 7-11. Upadhyay, R.S. and Bharat Rai (1983), “Extension Plant Pathology in Indian Agriculture”, XVIII Everyman’s Science: 12–16. Vander Plank, J.E. (1963), “The future of plant pathology”, Ann. Rev. Phytopath., 1: 1–4 Vander Plank, J.E. (1968), Disease Resistance in plants, Academic Press, New York, p. 206. Walker, C.J. (1969), Plant Pathology, Third Edition, McGraw-Hill, New York, p. 819. Wardlaw, C.W. (1961), Banana Diseases Including Plantains and Abaca, Longmans, London, p. 648. Weinhold, A.R. (1988), Trends in Plant Pathology—Significance for the Future, Phytopathology, 79: 33–37. Whetzel, H.H. (1929), “The terminology of phytopathology”, Proc. Inter. Congr. Pl. Sci., 2: Ithaca, 1204–1215. Woodhead, J, S.V. Ramamurty, M.B. Nanavati, M.A. Husain and WR Aykroyd (1945), The Famine Enquiry Commission, Final Report, Manager of Publications (Govt. of India), Delhi, p. 518. Zentmyer, G.A. (1994), “Plant Pathology–A 55 year Retrospective”, Ann. Rev. Phytophatol., 32: 19.

QUESTIONS 1. Define disease. 2. Distinguish the following pairs of terms : i. Simple interest disease and compound interest disease ii. Parasite and pathogen iii. Pathotype and serotype iv. Pathogenicity and pathogenesis v. Host and suscept vi. Primary inoculum and secondary inoculum vii. Penetration and infection viii. Disease cycle and life cycle ix. Symptoms and Syndrome x. Obligate and facultative parasites and saprophytes xi. Biotrophs and perthotrophs xii. Vertical resistance and horizontal resistance xiii. Pathodeme and pathotype xiv. Epidemiology and aetiology xv. Fungicidal and fungistat 3. Name some plant diseases that resulted in grave consequences for many nations. 4. Give definitions and examples of the following types of disease: i. Epidemic or epiphytotic disease ii. Sporadic disease iii. Endemic disease iv. Pandemic disease 5. What are the recent trends in phytopathological research and training worldwide?

2 History of Plant Pathology

It may be asked why it is necessary to have a historical account of plant pathology. A study of the history of any science helps to make the subject clearer. By studying the history of a science we get a better perspective of the subject; we come to know the contributions made in that field; the problems that were encountered and the manner in which they were tackled. We can follow the guidelines set by previous scientists while studying a particular branch of science. Whetzel (1918) has given a useful account in his book Outline of the History of Plant Pathology. Large (1940) has dealt with the subject in his remarkable book, The Advance of the Fungi. Keith (1959) has ably presented the historical development of plant pathology. Parris (1968) has discussed the chronology of plant pathology. Fish (1970) has given the history of plant pathology in Australia while that in India, Burma, and Ceylon (Sri Lanka) has been given by Das Gupta (1958) and Raychaudhuri et al. (1972). Orlob (1971) has discussed the history of plant pathology in the Middle Ages. Bennett (1973) has considered factors important for the growth of the science of plant pathology. Thurston (1973) gives an interesting account of some threatening plant diseases. There are several other reviews, such as Plant Pathology: Changing Agricultural Methods and Human Society by Ten Houten (1974). Akai (1974) has given a historical perspective of the science of plant pathology in Japan. Walker (1975) has given some highlights of plant pathology in the USA. Nolla, Manuel and Valiela (1976) have reviewed the history of plant pathology in South America, Central America, and Mexico. Fuchs (1976) has written a history of physiological plant pathology. Ubrizsy (1964) has discussed modern trends in plant pathological research. Landmarks in Plant Virology: Genesis of Concepts has been discussed by Markham (1977). Legacy for the millennium: A century of progress in plant pathology has been recently reviewed by sequeira (2000). It is interesting to note that the largest number of botanists specialize in mycology and plant pathology. This is due to the fact that fungi, bacteria and actinomycetes yield antibiotics; they have a role in the decomposition of organic matter, fermentation, nitrogen fixation, they are sources of food (mushroom culture), and lastly, some of these organisms are responsible for plant diseases which cause great annual losses throughout the world. It is estimated that in India there is an annual loss of Rs. 5000 crores as a result of plant diseases. In the USA the annual loss is estimated to be 9.1 billion dollars. The earliest known writings make references to the ravages of plant diseases. There can be little doubt that plant diseases preceded man on earth and that ever since he has practiced agriculture, they have taken their toll of crops. We must agree with the late Professor Whetzel (1918) of Cornell University, in saying that

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phytopathology like all natural sciences, had its beginings in the dawn of man’s civilization. There are references in the Vedas (1500 BC) to plant diseases and methods of control. The first book in which plant diseases have been discussed is Vraksha Ayurveda, written by Surapal in ancient India. There are references in the Old Testament of blasting, mildew, and insect pests. The Romans used to celebrate a festival, Robigalia, to ward off rust. The annual festival of Robigalia is reported to have been initiated by King Numa Pompilius probably around 700 BC, and was continued with modifications in the Christian era. Theophrastus, who lived from 370 BC to about 286 BC, was the first botanist to study and write about the diseases of trees, cereals, and legumes, although his approach was observational and speculative rather than experimental. He noticed that some kinds of plants were more susceptible to certain diseases than others. He was not aware that microorganisms caused diseases and it was generally accepted that fungi and various other organisms observed in association with diseased or decaying plants arose spontaneously from plants or from the environment. Comparatively little was added to the knowledge of botany or plant pathology for nearly two thousand years after Theophrastus. From the 5th century AD until the Renaissance there was little intellectual activity and not much was written about botany and plant pathology. In his writings on the plant pathology of that period, Whetzel (1918) states that one bright spot in that dark period was the work, in the 10th century AD, of an Arabian landed proprietor, Ibnal-Awam, who was said to have described accurately the symptoms of many diseases of trees and vines and to have given extensive consideration to their control. The period between 300 AD and 1300 AD is said to be the middle or ‘dark ages’ in history. There were petty wars, religious dogmas, and superstitions and there was practically no increase in the knowledge of plant pathology during that period. Then came the Renaissance from the 14th to the 16th century AD and it was during this period that printing (1440) and the microscope (1685) were invented. Anton van Leeuwenhoek a linen draper, surveyor, and wine gauger belonging to Delft, Holland, invented the compound microscope and Robert Hooke an English scientist, observed the cell for the first time under the microscope in 1660. THE HERBALISTS

The beginning of the revival of interest in botany is reflected in the writings of the herbalists and these are more important from the mycological standpoint. 1. Pinax Theatri Botanici (1923) written by the Swiss, Gaspard Bauhin, who attempted to compile all the plants known to him and his predecessors. 2. Nova Plantarum Genera (1729) written by Pier Antonio Micheli, an Italian, who cultured fungi on pieces of water melon. 3. Species Plantarum (1753) by C. Linnaeus (1707–78), a Swede, who is credited with the development of binomial nomenclature. He is often called the Father of Botany. THE SYSTEMATISTS

CH Persoon’s Synopsis Methodica Fungorum (1793) is the chief starting point for the nomenclature of the Uredinales, Ustilaginales, and Gasteromycetes. Persoon, a native of South Africa, and educated in Germany and Holland, did most of his work in France. Although a doctor of medicine, he devoted most of his time to the study of fungi.

History of Plant Pathology 23

Elias Magnus Fries (1794–1878) was Swedish and is regarded as the ‘Linnaeus’ of mycology. His Systema Mycologicum (1821–1832) is the chief starting point for the nomenclature of fungi. Fries was the son of a Swedish clergyman and was Professor of Botany, first at the University of Lund, Sweden and then later at Uppsala, where he succeeded Linnaeus. Fries regarded the rust and smut fungi as products of diseased plants. Systema Mycologicum has put the taxonomy of most fungi on a scientific footing more than any other earlier work. He was one of the most respected mycologists of his time. Matheiu Tillet (1714-1791), Director of the Mint at Troyes, France, and an amateur botanist and agriculturist, published an important paper on wheat bunt. In 1755 he tested the effect of dusting seed with the black mass contained in the diseased kernels. Thus, he demonstrated experimentally that the bunt of wheat was contagious and thought that the spores contained a poisonous entity. Tillet did not understand the full implications of his experiments. He, however, suggested the control of disease by treating seeds with salt and lime. It can be said that he was an experimenter who lived ahead of his times. Felice Fontana (1730-1805), an Italian physicist and naturalist, published a paper in the year 1767 in which he expressed the view that grain rust was a distinct parasitic entity. He distinguished between the red and black stages of wheat rust and made microscopic drawings. Benedict Prevost (1755-1819) who was a Swiss Professor of Philosophy at the Academy of France in 1807, observed the germination of the spores of the wheat bunt organism and conceived the idea that this organism penetrated the young wheat plant and was the actual cause of the disease. Provost’s experiments provided the first proof and interpretation of the role of a micro-organism in the causation of disease. Prevost also demonstrated the control of smut by steeping seeds in a solution of copper sulphate. His contemporaries did not accept his views until the devastating potato blight in 1845 which attracted the attention of mycologists and plant pathologists to plant diseases. Thus, Matheiu Tillet, Felice Fontana and Benedict Prevost were three experimenters who were definitely ahead of their times in their thinking with regard to the causation of plant diseases. THE BEGINNING OF MODERN PERIOD

In 1845, the devastating epidemic of the potato blight in North Europe and particularly in Ireland focussed attention on plant diseases and gave a great fillip to and stimulated interest in the subject of plant pathology. Much of the early literature on this disease is found in the Gardener’s Chronicle. Scientists at that time were unable to agree on the cause of the disease or to suggest a suitable remedy. The autogenetic theory of diseases was still dominant, although opposition to this was rapidly increasing. Dr John Lindley, editor of the Gardener’s Chronicle and Professor of Botany at University College, London, was the leader of the autogenetists. MJ Berkeley (1803-1889) who was then the most prominent British mycologist was at first somewhat reserved in supporting the parasitic theory, but advocated it strongly in 1846. Montagne in France, in 1845, described the fungus as Botrytis infestans. Morreu in Belgium, experimented with disease in 1845. He described the disease and fungus and regarded the fungus as the major cause of the disease, although he lacked convincing evidence. That the fungus was the real cause of disease and not the result, was proven experimentally by Speerschneider in 1857 and de Bary in 1861 and 1863 (Jones et al., 1912). Following the publication of the Origin of Species by Charles Darwin in 1859, evolution became accepted as a fact and the systems of classifications of both plants and animals were given a phylogenetic slant. Anton de Bary (1831-1888) became an outstanding figure in the botanical field. Much of his

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research was on fungi and the relation of parasitic fungi to their hosts. De Bary was born in 1831 in Frankfurt-am-Main, Germany. His mycological interests were more biological and physiological than systematic. From his investigations on life histories, parasites and saprophytes, the nature of lichens, and so on he made new and important discoveries. Heteroecism in the Uredinales was made clear by his experiments and he gave accounts of the development and sex in a number of Phycomycetes and Ascomycetes. He was the first to indicate the nature of obligate and facultative forms. His well-known text book, Morphologie und Physiologie der Pilze, Flechten, und Myxomyceten in 1866 and its second edition in 1884, records the broad classification of fungi. He is truly regarded as the ‘Founder of Modern Mycology’. A large number of students from different parts of the world came to his laboratory. More than sixty of them became prominent in their fields and carried his techniques and influences to many parts of the world. Famous among them were MS Woronin of Russia, O Brefeld of Germany, PA Millardet of France, HM Ward of England, WG Farlow of the USA and A Fischer of Switzerland. In his studies on the Sclerotinia disease of carrots and other vegetables, de Bary noted that the host cells were killed in advance of the pathogen and that the juice from the rotted tissue could break down healthy tissues of the host. On the other hand, if the juice was boiled it had no effect on the healthy tissues. De Bary concluded that the pathogen produced enzymes that brought about the degradation and killing of plant cells. De Bary’s noteworthy contribution to plant pathology were his studies on the Peronosporaceae and the diseases they incited, especially the late blight of potato, the discovery of heteroecism in the rusts and his work on Sclerotinia sclerotiorum (1886) which opened up an entirely new field of investigation, the physiology of parasitism. CONFIRMATION OF PREVOST’S WORK

In the 1840’s, the Tulasne brothers (Louis Rene Tulasne and Charles Tulasne) who were botanists at the Museum of Natural History in Paris, began extensive morphological studies on fungi. They made illustrated drawings of rusts, smuts, and ascomycetes that are regarded as classics in mycology. LR Tulasne (1815-1885) is often called the ‘Reconstructor of Mycology’. In 1847, the two brothers published studies on wheat fungus and confirmed the observations of Benedict Prevost with regard to the causal organism of the bunt disease of wheat. Julius Gotthelf Kuhn (1825-1910) was a contemporary of de Bary. He was initially a farm manager. He publised his famous text book for farmers in 1858 entitled The Diseases of Cultivated Crops: Their Causes and Their Control. This was the first text book to be published in which fungi were regarded as causal factors in plant disease. The diseases were grouped according to causes; unfavourable climatic and soil conditions; influence of animals; and parasitic plants. Nematodes were also included as they were known as plant parasites since the time of Needham (1774). Their economic importance at the end of the 19th century promoted nematological studies as a part of plant pathology (Raski, 1959). DOCTRINE OF SPONTANEOUS GENERATION

This theory held that living organisms could arise spontaneously from inanimate or non-living matter. In 1748, Needham, an English clergyman and naturalist, supported the theory of spontaneous generation. To prove his point he boiled meat in a corked flask. He demonstrated that the contents had spoiled due to micro-organisms. We now know that while he killed the vegetative bacteria, the bacterial spores were

History of Plant Pathology 25

not killed at the temperatures used. In 1775, Spallanzani, an Italian investigator challenged Needham and proved that the decay of meat by bacteria and other lower forms of life could be prevented by heating the material in a flask which was sealed in such a way as to exclude contamination from the air. His experiments supported his contention. Others failed to get similar results consistently. Nevertheless, the views put forward by Spallanzani at that time were not accepted. Antonie Laurent Lavosier (17431794) discovered the presence of oxygen in the air and its indispensability to sustain macroscopic forms of life. In 1775, he argued that this essential gas was vital for life and therefore, sealed flasks that did not permit the entrance of oxygen were incapable of allowing spontaneous generation to occur. Schwann (1837) and Cagniard Latour (1838) discovered the yeast cell and maintained that it caused fermentation. In 1839, Berselius, a Swedish chemist, strongly defended the mechanistic explanation of fermentation and he was supported by Liebig, a German agricultural chemist. It was in 1860 that Pasteur furnished irrefutable evidence that micro-organisms arise from pre-existing living entities and that fermentation is a biological phenomenon rather than a purely chemical one. It may be mentioned that the autogenetic theory of disease continued to be dominant throughout the first half of 19th century. The most influential proponents were Unger, Meyen and Liebig. Unger was an Austrian physician and professor of botany. He thought that fungi associated with plant diseases arose from the diseased plant because of abnormalities in plant juices and that they were products rather than the cause of diseases. His best known work on plant pathology was Die Exantheme der Pflanzen (1833). Thus, Needham, Lavosier, Liebig and several others could not interpret the results of their experiments correctly because of their reluctance to give importance to new scientific ideas. Pasteur was born in 1822 of humble parents in the village of Dole in France. He began his life as a chemist. He worked as a teacher at Strassburg and then at the Ecole des Beaux Arts in Paris. He never studied medicine, though his discoveries about the protozoan nature of the silkworm and the confirmation of the bacterial origin of anthrax buried forever the controversy over the theory of spontaneous generation. The germ theory in relation to diseases of man and animals was established in 1876 by Robert Koch. Koch was a German physician and Pasteur’s co-worker. The first final proof of the causal relation of the anthrax bacillus to anthrax disease was published by Robert Koch in Germany in 1876. Koch (1843-1910) was trained as a physician but turned his attention to the new science of bacteriology. He began his work by establishing laboratory procedures and techniques that would yield reproducible results. Koch established the principles of pure culture techniques although it was Lister, the English surgeon, who pioneered work in the field of aseptic surgery. He was impressed by Pasteur’s work and made it the basis of his own research. He was the first man to culture a bacterium capable of souring milk. In the medical field, Robert Koch discovered the bacterium that causes tuberculosis. He enunciated certain rules or criteria that should be satisfied before the identity of the disease-producing organism in a particular disease could be established. These rules are known as Koch’s postulates and can be briefly summarized as follows : 1. A specific organism must always be associated with a disease. 2. The organism has to be isolated in pure culture. 3. The organism must be identified. 4. The organism when inoculated must produce disease in a healthy susceptible host. 5. The organism must be isolated from the experimental host again in pure culture and its identity established. Brefeld (1875, 1881, 1883) in Germany worked on fungi and was mainly responsible for the early development of modern techniques of growing micro-organisms in a pure culture. Along with the improvements made by Koch, Petri and others his techniques are the basis for the pure culture methods

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currently employed. He was the pioneer in tracing the life cycles of the cereal smut fungi and their role in the causation of diseases. He made important contributions both to the pure culture of smut fungi and to the methods of inoculation. FOREST PATHOLOGY

Robert Hartig was born in Braunschweig in Germany in 1839. He was an enthusiastic and successful teacher. He is well known for his contributions to many aspects of forestry, botany and entomology. He is aptly called the ‘Father of Forest Pathology’. His foremost contributions are Wichtige Krankheiten der Waldaume in 1874 and Lehrbuch der Baumkrankheiten in 1882. The latter publication was an authoritative work for many years. DISCOVERY OF BORDEAUX MIXTURE

The discovery of this fungicide has an interesting story. It was discovered by Pierre Marie Alexis Millardet in 1882. Millardet was born in Montmery-la Ville in France in 1838. He studied medicine but was more interested in botany. He learnt botany from the great botanist, Hoftmeister, at Heidelberg and de Bary at Freiburg. He returned to France and obtained doctorates both, in medicine and science. In 1876, he joined the University of Bordeaux, France and served there till his retirement in 1899. Millardet was a man of broad interests. His contributions fall into three categories. 1. Studies on the morphology, physiology and systematic relationships of plants. 2. Investigation on the root aphid Phylloxera of the grape vines and hybridization experiments in order to have resistant plants. 3. Research on the downy mildew of grapes caused by Plasmopara viticola and its cure. The downy mildew of the vine was first reported in Europe in 1878. Millardet and Planchon discovered it at about the same time in France, where it had evidently been introduced from the USA somehow during the import of resistant root stocks of grape vines for root aphid Phylloxera. The disease spread rapidly and threatened the vineyards of Europe. Millardet began a thorough study of the disease and the methods of control. In 1882, he noticed that vines treated with a mixture of copper sulphate and slaked lime to prevent the grapes from being stolen, retained their leaves, whereas the untreated vines were defoliated. This fact suggested to Millardet a means of curing the disease. In 1883 and 1884, he performed extensive spraying experiments with many preparations of copper, calcium and iron salts, used singly and in various mixtures and also arranged tests by viticulturists. Both the seasons were dry and little mildew developed. Being a conservative scientist, Millardet preferred to delay the publication until he could recommend a thoroughly tested spraying programme. However, the news of his work spread and others began to publish the effects of copper preparations on mildew. In May, 1885, Millardet published his works and gave details of spraying with a mixture of copper sulphate and slaked lime, which was later known as the Bordeaux mixture. The downy mildew appeared in a severe form in 1885 and when people followed Millardet’s recommendation, spectacular results were obtained in controlling the disease. He, as well as others, rapidly improved spraying methods and studied numerous copper and other fungicidal preparations. The Bordeaux mixture emerged and for many years continued to be the most important and successful fungicide.

History of Plant Pathology 27

PLANT PATHOLOGY IN THE TWENTIETH CENTURY

The major trends initiated in the latter half of the 19th century progressed in several directions. The rediscovery of Mendel’s laws in the beginning of the century had an impact and the genetics of the host as well as the pathogen received the attention of plant pathologists. Some of the major trends were in the following areas : 1. Physiological plant pathology 2. Genetics of the host and the pathogen 3. Fungicidal research 4. Environment and its relation to plant disease 5. Nature of disease resistance in plants 6. Biochemistry and physiology of the diseased host plant 7. Molecular biology of pathogenesis and induced systemic resistance 8. Tissue culture in plant pathology 9. Biotechnological approach to disease control 10. Ecological studies of plant pathogens 11. Bacteriology 12. Virology 13. Mycoplasma group of pathogens, etc. These major trends are now discussed in brief. PHYSIOLOGICAL PLANT PATHOLOGY

The physiological trend in plant pathology can be divided into three aspects : 1. Studies on physiogenic diseases 2. Work on the indirect or secondary causal relationship of the environment to infectious diseases 3. Investigations into the physiology of parasitism Sorauer, born in Germany in 1839, initiated modern studies in physiogenic diseases. His best known work is Handbuch der Pflanzenkrankheiten which was written in 1909. Sorauer laid tremendous emphasis on physiogenic diseases. Prevost (1807) demonstrated experimentally and interpreted clearly the indirect or secondary causal role of the environment in the etiology of parasitic disease. HM Ward (1880) emphasized the role of the environment in the epidemiology of coffee rust. He may be considered the father of tropical plant pathology. He was the first to point out that the exclusive cultivation of a crop over unbroken areas was one of the most important prerequisites for an epidemic. The effect of copper fungicides against endophytic fungal pathogens was also demonstrated by him first and LR Jones et al. (1926) at Wisconsin initiated a new field of research on the effects of soil temperature and moisture and other environmental factors upon the empidemiology of the disease. de Bary (1886), in his last work, pioneered research on the physiology of parasitism. de Bary worked on Sclerotinia sclerotiorum and the disease it caused to carrots and other plants. He demonstrated that the pathogen killed the cells of the host in advance of the invading hyphae of the pathogen. He found that juice from rotted tissue could rapidly break down healthy host tissues. The capacity to break down tissues was completely lost after boiling. LR Jones did pioneering work on pectic enzymes secreted by

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soft rot bacteria. William Brown’s papers on the physiology of parasitism form the foundation of further research on the role of enzymes in plant pathogenesis. After the second world war mainly on the initiative of the American, Japanese, Soviet, English, and Indian researchers, the so-called physiological era commenced. Outstanding names of this era are JC Walker, KO Muller, JG Horsfall, AE Dimond, LP Miller, RG Ovens, T Akazawa, I Uritani, WH Fuchs, E Gaumann, RKS Wood, TS Sadasivasn, H Wheeler, JM Duniway, JA Kuc, F Fric, GF Pegg, R Heitefuss, RP Scheffer, K Rudolph, DF Bateman, RN Goodman, and IAM Cruickshank. GENETICS OF THE HOST AND THE PATHOGEN

In England, as early as 1905, RH Biffen began breeding cereals resistant to yellow rust. The genetics of the host plant and pathogen were studied extensively with regard to the inheritance of resistance and virulence, when Biffen’s rust resistant varieties succumbed to infection in Australia and the fact of genetically-bound disease resistance was doubted. EC Stakman, as early as 1914, contributed valuable information on delineating and cataloguing physiological races of the pathogen. It has been demonstrated by crossing formae specialis (Stakman et al., 1930) and pathotypes (Newton et al., 1930) of Puccinia graminis that the virulence of the pathogen is also inherited according to Mendel’s laws. The same is true for various other pathogens (Person and Sidhu, 1971). The demonstration by Biffen (1905) of the Mendelian nature of the inheritance became the starting point for the systematic genetic analysis of resistance. An increase in the knowledge of the sexuality of fungi by Blakeslee (1904), Craigie and Dodge (1927) enabled a genetic study of pathogenic fungi. Flor (1955) explained host-parasite interaction in flax rust caused by Melampsora lini. He gave the gene-for-gene hypothesis to explain this reaction. Van der Plank (1968) gave the concept of horizontal and vertical resistance in plants. Other pioneers in the field of breeding disease resistant varieties were WA Orton and LR Jones of the USA and BP Pal and KC Mehta of India. FUNGICIDAL RESEARCH

The earliest known fungicide was sulphur. The history of the development of fungicides is fascinating. In the 20th century, several biochemists and organic chemists along with plant pathologists started investigations on this subject. Fungicides in the 20th century, especially in the 1930’s were mercury or Bordeaux mixture. From the Bordeaux mixture developed the inorganic insoluble copper fungicides such as cuprous oxides or oxychlorides. The emphasis shifted from the oxides of copper to the organic fungicides, the organo-mercurial, the dithiocarbamates, and bisdithiocarbamates. WH Tisdale and I Williams (1934) revolutionized the study of organic fungicides by discovering alkyldithiocarbamates. Alkylene bisdithiocarbamates were discovered by Dimond, Heuberger, and Horsfall in 1943. Interest in the chemotherapy of plants has been generated and work on an interesting group of compounds, including chloranil, systemic fungicides, and antibiotics is of current interest to workers in this field. The mechanism of fungicidal action has been explored by Horsfall (1945, 1956), Sisler and Cox (1960), Sisler (1969), Rich (1960), Owen (1963) and others. In 1956, JG Horsfall published an authoritative book entitled Principles of Fungicidal Action. An informative book on systematic fungicides was edited by RW Marsh in 1972. Fry (1982) published a very useful book “Principles of Plant Disease Management”.

History of Plant Pathology 29

ENVIRONMENT IN RELATION TO PLANT DISEASE

LR Jones pioneered the study of the effect of soil temperature on cabbage yellows at Wisconsin. He developed soil temperature tanks to see the effect of soil temperature on the development of disease. For this type of experiment a thermostatically controlled water bath was devised into which water-tight vessels containing plants growing in soil or other substrates could be inserted. This apparatus is known as the Wisconsin soil temperature tank. Before the acceptance of the germ theory in relation to plant disease, the environment was considered to be the major factor. It is now well known that in cases where a parasitic micro-organism is part of the causal complex of a disease, the environment plays an important part. Experimental studies in disease development in relation to various environmental factors have now become quite extensive and the predisposing factors are considered to play an important role in the development of any disease. NATURE OF DISEASE RESISTANCE

The fact that many plants show a natural resistance to disease has long been used by plant geneticists in their breeding programmes. Rapid advances in modern biochemistry have now made it possible for plant pathologists to investigate the biochemical basis of this resistance. Valuable contributions in this field have been made by GL Farkas and Z Kiralay (1962), J Kuc’ (1963), I Uritani (1963) and others who investigated the role of phenolic compounds in natural chemical resistance. The biochemical nature of induced resistance is still shrouded in obscurity. It is an interesting and important phenomenon in both virus and bacterial diseases. A significant discovery by Loebenstein showed that as a consequence of viral infection, interferon-like substances are produced in the host plant. In infections by animal viruses similar antiviral substances appear in the host plant. Müller (1956) and later Cruickshank (1963) proposed that in the reaction to infections caused by microbes a mechanism leading to the formation of antibody-like materials operates in plants. This recognition led to a demonstration of the substances called phytoalexins which are currently known to be synthesized postinfectionally in the host plant and have a role in the tissue resistance phenomenon. An ever-increasing body of research on this class of compounds has been accumulating during the last few decades (Cruickshank, 1963; Kuc’, 1972, 1995) but more convincing evidence of how phytoalexins play a deciding role in host pathogen interaction leading to resistance is still lacking. BIOCHEMISTRY AND PHYSIOLOGY OF THE DISEASED HOST PLANT

In recent years, much of the earlier symptomatological approach to problems of plant disease has given way to an integrated study of cause and effect. It is now becoming increasingly clear that a sufficient knowledge of plant physiology and biochemistry is essential for interpreting the metabolic changes in plants during pathogenesis. As a matter of fact, biochemical and pathological trends in research started about 45 years ago and were founded on the physiological research of host-parasite relations. These modern trends intend to unravel the physico-biochemical processes of the diseased plant (such as photosynthesis, respiration, nitrogen metabolism, and growth). They aim to determine the true causes of cellproliferation, possibilities for the inhibition of virus multiplication, the mechanism of fungal toxicity,

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the mode of action of toxins; the role of hormones or hormone-like substances in pathogenesis, as well as to elucidate the virus biosynthesis. The importance of extracellular enzymes in the etiology of the disease is emphasized by workers currently interested in this area of investigation. MOLECULAR BIOLOGY OF PATHOGENESIS AND INDUCED SYSTEMIC RESISTANCE

The present era of plant pathology has seen much progress in the molecular biology of pathogenesis and induced systemic resistance (Vidhyasekaran, 1998). Recent advances in molecular plant pathology have paved way for the development of innovative techniques to manage crop diseases. Crop plants are endowed with numerous defence genes which encode various proteins responsible for synthesis and accumulation of defence arsenals in plants. The defence genes are involved in the production of antimicrobial compounds such as phenolics, phytoalexins, pathogenesis-related proteins (PR proteins) and active oxygen species or involved in the reinforcement of the cell wall by accumulating hydroxyproline-rich glycoproteins, callose, lignin and wall phenolics. Many of the proteins produced by defence genes are enzymes but some are not. Some of them show antifungal activity, some show elicitor-releasing activity, some increase the mechanical strength of the host wall and some others are involved in the release of second messengers and systemic signals (molecules). It is now believed that signals are required to activate defence genes which are commonly present in both susceptible and resistant plants. Signal molecules have been detected in fungi (Schaffrath et al., 1995, Vidhyasekaran, 1988a,b, 1993, 1997 and 1998), bacteria (He et al., 1993) and viruses (Culver and Dawson, 1991). These are produced by pathogens as well as non-pathogens and are released by the host enzymes when these pathogens come into contact with plants. The signal transduction system plays a key role in disease resistance. Thus the use of molecular genetic techniques have provided new insights into how plants defend themselves against attack. In turn this new understanding is suggesting novel ways for the control plant diseases. TISSUE CULTURE IN PLANT PATHOLOGY

Tissue culture has had the widest impact on the study of ‘crown-gall’. It has enabled plant pathologists to grow organs, tissues and free cells alive in vitro for almost an indefinite period. In the words of White (1968) “Tissue culture is a method of simplifying organisms in order that they may be handled more effectively”. In the early 1920s an off-shoot of conventional tissue culture and organ culture made a modest appearance and has since established itself as a method of choice in many areas of plant physiology, plant pathology, biological and medical research, biochemistry of drugs, antibiotics, hormones, virus research, etc. BIOTECHNOLOGICAL APPROACH TO DISEASE CONTROL

One of the frontier areas of plant pathology is the biotechnological approach to plant disease control. The genetic modification of organisms and application of modern techniques to study cellular and molecular processes is now expanding our knowledge of interactions between organisms and allowing a greater degree of control over biological processes. Specific genes may now be transferred between plants of different cultivars, lines, and species, from microbes to plants and from plants to microbes

History of Plant Pathology 31

using vectors such as plasmids, to integrate new characters into the plant genome (Issac, 1992). Transgenic plants are the current interest of both plant pathologists and agriculturists throughout the world. ECOLOGICAL STUDIES OF SOIL-BORNE FUNGAL PLANT PATHOGENS

Initially it was thought that knowledge of the life cycle of a plant parasite was sufficient to ensure its speedy control. Pathologists have now realized that a wider outlook is required which embraces the entire dynamics of microbial ecology, and that knowledge of life cycles is only a prerequisite to the proper understanding of a soil-borne fungus pathogen in relation to its environment. The surroundings of an organism in the soil are biologically complex and the interactions between the organisms themselves produces a balance. The most important worker in the field of root disease investigations was Dr. SD Garrett. To paraphrase Garrett (1970), his subject has opened for him, and for others too, “a window upon a world wider than that of the diseased plant”. This world is that of the living soil which is the source of all terrestrial plant life and therefore of direct concern to every human being and of interest to every biologist. Inside the soil, many kinds of living organisms are interacting continuously with one another and with the root systems of green plants. Thus, diseases of the root system cannot be studied in isolation as a simple interaction between the root system and the pathogen; hence, the root-disease investigator should be concerned with everything in the soil such as changes in the soil texture, temperature, moisture, pH, and other biological effects. BACTERIOLOGY

Thomas J Burrill (1839–1916) of the University of Illinois, USA, was the first to describe a bacterial disease of plants in 1878 and he showed that the fireblight of apples and pears was caused by the bacterium known as Erwinia amylovora. Several other plant diseases were described shortly afterwards. Wakker (1883) showed that the yellow disease of hyacinth was caused by a bacterium. Savastano (1887) worked on the olive knot disease and EF Smith worked on the bacterial wilt of cucurbits. Smith is regarded as an outstanding man who used the best available methods and standards in the study of bacterial diseases of plants and in establishing modern bacteriological trends in plant pathology. Smith’s name is still remembered as he resolved the controversy with the German bacteriologist, Alfred Fisher (1897,1899), who did not think that bacteria were the primary cause of disease in plants. The subject of bacteriology has now developed considerably and has its own techniques and specialists. Landmarks in the development of phytobacteriology have been discussed in 1984 by Starr and emerging problems in plant bacteriology have been critically discussed by Verma (1992). VIROLOGY

The German born Adolph Mayer (1886), worked in Holland on a serious disease of tobacco (tobacco mosaic) and this work can be said to be the alpha of virology. He showed that the juice from infected plants if applied to healthy plants, could reproduce the disease. In the same way EF Smith (1891), showed that the peach yellows was a contagious disease and could be bud-transmitted. In 1862, Dimitri

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Ivanowski a Russian, showed that the causal agent of tobacco mosaic could even go through a chamberland filter that withholds bacteria. It was Beijerinck, a distiguished Dutch microbiologist (1898) and Professor of Microbiology at the Technical University of Delft, Holland, who demonstrated that it could diffuse through an agar agar membrane and concluded that the tobacco mosaic was caused by a non-corpuscular contagium vivum fluidum which he called virus. In 1935, the first major contribution to the nature of viruses was made when Stanley obtained a crystalline protein which he thought to be gamma globuline. It was in 1938 that Bawden and Pirie discovered the real nature of the tobacco mosaic virus. In 1956, Gierrer and Schramm (1956) proved that the nucleic acid fraction of the virus was actually the infectious agent. Within the attacked cell, the virus somehow mobilizes nitrogenous and other compounds of the plant and replicates itself. The relationship between an insect and a virus was discovered by a Japanese farmer, Hashimoto, who worked in 1894 with the rice dwarf disease and the leaf hopper Nephotettix apicalis var. cinticeps. This information was however not available to the western world for quite some time (Smith, 1974). About 1907, three American workers—Ball, Adams and Shaw–proved the connection between the curly top of sugar beet and the leaf hopper Eutettix tenella. During the last few decades, much more information of the relationship of plant viruses with their vectors has accumulated. It is now clear that in addition to the insects which transmit plant viruses, other organisms such as mites, nematodes, worms, and fungal spores have all been associated as vectors. Other recent advances in the study of plant viruses include an increased knowledge of incomplete viruses and the discovery of satellite viruses (Kassanis, 1966) and ‘viroids’ (Diener and Raymer, 1967). Potato spindle tuber virus (Diener and Raymer, 1967) and the rice dwarf virus (Hiura et al., 1966) are shown to contain double stranded RNA. These are termed viroids. Viroids differ from viruses in the absence of a dormant phase (virions) and by genomes that are much smaller than those of viruses. The algal viruses which form a very distinct group of DNA viruses are another important and interesting field of research. The rod-shaped tobacco mosaic virus was first reconstituted from its protein and RNA components (Fraenkel-Conrat and Williams, 1955). Since then several spherical viruses have been reconstituted. “The tide of molecular biology has now reached the shores of plant virology” says Dr. Kenneth M Smith; and more precise knowledge of the ultrastructure of viruses has been obtained by electron microscopy and X-ray diffraction, techniques. A photographic rotation technique has been developed to increase the details of high resolution electron micrographs. The mechanism of replication of the viral nucleic acid and other constituents within the cells is being looked into critically, and in the case of the tobacco mosaic virus, the work of KK Reddi (1964) stands out prominently. By modifying the purine bases of RNA, Mundry (1959) succeeded in inducing TMV mutations; thus, he was the first to elucidate the biochemical mechanism of mutation. MYCOPLASMA

Before 1967, only fungi, bacteria, nematodes, and viruses were considered the main incitants of plant diseases. However, as early as 1898, Nocard and Roux had discovered another agent of diseases caused in animals which was similar to viruses in size but which could be cultured on artificial media. This group was known as mycoplasma. Mycoplasma mycoides, the causal agent of infectious pneumonia, had been cultured by that time. In 1967, Japanese scientists found that mycoplasma-like organisms could be responsible for most of the disease of the yellows type which were earlier thought to be caused by viruses in plants.

History of Plant Pathology 33

Mycoplasma-like organisms found in certain leaf hopper-transmitted diseases are morphologically similar to animal mycoplasmata, but Ploaie and Maramorosch (1969) were unable to grow the leaf hopper organisms in culture. The cells of mycoplasma lack a cell wall and hence are pleomorphic. They do have a cell membrane (10 nm unit membrane). They are very small in size (from 100 nm to 1 mm in diameter) and because of the lack of cell walls, can easily pass through bacteria-proof filters. They contain ribosomes and thin-stranded DNA and are sensitive to antibiotics of the tetracycline group. The reader may refer to an excellent review, Mycoplasmas as Plant Pathogens: Perspectives and Principles by Hampton (1972). There are some other reviews, by Ghosh and Raychaudhuri (1972), Whitecomb and Davis (1970) and Paul Khurana et al. (1988). Some of the important plant pathologists of the present era are GA Zentmyer of the University of California. Riverside, USA (Phytophthora diseases), CE Yarwood of the University of California, Berkeley, USA (powdery mildews), David Gottlieb (antibiotics), RS Ludwig of the Canada Department of Agriculture (toxins and plant diseases), NE Borlaug (composite varieties), JW Eckert (chemical control of post harvest diseases), K Maramorosch of the Waksman Institute of Microbiology, Rutgers University, New Brunswick, Canada (viruses and mycoplasmas), CS Holton of the US Department of Agriculture (smut fungi), GC Kent (cereal pathology), T Mizukami and S Wakimoto (epidemiology and control of leaf blight of rice), Vander Plank of Plant Protection Research Institute, Pretoria, South Africa (plant disease epidemics and control and principles of plant infection – theoretical aspects mainly), SD Garrett of Cambridge University, England (soil-borne root diseases and their control), HS Sisler of the University Maryland, USA (fungicides), NF Robertson of the University of Hull, England (growth processes in fungi), CJ Hickman of the University of Western Ontario, Canada (biology of Phytophthora zoospores), ME Gallegly of West Virginia University, Morgantown, USA (genetics of host pathogen interaction), Ralph Baker of Colorado State Univesity, Fort Collins, USA (mechanism of biological control of soil-borne pathogens), RN Goodman of the University of Missouri, USA (bacterial pathogens and physiology and biochemistry of plant diseases), Charles E Bracker of the University of California, Davis, USA (ultrastructure of fungi), RP Scheffer of Michigan State University, East Lansing, USA (host specific toxins), John L Lockwood of Michigan State University, East Lansing, USA (soil fungistasis), PE Waggoner, Director Connecticut Agriculture Research Station, New Haven (microclimate and plant disease), JC Walker of the University of Wisconsin (bacterial diseases and physiological plant pathology), DF Bateman of Cornell University, USA (Enzymes and tissue degradation), JM Daly of the University of Nebraska, Lincoln, USA (the physiology of diseased plant), H Wheeler of the University of Kentucky, USA (permeability alterations in diseased plants), I Uritani of Nagoya University, Japan (protein metabolism), GF Pegg of Wye College, University of London (growth regulatory metabolism of the diseased plant), J.A Kuc’ of the University of Kentucky, USA (phytoalexins), AH Ellingboe of Michigan State University, USA (genetics of host parasite interaction), R Heitefuss of the Institute fur Pflanzenpathologie und Pflanzenschutz der Universitat, Gottingen, Germany (nucleic acid metabolism of the diseased plant), RKS Wood of Imperial College, London (toxins and enzymes), F Grossman of the University of Hohenheim, Germany (systematic fungicides), IAM Cruickshank of the Division of Plant Industry, CSIRO, Canberra, Australia (phytoalexins), Michael Shaw of the Department of Biology, University of Vancouver, Canada (host, parasite relationships in rusts), G Ovens of the Boyce Thompson Institute for Plant Research, New York, USA (chemistry and physiology of fungicidal action) , J Dekker of the Agricultural University, Binnenhaven, Netherlands (acquired resistance to fungicides), GA Strobel of Montana State University, Bozeman, USA (toxins), S Akai of Kyoto University, Kyoto, Japan (host parasite relationships) etc.

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HISTORY OF THE DEVELOPMENT OF PLANT PATHOLOGY IN INDIA

Bagchee (1939) was the first to review the work on Indian forest mycology and plant pathology in India. Das Gupta (1958) gave the history of plant pathology and mycology in India till that period. The history of plant pathology in India has been reviewed by Raychaudhuri et al. (1972) and Raychaudhuri (1991). Reminiscences and observations on the growth of plant pathology in India were given by Saksena (1977). Mehta (1963) in his presidential address at New Delhi had discussed plant pathology in India— past, present and prospects. The role of plant pathology in India in crop production has been critically discussed by Raychaudhury (1968). Grover (1975) had critically discussed plant pathology in India-an introspection and prospects. Plant pathology, past, present and future in India has been discussed by Mehrotra and Aggarwal (1988). Nagarajan (2000) has recently discussed “Plant Pathology and Indian Agriculture—Past, Present and Future”. Patel and Kulkarni (1958) had reviewed the work of bacterial plant pathology in India in 1958. Phytobacteriology in India-past, present and future prospects were reviewed by Chand (1989). Verma (1992) has analysed the emerging problems in plant bacteriology. Chenulu (1984) has discussed, in his presidential address to the members of the Indian Phytopathological Society the status of plant virology in India, while Reddy (1990) discussed the development of virology in developing countries of the world. Some of the important events in the development of plant of pathology in India are as follows : 1. Barclay (1886) Uredinales of Simla. 2. Publications of two journals “Memoirs of the Department of Agriculture” and “Agriculture Journal of India”. 3. Appointment of EJ Butler in 1901 as Cryptogamic Botanist to the Government of India later transferred to the then Imperial Agricultural Research Institute established in Pusa in 1905 as the first Imperial Mycologist. Butlers work on Phytophthora blight of potato from Assam, Bengal and Eastern Himalyas and Nilgiris in 1903 and fusarial wilt of pigeonpea caused by a new species of Fusarium in 1906 and 1910 were pioneering contributions to phytopathology in India. Along with Hayman he published a note in 1906 in the Memoirs of the Department of Agriculture on the wheat rust problem. Elsewhere with HP Sydow, he commenced a series of articles which ultimately led to the Cryptogamae Indiae Orientalis (National herbarium) in 1905 and the publication of Fungi of India with G B Bisby in 1931. This is being constantly updated by Vasudeva (1960, 1962), Bilgrami et al. (1979, 1981, 1991), Tandon and Chandra (1964), Sarbhoy et al. (1974, 1994) and Tilak and Ramchandran (1970). His book ‘Fungi and Diseases in Plants’ became a classic for Indian phytopathologists. 4. Among Butler’s contemporaries, F J F Shaw’s contribution in 1912 along with S L Ajrekar on the genus Rhizoctonia with special reference to its morphology and parasitism was one of the landmarks in plant pathological research in India (Raychaudhuri, 1991). The following are important plant pathologists of past and present era in India. D D Cunninham, (1889) identified the causal organism of red rust of tea in Assam caused by Cephaleuros virescens. W McRae, (bud rot of palm) made a detailed study of Phytophthora diseases of rubber in South India. S Sundaraman, (1922) reported Pyricularia oryzae on rice for the first time from India. B B Mundkur (1948) laid the foundations of the Indian Phytopathological Society and later published a book in 1949 entitled’ Fungi and Plant Disease. Other significant contributions of Mundkur include the monograph on Ustilaginales of India and the Supplements to Fungi of India and Genera of

History of Plant Pathology 35

Rusts in collaboration with Dr. M J Thirumalachar. K C Mehta (1940) published a monograph entitled Further studies on cereal rusts in India and did pioneering work on the wheat rust problem in our country. R Prasad contributed to cereal rusts, linseed rusts, alternaria blight of wheat caused by A. triticina. RH Dastur studied the genus Phytophthora, cotton wilt, foot rot of betel, ripe rot of bananas, stem breaking of cotton, pink disease of citrus, anthracnose and Nematospora on cotton, and black thread of Hevea. Some of the other important plant pathologists of India, past and present, are as follows: SN Das Gupta (black tip of mango), BL Chona (sugarcane diseases), RS Vasudeva (root rot of cotton, and viral diseases of potato), JC Luthra and A Sattar (gram blight and wilt, solar heat treatment against loose smut of wheat), BN Uppal (cereal smuts, downy mildews of millets and maize), M Mitra (Helminthosporium diseases, discovered the new bunt of wheat caused by Neovossia indica), KPV Menon (coconut diseases), S Y Padmanabhan (diseases of rice), SB Chattopadhyay (diseases of rice and revised the book entitled Fungi and Plant Disease originally written by BB Mundkur), K Bagchee (forest pathology), B K Bakshi (forest pathology), MJ Thirumalachar (monograph Ustilaginales of India, Uredinales of the World, Cercosporae, Physoderma, Cephalosporium etc. At Hindustan Antibiotics Ltd., Pimpri, Pune, he discovered a number of antibiotics such as Haymycin, Aureofungin, Antibiotic 226, Antiamoebin etc.), MJ Narasimhan (Phytophthora diseases, sandal-spike, downy mildews of grasses and cereals , heteroecism in rusts, entomogenous fungi and biocontrol of insect pests). TS Sadasivan (established the school of Mycology and Plant Pathology at Madras University, Madras, (now Chennai) and it was here that the foundations of research on soil-borne pathogens was laid and the physiology of hydromycotic wilting, specially cotton wilt, was investigated and the control of wilt diseases by trace element amendment was tried), R Kalyansundaram (wilt diseases, fusaric acid as a wilt toxin), CS Venkata Ram (tea diseases), R N Tandon (physiology of fungal pathogens and post harvest diseases of fruits and vegetables), V Agnihothrudu (tea diseases), R P Asthana (control of smuts of millets), C V Subramanian (wilt of cotton), N Prasad (fusarium diseases and Phytophthora blight), D Suryanayana (downy mildew diseases), T S Ramakrishnan (contributions on genera Pythium, Phytophthora, Colletotrichum and the rusts and his monograph on Diseases of millets published by the ICAR) MK Patel (bacterial diseases, advocated a new family Phytobacteriaceae to include all phytopathogenic bacteria), A P Misra (Helminthosporium diseases), S P Raychaudhuri (viral diseases of crop plants), PR Mehta (diseases of cereals and millets and plant protection), H K Saksena (Rhizoctonia diseases and gram rust), MK Hingorani (bacterial diseases), L M Joshi (wheat rusts), M N Kamat (ascomycologist, control of plant diseases and author of “Introductory Plant Pathology)”, R K Saksena (cytology of Pythiaceae and ecology of soil fungi), SB Saksena (mycologist of eminence, discovered Saksenea which is a human pathogen, root diseases and biocontrol), DN Srivastava (bacterial blight of paddy and diseases of fruits and vegetables), S Sinha (rusts), YL Nene (khaira disease of rice and authored Fungicides in Plant Disease Control), MM Payak (maize diseases), S B Mathur (seed pathology), VP Bhide (bacterial diseases), SP Kapoor (viral diseases), Pushkarnath (potato diseases), G Rangaswami (bacterial diseases, authored Diseases of Crop Plants in India, Bacterial Plant Diseases in India), A Mahadevan (physiological plant pathology), Y S Kulkarni (bacterial diseases of plants), HC Arya (downy mildew of pearl millet, axenic culturing of Sclerospora graminicola), BP Chakravarty (bacterial diseases), D Ganguly (Helminthosporium disease of rice), BB Nagaich (viral diseases), RP Purkayastha (phytoalexins and induced resistance), VP Agnihotri (Pythium, sugarcane diseases), V Muniyappa (viral diseases), Anupam Varma (viral diseases), TK Nariani (viral diseases), JP Verma (bacterial diseases, specially bacterial blight of cotton), RP Thakur

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(diseases of millets), HS Shetty (induced resistance in pearl millet), Chtreshwar Sen (biocontrol of plant diseases), Hari Om Agrawal (viral diseases), S Mukhopadhyay (viral diseases), AN Mukhopadhyay (biocontrol of plant pathogens), KS Bhargava (viral diseases), RK Hegde (Phytophthora diseases of arecanut, coconut and Piper nigrum), Kishan Singh (sugarcane diseases), Abrar Khan (nematode diseases of vegetables and control), MV Pagvi (Indian Ustilaginales, Physoderma, Tapharina maculans and rusts), VR Reddy (Viral diseases), KS Bilgrami (mycotoxins), JN Chand (bacterial diseases), HS Sohi (diseases of ornamentals and fruit crops and mushroom culture), SK Saxena (nematode diseases), KM Safeeulla (diseases of pearl millet), RS Singh (organic amendments and control of soil borne pathogens, author of the books, Plant Diseases, and Diseases of Vegetable Crops), MV Naydu (viral and bacterial diseases), CD Mayee (groundnut diseases, sunflower diseases and plant disease forecasting), Dharam Vir (fungicides), MS Chatrath (fungicides), SJ Singh, (viral diseases), RJ Singh, (rice diseases), YS Ahlawat (viral diseases), KC Alexander (sugarcane diseases), RS Dwivedi (soil mycology and root diseases), MW Khan (powdery mildews), S Maiti (betelvine diseases), SM Paul Khurana (viral diseases of potato), GS Shekhawat (bacterial diseases of potato), Uma Kant (insect galls and other teratomas), A Narain (minor diseases of rice and toxins), R Jeyarajan (biocontrol and viral diseases), VN Pathak (post-harvest diseases of fruits and vegetables), AK Sarbhoy (fungal taxonomy and Rhizoctonia diseases), B L Jalali (mycorrhizae and integrated disease management), DV Singh (bunts of wheat), JS Grewal (diseases of pulse crops), KG Mukerji (mycorrhiza and biocontrol), DJ Bagyaraj (mycorrhiza and biocontrol), C Manoharachary (post-harvest diseases and mycorrhiza), Sudhir Chandra (post harvest pathology, mycorrhiza and biocontrol), JS Chauhan (diseases of oil seed crops), YR Sarma (diseases of plantation crops), PK Koshy (nematode diseases in plantation crops), Bharat Rai (wilt diseases of pulse crops), BP Singh (viral diseases), SS Sokhi (vegetable diseases), VK Gupta (diseases of apples), S Nagarajan (wheat diseases), MP Srivastava (post-harvest pathology), HN Verma (viral diseases), RK Grover (fungicides), P Ramarao (Soil-borne diseases), P Vidhyasekaran (physiological plant pathology), RA Singh (rice diseases), JS Jhooty (powdery mildews), BM Singh (plant disease management), UP Singh (diseases of pulse crops), V Muniyappa (viral diseases) SS Chahal (pearl millet diseases), SS Aujla (wheat diseases), TN Lakhanpal (tree diseases), BP Singh (potato diseases), RD Parasher (bacterial diseases), Gopal Swarup (nematology), Amerika Singh (wheat diseases), K Jindal (bacterial diseases), JL Kaul (diseases of fruits), BN Johri (mycology, mycorrhiza), WM Khan (powdery mildew), Satyavir (sugarcane diseases), YS Ahlawat (viral diseases), D Kumar (mycorrhiza), SB Sharma (nematode diseases), D Lalitha Kumari (fungal biotechnology of antagonistic organism), MN Khare (pulse diseases), B Chandra Mouli (tea diseases), V Mariappan (rice diseases), VR Mali (viral diseases), BL Chopra (diseases of cotton), SP Kapoor (viral diseases), KM Vyas (soilbone diseases), DK Arora (soil-borne diseases and biocontrol). Trends in Teaching and Training in Plant Pathology in India

Plant pathology in India began with the establishment in 1905 of the Agricultural Research Institute at Pusa, Bihar now called the Indian Agricultural Research Institute and locate in Delhi and the appointment of E.J. Butler as the first Indian mycologist. The first Indian Universities that were established in 1857 at Calcutta, Madras and Bombay emphasized the taxonomy of fungi. Plant pathology as a university science became established at Lucknow, Allahabad and Madras Universities (founded in 1921,

History of Plant Pathology 37

1887, and 1857, respectively), only in the 1930s. There are now 29 agricultural universities and 4 deemed universities in our country. Work on plant pathology is carried out at various agricultural research institutes in the country, agricultural universities, and botany departments of traditional universities. The need to bring about a rapid increase in food production in the post-independence period necessitated re-examination of the prevailing system of agricultural research and education. Under the existing system agricultural research and education was managed by the state departments of agriculture. For purposes of examination and award of degrees, the colleges of agriculture were affiliated to conventional or traditional universities which also controlled the syllabi and courses of reading. It was felt that these institutions and procedures oriented to the needs of the past could not meet the requirements of the present day. It was around this time that the agricultural universities came into existence, which could integrate research and education extension with education. The Indian Agricultural Research Institute, New Delhi, was the first to adopt the new system when the present post graduate school was set up in 1958. It was in Uttar Pradesh that the first agricultural university was set up in 1960. The establishment of the agricultural universities was an important landmark in the development of agricultural education and research in our country and which revolutionized the process of education of agricultural sciences in our country including the subject of plant pathology. In the beginning, for a Master’s degree in plant pathology, the course work-content emphasized mycology and major diseases caused by fungi, bacteria and viruses. Now in addition to these courses, well-planned courses are offered in specialized areas such as epidemiology, forecasting and loss assessment, physiology and biochemistry of plant infection, soil plant disease relationships, phytopathological techniques, seed pathology, nematology, chemistry of fungicides. A student doing his Masters programme or Ph.D. in plant pathology is now up to date not only in his area of specialization but also in supporting minor subjects. A young plant pathologist is now better equipped to deal with problems in the field. In these agricultural universities extension in education became an important part which brought farmers and scientists in the agricultural universities closer and made it possible to transmit practical knowledge to the farmers which led to awareness among the public of the role of plant pathological science in ameliorating problems related its plant diseases. Extension is a very important part of pathological science. It puts the scientific worker to test. With feedback from the community, the plant pathologists learns his mistakes and can rectify them. Extension plant pathology in Indian agriculture was critically evaluated by Upadhyay and Bharat Rai (1983). Extension activities in plant pathology are better organized today than they were 40 years ago. The whole concept of extension has undergone a change. Many agricultural universities hold farmers fairs and there is no substitute for developing closer links between farmers and plant pathologists. Extension pathologists are doing very meaningful and valuable work. Trends in research and teaching in plant pathology in India have been discussed by Saksena (1977). From 1977 there has been a remarkable shift in the areas of research in plant pathology. In the beginning the emphasis was on taxonomic aspects of pathology. Nene (1987) has critically suggested various facets of plant pathology in India and plant pathology in 2000 AD. PLANT DISEASE CLINICS

According to Barnes (1994) the evolution of the plant disease clinic arose from a perceived need for an organized systematic, professional effort to assist in the identification of plant disease problems. In the

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United States of America for many years, most states operated a plant disease clinic through their departments of plant pathology (Agrios, 1997). Growers, country extension agents and homeowners would send diseased plants, soil from areas infested with diseased plants, and sometimes insects, to the plant disease clinic and the pathogen or insect would be identified and some suitable control measures suggested by extension plant pathologists. Barnes (1994) has reviewed the role of plant disease clinics in disease diagnosis and education in the North American perspective. The plant disease clinic frequently represents “the tie that binds”, bridging various aspects of basic and applied features of plant pathology into a meaningful, functional, responsive, accountable component of agriculture (Barnes, 1994). Earlier plant disease clinics were manned mainly by students of MSc of Agriculture or Ph.D. students who would identify the diseases with the help of some basic facilities such as dissecting microscope, culture dishes, test tubes, surface sterilizing agents and culture media. Viral disease identification was made by symptomatology but after 1970, greater attention was paid to these plant disease clinics which have now facilities for viral disease diagnosis, host range tests, serological tests, cell inclusion identification, electron microscopy, and dot-blot assays of radioactive or colour production DNA probes. In the most agriculturally-advanced countries such as the USA modern computers with data bases, videodisc facilities, and E-mail for transmitting of the results of the diagnosis and recommendations for the management of the disease, now exist. In our country also the plant disease clinics have now come up in almost all the agricultural universities when a modest beginning was made by the Punjab Agricultural University, Ludhiana, some time back. Some private plant disease clinics have come up in the United States of America. We do not have such a system so far in our country. It may be emphasized that the plant disease clinics have a significant contribution to make in the education of students of plant pathology and perform a critical function by providing diagnostic capabilities that are essentially unavailable elsewhere as well as unique and varied educational opportunities. These have an important role in the transfer of technology as far disease management is concerned, from lab to land. According to Agrios (1991, 1997), plant pathology, unlike its sister sciences of medicine and veterinary sciences, has yet to develop a teaching and training programme that will produce practitioners, physicians and veterinaries who can be professional enough to identify all types of causes of disease and injury to plants and of making recommendations to control or manage these diseases. In some of our agricultural universities plant disease clinics have been established based on the concept that there can be doctors in plant pathology similar to human or animal pathology. Education and Information Technology in Plant Pathology

According to Agrios (1997) numerous advances have been made in developing, collecting, organizing and transmitting information for use by researchrs, extension pathologists, teachers, and students of plants pathology. Ariena and Bruggen (1991) have discussed the importance of the computer-based diagnosis of plant diseases. Computers have provided the capacity to simulate epidemics and their modelling. Some of the facilities available now are electronic mail (E-mail), videodiscs, and use of videotapes and access to satellites to uplink and downlink television. In the western countries of the world computers are being used more and more for database management and are being made use of in

History of Plant Pathology 39

diagnostic clinics. However, in our country we have to develop these areas more so that computer based diagnosis of plant diseases is available not only for diagnosing a diseases but also suggesting a suitable control strategy.

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Koch, R. (1876), “Die Aetiologie der Milzbrand-Krankheit, begrudet auf die Entwick lungsgeschichte des Bacillus anthracis”, Beiter. Biol. Pflanz., 2: 277–310. Kuc, J. (1963), “Role of phenolic compounds in disease resistance”, in: Perspectives of Biochemical Plant Pathology, S. Rich (Ed.), Coon. Agr. Exp. Sta. (New Haven). Bull., 663: 20–25. Kuc, J. (1972), “Phytoalexins”, Ann.Rev.Phytopath., 10: 204–232. Kuc, J. (1995), “Phytoalexins, stress metabolism, and disease resistance in plants”, Ann. Rev. Phytopath., 33: 275–297. Kühn, J.G. (1858), Die Krankheiten der kulturgewächse, ihre ursachen und ihre Verhütung, G. Bosselmann, Berlin, p. 312. Large, E.C. (1940), The Advance of the Fungi, Henry Holt, New York, p. 488. Large, E.C. (1958), The Advance of the Fungi, Jonathan Cape, London, p. 488. Linnaeus, C. (1753), “Species Plantarum”, Impensis L Salvii, Holmiae, 2 Vols. Markham, R. (1977), “Landmarks in plant virology: Genesis of Concepts”, Ann. Rev. Phytopath., 15: 17–39. Marsh, R.W (Ed.) (1972), Systemic Fungicides, Longman, London, p. 321. Mayer, A. (1886), “Ueber die Mosaikkrankheit des Tabaks”, Landwirtsch. Vers. Sta., 32: 451–467. (English translation by J. Johnson, in: Phytopath. Classics, No. 7, 1942). McCarthy (1978), A History of our Own Times, Harper and Brothers, New York, 1: Chapter 15. Mehrotra, R.S. and Ashok Aggarwal (1994), “Plant Diseases caused by Fungi”, Chapter 14, in: History of Botany, B.M. Johri (Ed.), IBH and Oxford Publications Co., New Delhi, 255–294. Mehta, P.R. (1963), “Plant Pathology in India—Past, present and prospects”, Indian Phytopath., 16: 1–7. Micheli, P.A. (1729), Nova Plantarum Genera, Typsis B. Paperinii, Florentiae, p. 234. Miura, K.I., I. Kimura and N. Suzuki (1966), “Double-stranded ribonucleic acid from rice dwarf virus”, Virology, 28: 571–579. Müller, K.O. (1956), Einige einfache Versuche zum Nachweis von Phytoalexinen”, Phytopath. Z., 27: 237–254. Mundry, K.W., (1959), “The effect of nitrous acid on tobacco mosaic virus: mutation not selection”, Virology, 9: 722–726. Nagarajan, S. (2000), “Plant Pathology and Indian Agriculture—Past, Present and Future”, Indian Phytopath., 53(2): 121–128. Needham, T. (1744), “Some microscopical observations on the farina of the red lily and of worms discovered in smutty corn”, Phil. Trans. Roy. Soc., London, 42: 641–643. Nene, Y.L. (1987), “Indian Plant Pathology – 2000 A.D.”, Indian Phytopath., 40: 11–13. Newton, M., T. Johnson and A.M. Brown (1930), “A preliminary study on the hybridization of physiologic forms of Puccinia graminis tritici”, Sci. Agr. , 10: 721–731. Nolla, J.A.B., V. Manuel and Fernandez Valiela (1976), “Contribution to the history of plant pathology in South America, Central America and Mexico”, Ann. Rev. Phytopath., 14: 11–51. Orlob, G.W. (1971), “History of plant pathology in the middle ages”, Ann. Rev. Phytopath., 9: 7–21. Owen, R.G. (1963), “Chemistry and physiology of fungicidal action”, Ann. Rev. Phytopath., 1: 77–100. Parris, G.K. (1968), A Chronology of Plant Pathology, Johnson and Sons, Starkville, Mississippi. Patel, M.K. and Y.S. Kulkarni (1953), “A review of bacterial plant disease investigation in India”, Indian Phytopath., 6: 131–140. Person, C. and G. Sidhu (1971), Genetics of host-parasite interrelationship, Mutation Breeding for Disease Resistance, IAEA, Vienna, pp. 31–38. Persoon, D.C.H. (1801), Synopsis methodica fungorum sistens enumerationem omnium nucusque detectarum specierum, cum previbus descriplionibus nec non synonymis et observationibus selectis, 2 vols., Gottingen. Ploaie, P. and K. Maramorosch (1969), “Electron microscopic demonstration of particles resembling mycoplasma or psittacosis-lymphogranuloma-trachoma group in plants infects with European yellow-type diseases”, Phytopath., 59: 536.

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Prevost, B. (1807), Memoire sur la cause immediate de la carie ou charbon des bles et de plusieurs autres maladies des plantes’ et sur les preservatifs de la carie, Bernard, Paris, p. 80. (English translation by G.W. Keitt, in: Phytopath. Classics, No. 6, 1939). Raski, D.J. (1959), “Historical highlights of nematology”, in: Pl. Pathol.- Problems and Progress, 1908-1958, Madison, University of Wisconsin Press, pp. 384–394. Raychaudhuri, S.P. (1967), “Development of mycological and plant pathological researches, education and extension work in India”, Rev. Appl. Mycol., 46: 577–583. Raychaudhuri, S.P. (1991), “Development of Mycological and Plant Pathological Work in India”, Indian J. Mycol. Plant Pathol., 21: 14–26. Raychaudhuri, S.P., J.P. Verma, T.K. Nariani and Bineeta Sen (1972), “The history of plant pathology in India”, Ann. Rev. Phytopath., 10: 21–36. Reddi, K.K. (1964), “Studies on the formation of tobacco mosaic virus ribonucleic acid- V: Presence of tobacco mosaic virus in the nucleus of the host cell”, Proc. Nat. Acad. Sci., 52: 397–401. Reddy, D.V.R. (1990), “Plant virology in developing countries”, Indian Phytopath., 43: 331–339. Rich, S. (1960), “Fungicidal chemistry”, in: Pl. Pathol. – An Adv. Tre., J.G. Horsfall and A.E. Dimond (Eds.), Academic Press, New York, 2: 553–602. Saksena, H.K. (1977), “Reminiscences and observations on the growth of Plant Pathology in India,” Indian Phytopath., 30(2): 165–175. Savastano, L. (1887), “Tuberculosi iperplasie e tumori dell’ olivo I. II. Memoria”, Filli Ferrante, Napoli, p. 131. Schaffrath, U., H. Scheinpflug and H. Reisner, (1995), “An elicitor from Pyricularia oryzae induces resistance response in rice: Isolation, characterization and physiological properties”, Physiol. Mol. Plant Pathol, 46: 293–307. Sequeria, L.(2000), “Legacy for the millennium: A century of progress in Plant Pathology”, Ann. Rev. Phytopath., 38: 1–18. Sequeria, L.(1986), “Facing the issues of Plant Pathology”, Phytopath., 17: 24–28. Sisler, H.D. (1969), “Effect of fungicides on protein and nucleic acid synthesis”, Ann. Rev. Phytopath., 7: 311– 330 Sisler, H.D. and C.E. Cox (1960), “Physiology of fungitoxicity”, in: Pl. Pathol. – An Adv. Tre., J.G. Horsfall and A.E. Dimond (Eds.), Academic Press, New York, 2: 507–544. Smith, E.F. (1891), “Additional evidence on the communicability of peach yellow and peach rosette”, U S Dept. Agr. Div. Vegetable Pathol. Bull., 1: 65. Smith K.M. (1974), Plant Viruses , Second edition, Chapman and Hall, London, p. 211. Sorauer, P. (1909), Hanbuch der Pflazenkrankheiten, third edition, P. Parey, Berlin, 1: 891. [English translation by Frances Dorrance (1922), Record Press, Wilkes Barre, Pennsylvania.] Stakman, E.C., M.N. Levine and R.U. Cotter (1930), “Origin of physiologic forms of Puccinia graminis through hybridization and mutation”, Ann. Sci. Bot., 10: 707–720. Stanley , W.M. (1935), “Isolation of a crystalline protein possessing the properties of tobacco mosaic virus”, Science, 81: 644–645. Starr, M.P. (1984), “Landmarks in the development of phytobacteriology”, Ann. Rev. Phytopathol, 22: 169–188. Ten Houten, T.G. (1974), “Plant pathology: changing agricultural methods and human society”, Ann. Rev. Phytopath., 12: 1–3. Thurston, H.D. (1973), “Threatening plant diseases”, Ann. Rev. Phytopath., 11: 27–53. Tillet, M. (1755), Dissertation on the “Cause of the corruption and smutting of the kernels of wheat in the head”, translated from the French by H.B. Humphrey in Phytopath. Classics, No. 5, American Phytopathological Society, Ithaca, New York, p. 189. Tisdale, W.H. and I. Williams (1934), Disinfectant, US Patent No. 1, 972, 961. Tulasne, L.R. and C. Tulasne (1847), “Memoire sur les ustilaginees comparees aux Uredinees”, Ann. Sci. Nat. , 7(3): 12–127. Ubrizsy, G. (1964) , “Modern trends in plant pathological research”, in: Host-Parasite Relation in Plant Pathology, Z. Kiraly , and G. Ubrizsy (Eds.), Rubber Research Institute for Plant Protection, Budapest, Hungary, pp. 1–7.

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Unger, F. (1833), Die Exantheme der Pflanzen, C. Gerold, Vienna, p. 422. Upadhyay, R.S. and Bharat Rai (1983), “Everman’s Science”, 12–16. Uritani, I. (1963), “The biochemical basis of disease resistance induced by infection”, in: Perspectives of Biochemical Plant Pathology, Saul Rich (Ed.), The Connecticut Agricultural Experiment Station, pp. 4– 20. Vander Plank, J.E. (1968) Disease Resistance in Plants, Academic Press, New York, p. 206. Verma, J.P. (1992), “Emerging Problems in Plant bacteriology”, India Phytopath., 45(2): 159–171. Vidhyasekaran, P. (1988a), “Physiology of disease resistance in plants”, Vol. I, CRC Press, Florida, pp. 149. Vidhyasekaran, P. (1988b), “Physiology of disease resistance in plant”, Vol. II CRC Press, Florida, pp. 127. Vidhyasekaran, P. (1993), “Defence genes for crop disease management”, in: Genetic Engineering, Molecular Biology, and Tissue Culture for Crop Pest Management, P. Vidyasekran (Ed.), Daya Publishing House, Delhi, 17–30. Vidhyasekaran, P. (1997), Fungal Pathogenesis in Plants and Crops. Molecular Biology and Host Defence Mechanisms, Marcel Dekker, New York, pp. 553. Vidhyasekaran, P. (1998), “Molecular biology of pathogenesis and induced systemic resistance,” Indian Phytopath., 5(2): 111–120. Wakker, J.H. (1883), “Vorlaufige Mittheilungen unber Hycinthenkrankheiten”, Botan. Centr. 14: 315–317. Walker J.C. (1975), “Some highlights in plant pathology in the United States”, Ann. Rev. Phytopath., 13: 15–31. Ward, H.M. (1888), “A lily disease”, Ann. Bot. , London. 2: 319–376. Weinhold, A.R.(1988), “Trends in Plant Pathology-significance for the future,” Phytopath., 79: 33–37. Whetzel, H.H. (1918), An Outline of the History of Phytopathology, Saunders, Philadelphia, p. 130. Whitcomb, R.F. and R.E. Davis (1970), “Mycoplasma and phytarboviruses as plant pathogens persistently transmitted by insects”, Ann. Rev. Entomology, 15: 405–464. White, P.R. (1968), “Tissue culture in the service of plant pathology”, Indian Phytopath., Fifth Mundkur Memorial Lecture, 21: 14–22. Woodham-Smith, C. (1962), The Great Hunger (reprinted as a four-square-paperback), Hamish Hamilton, London.

QUESTIONS Mention five important milestones in the development plant pathology. What have been the three great controversies of mycology and plant pathology? Name any three workers who ‘lived ahead of their times”. List five major breakthroughs in phytopathology during the 20th century and the scientists responsible for them. 5. Name five contemporary scientists of plant pathology. 6. Name the person who introduced plant pathology in India. Mention his important contributions. 7. Mention the most important contribution of the following : i. L. Spallanzani ii. A. Leeuwenhoek iii. P.A. Micheli iv. M. Tillet v. B. Prevost vi. A. de Bary vii. T.J. Burrill viii. M.S. Woronin ix. O. Brefeld x. L. Pasteur xi. P.A. Millardet xii. H.M. Ward xiii. J.G. Kühn xiv. W.G. Farlow xv. R.Koch xvi. R. Hartig

1. 2. 3. 4.

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xvii. A. Mayer xviii. D.Ivanowski xix. V. M. Biejerinck xx. E.Gäumann xxi. W.Brown xxii. K.O. Müller xxiii. S.A. Waksman xiv. E.J. Butler xxv. R.H. Biffen xxvi. E.C. Stakman xxvii. J.E. Vander Plank xxviii. J.C. Walker xxix. J.G. Horsfall xxx. S.R. Bose xxxi. T.S. Sadasivan xxxii. K.C. Metha xxxiii. J.F. Dastur xxxiv. M.J. Thirumalachar xxxv. R.N. Tandon xxxvii. B.B. Mundkur xxxvi. T.O. Diener xxxviii. S.P. Raychaudhuri 8. What are the recent trends in phytopathological research and training worldwide?

3 Pathogenesis

PENETRATION AND ENTRY BY PLANT PATHOGENS Contact

Pathogenesis caused by an infectious agent begins as soon as the pathogen comes into contact with any portion of a susceptible plant. Most of the bacterial and fungal pathogens come into contact with their hosts accidently in the form of wind-borne spores or propagules. Some fungi, many bacteria, and most of the viruses are carried to their host plants by insects, nematodes, and other vectors. Motile propagules of fungal pathogens are attracted to root exudates such as the zoospores of many plant pathogens (Hickman and Ho, 1966). Attraction and accumulation of zoospores to the root in the soil just behind the root tip has been demonstrated [Fig. 3.1 (a,b) ] by Mehrotra (1970) and evidence of the attraction of nematodes to the root has been ably summarized by Webster (1969). Interesting research by Kerr and Flentje (1957) demonstrated that the hyphae of the fungus Rhizoctonia solani aggregated on the outside of cellophane bags in contact with the roots indicating that root exudates induce the formation of an infection cushion by the fungus. Recognition between Host and Pathogen

It is still not clear as to how pathogens recognize their hosts and vice versa. Recognition plays a central role in interactions between plants and their pathogens. Pathogens must be able to recognize the presence of the host plant in their environment and often must recognize specific surface features of their host in order to effect successful penetration and infection. Successful pathogens must also be able to recognize and overcome plant defence responses. Since plants are sessile they must in return, have evolved sophisticated mechanisms to detect the multitude of potential pathogens in their environment and to activate diverse defence mechanisms. The mutual signalling between plant host and potential pathogens has been the focus of extensive researches during the last few decades. The emphasis has been on identifying and characterizing the molecules involved in recognition between the host and the pathogen.

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Fig. 3.1

Accumulation of zoospores on root tip: (a) in petridish (b) in soil-encysted zoospores with their germ tubes fluorescing under the fluorescent microscope

Host components acting as signals for activation of pathogens are numerous. These may include fatty acids of the plant host cuticle that activate production of cutinase enzymes which break down cutin of the host galacturonan molecules of pectin which stimulate the production of pectin lyase enzymes by the fungal or bacterial pathogen. Then there are phenolic compounds, such as which stimulate the activation and germination of propagules of some pathogens, and phenolics and sugars released from plant wounds that activate a series of genes in certain pathogens leading to infection. The term elicitor was originally used to refer to molecules and other stimuli that induced the synthesis and accumulation of antimicrobial compounds (phytoalexins) in plant cells, but is now commonly used for molecules that stimulate any plant defence mechanism. Examples of plant defence mechanisms induced by elicitors include the synthesis and accumulation of antimicrobial phytoalexins, the production of glycosyl hydrolases capable of attacking surface polymers of pathogens, the synthesis of proteins that inhibit degradative enzymes produced by pathogens, the production of activated oxygen species

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(oxidative burst), and the modification of plant cell walls by the deposition of callose hydroxy prolinerich glycoproteins and /or lignin. Pathogen components that act as elicitors of recognition by the host plant and subsequent mobilization of plant defences are still poorly known. The induction of these plant defence reactions or otherwise is mediated by an initial recognition process between plant and pathogen, which involves detection of certain unique structural features of incompatible or compatible pathogens by recognition molecules in plants thereby setting off a cascade of biochemical events leading to resistance or susceptible reaction. When the initial signal by the pathogen favours its growth and development, disease may be induced, if the signal suppresses the growth and development of the pathogens disease does not occur. On the other hand, if the initial recognition elicitor received by the host triggers a defence reaction, the growth of the pathogen and its activity is slowed down or will not develop at all. If the elicitor either suppresses or bypasses the defence reactions of the host, disease may develop. Daly (1984) has reviewed the role of recognition in plant disease. Plant-microbe interactions, molecular and genetic perspectives have been brought over in an edited volume by Kosuge and Nester (1984). Microbial elicitors and their receptors in plant have recently been reviewed by Hahn (1996). The molecular mechanism of fungal pathogenicity to plants has been reviewed by Schaffer (1994), while molecular biology of pathogenesis and induced systemic resistance has been reviewed by Vidhyasekaran (1998). Host Penetration and Infection

When a pathogen comes in contract with a host plant, a complex of interacting factors including temperature, moisture, susceptibility of plant tissues, effects of other micro-organisms present, aggressiveness of the pathogen, and so on, determine whether infection will occur or not. Pathogenicity is the property of a micro-organism whereby it becomes a part of the causal complex. Pathogenesis is a process or the chain of events by which disease development takes place. The events of pathogenesis will be discussed under the following heads: 1. Prepenetration 2. Penetration 3. Postpenetration or further colonization of plant by the pathogen PREPENETRATION

In fungal pathogens, this includes spore germination and growth of the resulting tube on the surface of the host plant. Germination is essentially the change from a low metabolic rate to a high metabolic rate and involves a change from near-dormancy to intense activity; for this an energy source is needed such as a carbohydrate or fat reserve in the propagule. Fungal invasion is chiefly by germ tubes or structures derived from them. There are two well-known situations involving hyphae acting in a concerted way to achieve host penetration. Hyphae of Rizoctonia solani often aggregate to form an infection cushion from which multiple penetration occurs by means of appressoria and penetration pegs. This is shown in Fig. 3.2 (a, b). The other example of penetration is the penetration of intact root periderm by the rhizomorphs of Armillaria mellea which is shown in Fig. 3.3. In the ectotrophic infection habit, among the specialized pathogens such as Gaeumannomyces (Ophiobolus) graminis, the fungus progresses

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epiphytically over the root system as a sparse network of dark hyphae known as runner hyphae from which hyaline branches quickly penetrate and infect the underlying cortex [Fig. 3.4 (a, b)]. The entry of bacteria and viruses is achieved in various ways. According to the nature of the spore and the environmental conditions, the germination of fungal spores also occurs in various ways. Sporangia of Chytridiomycetes, Oomycetes and parasitic slime moulds produce zoospores in wet conditions, and germ tubes in dry conditions, while those of some others appear always to germinate by zoospores (e.g. Plasmopara viticola) or always by germ tubes (e.g. Peronospora sp). Teliospores of

Fig. 3.2

(a) Formation of infection cushion by crucifer isolate of R. solani on radish hypocotyl (b) production of lobate appressoria by solanaceous isolate of R. solani on tomato hypocotyl

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Fig. 3.3

Apical region of rhizomorph of Armillaria mellea in longitudinal section: (a) mantle of filamentous hyphae (b) central conducting cells (c) cells of the rind (d) boundary of enveloping mucilaginous layer

Fig. 3.4

Ectotrophic infection of Gaeumannomyces (Ophiobolus) graminis: (a) in surface view (b) in longitudinal section

Uredinales and Ustilaginales normally germinate by producing a promycelium on which basisdiospores, (sporidia) are borne. Budding and fission are characteristics of some yeasts and bacteria, although conidia, ascospores (e.g. Taphrina sp.) and basidiospores (Ustilaginales) may bud under certain condition.

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Some spores germinate easily on release and sometimes even before release, while resting spores have a dormancy period during which they do not normally germinate. There can be chemical and biological factors inhibiting or stimulating germination. The physiology of spore germination will not be discussed here nor the requirements for spore germination (such as moisture, temperature, light, pH, oxygen, carbon dioxide and biological factors). The behaviour of germ tubes growing on a plant surface depends on many factors. In order to procure food, plant pathogens have to establish an intimate connection with their hosts; this means that they have to enter into tissues or send haustoria into surface cells. Entry is mostly affected by the germ tube from spores but can also often be accomplished by mycelial hyphae. The pathogen invades plants through natural openings (stomata, lenticels, nectarthodes, hydathodes), through wounds (natural or due to insects or nematodes), or by direct penetration. While some enter in one way only, others enter in more than one way. In Uredinales, the basidiospores penetrate directly while uredospores generally but not invariably, invade through stomata. Botrytis cinerea may invade directly or through wounds and some fungi which normally grow through stomata may penetrate directly under certain conditions. Gaumann (1950) deals in an exhaustive manner with the penetration and infection processes in various host-parasite combinations. The infection of plants by fungi is reviewed by Eide (1955) and Wood (1967). The cytology of virus infections and virus transport has been discussed by De Zoeten (1977). Physiological and cytological aspects of the bacterial infection process have been reviewed by Goodman (1977). The cytology of penetration and infection by fungi has been reviewed by Aist (1976). ENTRY THROUGH NATURAL OPENINGS

The process by which fungi enter through stoma will be illustrated by the stem rust fungus, Puccinia graminis tritici (Fig. 3.5 a). Urediniospores of the pathogen will germinate in water on a leaf surface in an hour under optimum conditions. The germ tube from the uredospore grows over the leaf and approaches the stoma. The protoplasm of the germ tube accumulates at the tip to leave the older parts empty. On reaching the stoma, the end of the germ tube swells to form an appressorium in the stomatal aperture. The empty germ tube is cut off by the formation of a cross wall. From the appressorium a blade-like wedge grows through the stomatal slits and swells inside to form a substomatal vesicle into which the contents of the appressorium pass, leaving the latter empty. One or more hyphae grow from the vesicle towards the cell lining the substomatal cavity and after making contact, proceed to penetrate the cell wall. After penetration a haustorium is produced in the cell. In disease caused by obligate parasites, facultative saprophytes, and in a few caused by facultative parasites, there is no massive penetration of the cell cavities of the host by ordinary hyphae of the parasite. Instead, the cell wall is penetrated by short branches which usually grow to a limited extent in the lumen of the cell before their ends are transformed into structures called haustoria, which may be simple or relatively complex in form depending on the parasite. Haustoria are presumed to have an absorptive function and are the main source of metabolic exchange between the host and parasite. A detailed account of haustoria will be given later. There are variations in the behaviour of the germ tube at the time of penetration through the stomata, as with Peronospora destructor infecting onion leaves in which the germ tube continues to grow after the formation of the first appressorium (Fig. 3.5 b.) On the other hand, zoospores of a species of Phytophthora infecting fennel (Foeniculum vulgare), almost always enter through stomata while Phytophthora infestans almost always enters plants directly,

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but at times through stomata after the formation of an appressorium. In Pseudoperonospora cubensis (Fig. 3.5 c) the zoospores swim along the boundary lines of the epidermal cells towards the stomata and encyst above the lines separating the guard and epidermal cells. Hyphae penetrate the stomatal aperture and swell to form a substomatal vesicle from which in turn, other hyphae may grow to form haustoria in the adjacent cells of the leaves. Mycosphaerella musicola (Fig. 3.5 d) responsible for Sigatoka disease of bananas, enters leaves in a similar manner as do the rusts. The germ tube from a conidium grows slowly over the leaves for a few days before its penetration. This indicates that it can withstand a period of desiccation. A small structure called a stomatopodium is formed over the pore of a stoma. A hypha then arises from it which grows into the substomatal chamber and swells to form a vesicle, which in turn gives rise to hyphae which invade palisade tissues.

Fig. 3.5 Penetration through natural openings

Leaf infecting fungi-imperfecti generally enters through stomata. Some examples are Cladosporium fulvum infecting tomato; Cladosporium cucumerinum infecting cucumber and Stemphylium solani infecting tomato. Members of Sphaeropsidales entering through stomata are Phoma trifolii and Phoma herbarum var. medicaginis on Trifolium pratense and Medicago sativa, respectively. These fungi also enter by direct penetration under certain conditions. The mode of entry into plants by Sclerotinia cinerea which causes stone fruits to rot depends on the host species infected. It enters plums through the stomata, apricots through the stomata or cuticle, peaches through hair sockets and cherries and nectarines through the cuticle. Stomata are the most important and sometimes, the only pathways of entry for many parasitic bacteria such as Pseudomonas tabaci on tobacco, Erwinia amylovora infecting apples and pears, Xanthomonas campestris pv. phaseoli infecting Phaseolus spp., Xanthomonas campestris pv. malvacearum infecting cotton and Pseudomonas phaseolicola infecting Phaseolus spp. Bacteria enter stomata only through films of water that extend from outside through the stomatal aperture into the inside of the shoots. Because of this the water content of shoots is of great importance in determining whether or not they will

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get infected. The stomatal morphology may in fact determine whether successful infection will take place or not. In case of citrus fruits, the conformation of the cuticle around the stoma either prevents or allows the passage of the water droplet containing the bacterial pathogen, Xanthomonas campestris pv. citri. Lenticels

In many dicotyledonous plants, a protective layer develops as the epidermis becomes redundant. This is called the periderm. The periderm also develops on surfaces exposed by abscission of plant parts or on surfaces produced by the wounding of tissues. It also develops on the surface of storage tissues such as potato tubers. Streptomyces scabies, the cause of the common scab of potato, enters young developing tubers through stomata or lenticels. Oospora pustulans which causes the skin scab of potato tubers also enters through lenticels. Penicillium expansum which is responsible for storage rot of apples and Gloeosporium perennans, the cause of another storage rot, also enters through stomata or lenticels. Nectria galligena, the cause of apple canker, can enter twigs through lenticels. Lenticular wound penetrations are somewhat similar, cork being involved in both and many lenticular invaders can also enter through wounds, particularly soil-borne pathogens, such as S. scabies, Armillaria mellea, Spongospora subterranea, Phymatotrichum omnivorum, and Pectobacterium carotovorum. Nectarthodes, Hydathodes and Trichomes

Erwinia amylovora enters through nectaries of apple and pear flowers. In pears, nectaries occur in saucer-shaped tissues between the point of emergence of the style and stamens. Droplets of nectar are exuded through stoma-like nectarthodes at the bottom of depresions on the surface of the nectaries. Within 24-28 hours of adding suspensions to open pear blossoms, the bacteria are found locally in great numbers beneath the nectarthodes. Hydathodes are structures through which water is discharged from the interior of leaves, usually more or less directly from bundles of terminal tracheids which end in thin-walled parenchyma known as epithem. Hydathodes are the only means of entry for certain bacteria. Bacteria which enter through hydathodes are Erwinia amylovora and Xanthomonas campestris. In high humidity bacteria accumulate in water from hydathodes and are drawn into epithem and tracheids, as the water recedes when conditions reduce water congestion, for example, in rice leaves and Xanthomonas campestris pv. oryzae. Baker et al. (1995) have reported that Botrytis cinerea colonizes necrotic tissue around the hydathodes of Methiola incana; this necrosis (tip-burn) perhaps resulting from high salt concentrations due to the evaporation of the hydathode exudate. Corynebacterium michiganense, which causes the bacterial canker of tomato plans, often enters through trichomes. DIRECT PENETRATION

Direct penetration of plants has been known for many years. de Bary (1886) was the first to study critically the nature of direct penetration by Sclerotinia sclerotiorum. He laid emphasis on the relation-

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ship of nutrients in the infection drop. The physicochemical characteristics of cuticles and cell walls are important factors in direct penetration. Structure of Cell Walls and Cuticles

According to Dickinson (1960), the external layers lining the intercellular spaces and between the adjacent cells (middle lamella) consist largely of pectic substances, particularly calcium and magnesium pectates, and can be disintegrated by the fungal and bacterial pathogens which produce pectolytic enzymes. Proteins have also been reported to occur in the middle lamella. Within this is a primary cell wall consisting largely of cellulose embedded in a matrix of pectic substance and hemicelluloses. The cellulose may be present as micelle. Other substances such as proteins, lignin, suberin, and cutin have been reported in the primary cell wall. In some tissues a secondary cell wall consisting chiefly of cellulose is laid down within the primary wall. The cuticle is a non-cellular membrane which covers most of the aerial parts of the higher plants. The outer waxy layer is hydrophobic and largely impermeable to water but the inner layer can absorb moisture, and swell and force the wax aggregates apart. The innermost layer of the cuticle passes gradually into a predominantly pectic layer which in turn merges into the largely cellulose outer walls of the epidermal cells. The intact or uninjured plant surface or epidermis which is covered by a well-defined layer of cuticle, is penetrated directly by a mechanical process. In most cases, penetration is preceded by the formation of a special structure, the penetration hypha or appressorium which exerts a mechanical pressure on the intact wall of the host. An infection peg, arising from the appressorium pierces the host wall to cause infection. The penetration of the cuticle is regarded by many as purely mechanical although there is evidence of the chemical degradation of the cuticle. Many root pathogens accumulate on the surface of roots, hypocotyl, and other subaerial parts and penetrate directly. The penetration may or may not be preceded by the formation of an appressorium. McKeen and Rimmer (1973) have shown that barley leaves induce the conidial germ tubes of an Erysiphe graminis to grow longitudinally on leaves and stimulate the production of an appressorium. The papilla, a result of host reaction to the fungus, begins to form in the epidermal cell below the appressorium-arm as the appressorial infection pore develops. The tip of the blunt infection peg lacks a cell wall and the host epidermal wall dissolves only in the region of the tip. Mechanical pressure is thought to force the peg through the host cell wall and papilla and to invaginate the cytoplasm. According to Yang and Ellingboe (1972), the primary infection of wheat and barley by P. graminis consists of a number of morphologically indentifiable stages of development, that is, spore germination, formation of appressoria, penetration into host cells, formation of haustoria in host cells and formation of elongating secondary and tertiary infections. The wax layer of the cuticle surface is a major factor for the formation of appressoria. The direct penetration of epidermal cells by fungi has been described by Dickinson, (1960), Gaumann, (1950), Butler and Jones, (1949) and others. In direct penetration of a leaf, the infection hypha (peg or thread) which arises from the underside of an apressorium has to pass through the cuticle and underlying tissue and sometimes through the thickened outer wall of the epidermal cell, although at times it passes between the epidermal cells. The process has been studied in many fungi, including Cladosporium carpophilum [Fig. 3.6 (a, b)] on almond, Venturia inaequalis and Venturia pirina on pear, Phytophthora infestans on potatoes, Puccinia graminis on barberry, Puccinia psidii on pimento

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and Ravenelia humphreyna on Caesalpinia. The smut fungi usually penetrate directly into young ovaries, shoot buds, young seedlings or young tissues such as the loose smut of wheat and barley; sugarcane smut (Ustilago scitaminea): bunts of wheat; flag smuts of wheat and rye; covered smuts of barley and oats and common smut of maize (Fig. 3.6 c). Batts (1956) observed the direct penetration of the ovary by the loose smut pathogen.

Fig. 3.6

Direct penetration of the cuticle. Penetration of almond leaves by germ tubes of Cladosporium carpophilum, the penetration hypha passing (a) between epidermal cells (b) passing into the epidermal cell (c) direct penetration of corn cells by the smut fungus, Ustilago maydis, the germ tube penetrating directly through an epidermal cell and making contact with host nucleus

In the case of infection due to Erysiphe graminis on wheat leaves, a fine stylar process from the germ tube (or from a small subapical appressorium) pierces the cuticle, and in its passage through the cellulose layer it is preceded by a local thickening of the layer into a papilla which it eventually pierces at the apex. Inside the host cell it becomes dilated into a branched haustorium (Fig. 3.7).

Pathogenesis

Fig. 3.7

55

Germination of conidia of Erysiphe graminis on the epidermis of wheat: (a) germination of conidium (b) a fine stylar process from the germ tube (or from a small subapical appressouirm) pierces the cuticle (c) local thickening of the layer to form a papilla (d) papilla eventually pierces at the apex (e) dilatation inside the host cell (f) development of a lobed haustorium (g) branched haustorium and invagination of the lining layer of cytoplasm (x2000). (From S.A.J. Tarr, Principles of Plant Pathology, The Macmillan Press, London and Basingstoke, 1972.)

The initiation, formation and action of appressoria are integral parts of the infection process of many parasitic fungi. The formation of appressoria may be obligatory for infection, while sometimes it may be unnecessary. Appressoria have been discussed in a review article by RW Emett and DG Parbery (1975). The term appressorium was introduced by Frank (1883) for spore like “organisms” formed on the germ tubes of Colletotrichum lindemuthianum, Polystigma rubrum, and Fusicladium tremulae. He believed that appressoria were adhesive discs attaching a parasitic fungus to its host during the early stages of infection. Hasselbring (1906) confirmed Frank’s belief by experimenting with Gloeosporium fructigenum. He rejected the interpretation of many early authors that appressoria were also spore-like in function. The conflict of ideas about the nature of appressoria has been reviewed by Emmett (1969). Appressoria may be simple or compound. Some fungi produce appressoria at different stages of growth; some produce more than one kind. The dematiaceous epiphytic parasites mostly produce appressoria on germ tubes identical to those produced later on hyphae. In the members of Erysiphaceae, most species produce appressoria which are different in form on hyphae than those on germ tubes, each type being morphologically constant for a species. Emmett and Parbery (1975) have surveyed the variety of appressoria formed by various ecological and phylogenetic groups of fungi. The different types can be divided into two basic groups: simple appressoria formed from a single modified cell and compound appressoria made up of many cells. Traditionally, only the structures produced on germ tubes are known as appressoria, while adhesion and penetration structures produced on hyphae have been described as “hyphopodia”. The main role of appressoria is the direct penetration of a host. To achieve this, appressoria must attach themselves firmly to the host surface and produce infection pegs that pierce the cuticle and usually the epidermal cell wall. Appressoria produced by germinating urediniospores of rusts and

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Mycosphaerella citri penetrate through stomata. Few authors disagree that this is the primary role of the appressorium, but there are suggestions that appressoria may play a secondary role, and not the auxiliary role (Emmett and Parbery, 1975). Simple appressoria occur terminally on germ tubes but may be terminal, lateral or intercalary when produced by mycelial hyphae. Appressoria produced on germ tubes are epiphytic and three types can be distinguished. The first “proto appressoria” are little more than slightly swollen, hyaline, germ-like apices adhering to the host and giving rise to an infection peg. They are rarely delimited by a septum and occur in Venturia inaequalis, Pythium spp. and various other fungi. Hyaline appressoria occur in the Erysiphales, Uredinales and other species in the second group. They become swollen and are usually delimited from the germ tube by a septum. The third type, the dark appressoria, may be sessile or subtended by a germ tube from which they are delimited by one or more septa. Much has been written on the ways in which infection hyphae penetrate the cuticle, particularly as to whether penetration is a totally physical process or cuticle softening enzymes are involved. It may be that slight localized softening of the cuticle occurs around the infection hypha, facilitating penetration. In the presumed absence of cuticle-softening enzymes, it is probable that penetration is largely by a physical force. Resistance of the leaves of Berberis and Odostemon spp. to basidiospores of Puccinia graminis was partly associated with their resistance to the combined thickness of the epidermal walls and cuticle (Melander and Craigie, 1927). Cuticle thickness has been correlated with resistance to foot rot of peas caused by Ascochyta pinodella. Butler and Jones (1949) reported that older barley leaves with cuticles 2.5-5 mm thick were more resistant to mildew as compared to younger leaves, with cuticles 0.4-1.5 mm in thickness. Resistance of the strawberry varieties to Sphaerotheca macularis was shown both by Peries (1962) and Jhooty and McKeen (1965) to vary with cuticle thickness. Penetration of the Cell Wall

After passing through the cuticle, the infection hypha has to grow through the outer wall of the epidermal cells which consists of a network of cellulose microfibrils in a matrix consisting principally of pectic materials. The infection hyphae of some fungi pass between the radial walls of the epidermal cells without entering them. In Diplocarpon rosae, the cause of the black spot of rose leaves—the mycelium—develops between the cuticle and epidermis and enters the cells of the latter. The penetration of the walls is largely enzymic. The infection hyphae grow quickly through the softened materials. Pectolytic enzymes are important in this but there is doubt as to the part played by cellulases. Penetration of the cell walls may occur within a minute and is probably a combination of physical pressure and enzymic softening. Swelling of the invaded cell wall and of walls some distance away may occur due to the diffusion of enzymes. Such swellings vary considerably in extent, ranging from sharply localized (Ustilago segetum tritici on wheat; Batts, 1955) to pronounced thickening which almost obliterates the lumen of the cell, as in the case of Venturia inaequalis attacking the epidermal cells of the stem (Marsh and Walker, 1932) of apple. Infection can be as readily caused by mycelial hyphae of many fungi as on germ tubes from spores. In (Armillaria), a root parasite of many trees, infection is restricted to the rhizomorphs. The germ tubes of aeciospores of Puccinia menthae (mint rust) produce infection structures resembling appressoria when exposed to thymol at its own saturated vapour pressure on glass but not on glass in the absence of thymol. Thymol also reduces spore germination and elongation of germ tubes. It is

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thought that it acts as a morphogenetic switch in bringing about the formation of appressoria (Cox, 1969). Studies on different strains of Corticium solani on the roots, stems, and leaves of several plants by Flentje and his co-workers in Australia (Flentje, 1957: Kerr and Flentje, 1957) indicated that appressoria developed on the susceptible but not on resistant plants. The formation of appressoria is probably affected by the nature of the surface on which the hypha grows and also by diffusable substances which cause the arrested growth and multiple branching of side branches leading to the development of appressoria. In the case of infection by Rhizoctonia solani, infection cushions are seen on the roots or hypocotyls of the infected plants. Multicellular infection cushions are produced from a branch hypha in which normal elongation has ceased but prolific side branching now takes place. Strains of Rhizoctonia solani, non-pathgenic to seedlings of a particular species, failed to become attached to the hypocotyl and organize the infection cushions. ENTRY THROUGH WOUNDS

All bacteria, most fungi, some viruses, and all viroids can enter plants through various types of wounds. Penetration of viruses, mollicutes, fastidious bacteria, and protozoa through wounds depends on the deposition of these pathogens by their vectors in fresh wounds created at the time of inoculation. Many so-called low grade pathogens are not able to penetrate the host surface in the absence of a wound. However once they are in the plant, they are often able to cause severe disease symptoms. Wounds may be natural or they may be caused by the activities of man or other agencies, such as insect bites or nematodes. Many horticultural and agricultural practices involve accidental or even deliberate wounding. Entry through natural wounds (lenticels and abscission areas) has been dealt with earlier. Other natural wounds are those that occur due to the emergence of laterals from main roots. This is a means of entry for the tobacco root-rot fungus, Thielaviopsis basicola, and for Fusarium solani f. phaseoli into bean roots. Many important forest pathogens also enter through wounds. Heterobasidion annosus, a destructive pathogen of conifers, naturally colonizes wounds caused by high winds, snow or other natural agencies. It becomes a very damaging pathogen in plantations where it enters through the stumps left after felling. Man’s activities are responsible for many of the wounds through which fungal penetration occurs. Many fruit and vegetable products are quite succulent when they are ripe for marketing. In this condition they are most susceptible to wounds by rough and careless handling. The Rhizopus rot of sweet cherries and peaches develops in wounds in this manner. The size of the wound necessary for the infection of oranges by Penicillium digitatum varies with the degree of maturity and variety of the fruit. Wound need to be deeper for the infection of Valencia oranges than for Naval and Jaffa oranges and also for the infection of immature fruits than for mature ones. An unusual type of entry of a variety of micro-organisms occurs through wounds in flax and maize seeds. The damage is caused mechanically during or after harvesting. Somewhat similar is the infection of wheat seedlings by Cephalosporium gramineum which enters through wounds caused by the breaking of the pericarp and coleorhiza as the root emerges on germination (Otieno, 1962). Wounds caused by insects are important in many diseases. In some diseases, the relationship between the insect and parasite is non-specific while in others the relationship may be more specific, the distribution of the disease coinciding with the appearance of a particular insect species.

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Among the diseases caused by bacteria, vascular wilt of cucurbits is one where the relationship is highly specific. This wilt is caused by Erwinia tracheiphila which overwinters in hibernating adults of the cucumber beetles, Diabrotica vittata and D. duodecimpunctata. When adults feed on plants, they introduce the bacterium into the vascular system where it spreads to cause a typical vascular disease. There is a similar relation in bacterial wilt and Stewart’s leaf blight of corn caused by Xanthomonas stewartii. The bacteria overwinter in the intestinal tracts of insects, in this case the flea beetles (Chaetocnema pulicaria and C. deniticulata), and plants become infected when beetles begin to feed in spring. In another disease the relationship is more casual, for example, in fire blight of apple and pear leaves caused by Erwinia amylovora where the bacteria overwinter in cankers. With the return of suitable growing conditions in spring, bacteria multiply in the cankers and are carried to the blossoms by insects. A large number of fungal diseases are disseminated by insects and are instrumental in their entry into the host. In the ergot disease of rye caused by Claviceps purpurea, conidia are disseminated by species of Diptera and Coleptera. In oak wilt caused by Ceratocystis fagacearum, the relationship between the fungus and insects is also relatively non-specific. A variety of insects also causes feeding wounds, through which species of Sclerotinia invade stone and pome fruits. In the well-known Dutch elm disease, there is a highly specific relationship between the fungus and insect vector. The pathogen Ceratocystis ulmi is carried and transmitted to elm trees by feeding beetles (Scolytus scolytus, S. multistriatus). Conidia are introduced directly into the sap wood as the beetles feed on the bark. In this example, the feeding tunnels not only breach the external protective layers of bark but also provide direct access to the vascular elements in which the pathogen can flourish. WOUNDS CAUSED BY OTHER FUNGI

Resistance to infection can be modified by other fungi. Tubers highly resistant to Phytophthora infestans become susceptible when the parasite enters through wounds caused by Spongospora subterranea. The apple canker fungus Nectria galligena may enter through lesions on a stem caused by Venturia inaequalis. Similarly, Fusarium species which rot potato tubers may enter through wounds caused by Spongospora subterranea and Phytophthora infestans. WOUNDS CAUSED BY NEMATODES

It is now well known that infection of plants by some bacteria and fungi depends on or is increased by damage caused by nematodes. Wilt of solanaceous plants caused by Pseudomonas solanacearum is introduced by Meloidogyne incognita var. acrita. In the case of yellow ear rot of wheat caused by the bacterium Clavibacter (Corynebacterium) tritici, another nematode Anguina tritici plays an important part in the transmission and infection process. Several vascular wilt pathogens enter into the vascular plants through the injuries caused by the attack of nematodes; for example, Fusarium oxysporum f. sp. vasinfectum gains entry into the roots of susceptible cotton plants through the injuries caused by the root-knot-nematode, specially Meloidogyne species.

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ENTRY THROUGH ROOT HAIRS

Root hairs are particularly vulnerable to invasion by pathogens. Among the pathogens which enter through root hairs are Plasmodiophora brassicae (finger and toe disease of crucifers), Phymatotrichum omnivorum (Texas root rot of cotton), Fusarium oxysporum f. sp. lini (flax wilt), F. oxysporum f. sp. conglutinans (cabbage yellows). The mode of entry by Plasmodiophora brassicae appears to be quite unique. This process involves several unique structural features. Zoospore of the pathogen encyst on the wall of a root hair. The contents of the zoospore are injected into the host cell by a fine puncture or hole made in the cell wall of the host. The puncture is the result of the production of a bullet-shaped structure which suddenly forces its way from within the cyst through the wall into the interior of the root hair cell. This is an example of mechanical penetration and the actual infection process takes only a few seconds (Williams et al., 1973). Another interesting example of entry through root hairs is by the root nodule bacterium (Rhizobium) of leguminosae which enters through root hairs in the form of an infection thread. ENTRY THROUGH BUDS

Some rust fungi, including Uromyces pisi enter through unfolding buds. Taphrina cerasi attacks cherry buds and causes them to develop into typical witche’s brooms. The peach leaf curl fungus T. deformans enters its host through the unfolding leaf-buds. An unusual mode of entry is that of the pathogen F. oxysporum f. sp. pisi which enters through cotyledonary bundles which are exposed when cotyledons are rotted by soil saprophytes (Hepple, 1963). DEVELOPMENT INSIDE HOST TISSUE

Following penetration there are very wide variations in the pattern of development and colonization of host tissues. In certain pathogens such as powdery mildews, the growth of the pathogen is superficial (i.e. ectotrophic). The mycelial development of the apple scab fungus (Venturia inaequalis) is restricted to the subcuticular layer of the leaf and fruit, at least until the onset of general tissue senescence. Some parasitic fungi colonize the interior of the plant and also produce an external aerial mycelium which grows superficially and enters the host repeatedly, for example, Corticium solani responsible for stem canker of potato and G. (Ophiobolus) graminis responsible for take all diseases of wheat. Many pathogens colonize the parenchyma of mesophyll and cortex; their further spread into the vascular bundles is prevented by the endodermis, although there may be an entry into the pith. In Taphrina the hyphae are entirely intercellular, obtaining nutrients through the membranes of the adjacent cells and this indirect method perhaps results in some degree of systemic development. The hyphae of less specialized pathogens are often intracellular, progressing with branches from cell to cell until the tissue is more or less completely colonized. Toxins and enzymes may be secreted and bring about rapid softening and necrosis of tissues ahead of the pathogen. Some parasites enter living cells, which however, may die soon after penetration. In diseases caused by obligate parasites and facultative saprophytes and in a few caused by facultative parasites, the cell wall is penetrated by short branches which usually grow to a limited extent in the lumen of the cell before their ends are transformed into structures which are simple-to-relatively

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complex in form in different parasites. These structures are haustoria, they are presumed to have an absorptive function and are the main means of metabolic interchange between the host and parasite. Haustoria almost always occur in parenchyma but in most powdery mildews they are confined to epidermal cells. A comprehensive account of haustoria, as revealed by a light microscope, is given by Fraymouth (1956) for downy mildews, for rusts by Rice (1927), for powdery mildews by Smith (1900) and Hirata (1937). Haustoria of Phytophthora infestans were described by Blackwell (1953). The general picture revealed by light microscopy has been confirmed by electron microscopy. A more substantial account of the fine structure of haustorium is that of Ehrlich and Ehrlich (1963) for Puccinia graminis tritici on wheat. The haustorium originates as a slight bulge on the inside wall of the haustorium mother cell and it becomes closely appressed to the host cell wall at the point it is to be penetrated. Here there is formed a conical opening, wide at the base and tapering at the apex, through which the haustorium passes. As it emerges, it is bound only by a membrane continuous with that of the haustorium mother cell. The fact that the haustorium is not surrounded by a cell wall at this stage suggests that penetration is primarily a chemical and not a mechanical process and that the haustorium mother cells produce substances that dissolve both their own walls and those of the host cells. After penetration, the haustorium elongates to form a neck around which a wall is formed. The apex of the neck expands to the characteristic final structure. There is some (but not conclusive) evidence of the invagination of host plasma membrane by the developing haustorium. The mature haustorium containing nuclei, mitochondria, and endoplasmic reticulum is more complex than the haustorium mother cell. The plasma membrane is invaginated at certain places. Outside is the cell wall which is thick and probably consists of at least two zones. Between the cytoplasm of the host and the wall of the haustorium is an encapsulation or “zone of apposition”, which is not found around the parent hyphae or around the developing haustorium. The encapsulation is enclosed by a membrane which may be the host plasma membrane or a new structure. Ehrlich and Ehrilich (1963) interpret this to mean that haustorial protoplasm probably oozes out through ultramicroscopic pores in the walls and comes in intimate contact with the protoplasm of the host cell. At some points, the surface of the encapsulation is disrupted by small vesicles which project into the host cytoplasm and which contain particulates similar to those of encapsulation. These vesicles or secretory bodies are formed in the host cytoplasm, which then move to the encapsulation and discharge their contents. Encapsulation is also well developed in downy mildews. Berlin and Bowen (1964) suggest that this term be used instead of sheath which was used by Fraymouth (1956) and earlier workers, and that sheath should be applied to the inward projection of the host wall which surrounds the base of the haustorium as a collar. This is illustrated in Fig. 3.8 which shows diagrammatically the complex of host cell wall and haustoria for Peronospora manshurica on leaves of soybean (Peyton and Bowen, 1963) and for Albugo candida on leaves of radish [Berlin and Bowen, 1964 (Fig. 3.9)] The basic structure is similar in both. The only important difference is the absence of a fungal cell wall around part of the neck beneath the head of the haustorium and the absence of nuclei in Albugo candida.

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Fig. 3.8

Diagram of the structure of Peronospora manshurica infecting Glycine max as revealed by electron microscopy. The cytoplasm (FC) of the fungus is bounded by plasma-membrane (FP), lomasomes (LO) and cell wall (FW). The host cell shows vacuole (V), cytoplasm (HC), plasma membrane (HP), and cell wall (HW), ending in sheath (S). A zone of apposition (Z) Separates haustorium from host plasma membrane. Invaginations (sec) of host plasma membrane and vesicular host cytoplasm suggest secretion by host cell. (Adapted from G.A. Peyton and C.C. Bowen, Am. J. Bot., 50: 787-797, 1963.)

Fig. 3.9

Diagram of structure of haustorium of Albugo candida infecting Raphanus sativus as revealed by electron microscopy. Cytoplasm (FC) is bounded by plasma membrane (FP), lomasomes (LO) and cell wall (FW). The host cell shows vacuole (V), cytoplasm (HC), plasma membrane (HP), cell wall (HW) which ends in a sheath (S). An encapsulation (E) separates haustorium from host. Invaginations (sec) of host plasma-membnrane may be secretory sites. (Redrawn after modification from J.D. Berlin and C.C. Bowen, Am. J. Bot., 51: 445–452, 1964.)

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REFERENCES Aist, J.R. (1976), “Cytology of penetration and infection-Fungi”, in: Encyclo. of Pl. Physiol (New series), R. Heitefuss and P.H. Williams (Eds.), Springer-Verlag, Berlin, 4: 197–221. Baker, K.F., D.A. Matkin and L.H. Davis (1954), “Interaction of salinity injury, leaf age, fungicide application, climate and Botrytis cinerea in a disease complex of column stock”, Phytopath., 44: 39–42. Batts, C.C.V. (1955), “Observations on the infection of wheat by loose smut (Ustilago tritici)”, Trans. Brit. Mycol. Soc., 38: 465–475. Berlin, J.D. and C.C. Bowen (1964), “The host-parasite interface of Albugo candida on Raphanus sativus” Am. J. Bot., 51: 445–452. Butler, E.J. and S.G. Jones (1949), Plant Pathology, Macmillan, New York, p. 979. Cox, P.G.(1969), M.Sc. Thesis, Univ. of Exeter. Daly, J.M. (1984), “The role of recognition in plant disease”, Ann. Rev. Phytopathol. 22: 273–307. De Zoeten, G.A. (1977), “ Cytology of virus infection and virus transport”, in: Encyclo. of Pl. Physiol. (New series), R. Heitefuss and P.H. Williams (Eds.), Springer-Verlag, Berlin, 4: 129–149. De Bary, A. (1886), “Ueber einige Sclerotinien and Sclerotien Krankheiten”, Bot. Ztg., 44: 377–474. Dickinson, S. (1960), “The mechanical ability to breach the host barriers”, in: Pl. Pathol.-An Adv. Tre., J.G. Horsfall and A.E. Dimond (Eds.), 2: 203–232. Ehrlich, H.G. and M.A. Ehrlich (1963), “Electron microscopy of the host parasite relationships in stem rust of wheat”, Am. J. Bot., 56: 123–130. Eide, J.C. (1955), “Fungus infections of plant”, Ann. Rev. Microbiol., 9: 297–318. Emmett, R.W. (1969), “Appressoria”, B. Agr. Sci. (Hons.) Thesis, Univ. of Melbourne. Parkville, Victoria, p. 180. Emmett, R.W. and D.G. Parbery (1975), “Appresoria”, Ann. Rev. Phytopath., 13: 147–167. Flentje, N.T. (1957), “Studies on Pellicularia filamentosa (Pat.) Rogers-III: Host penetration and resistance and strain specialization”, Trans. Brit. Mycol. Soc., 40: 322–336. Foister, C.E., A.R. Wilson and A.E.W. Boyd (1952), “Dry rot disease of potato-1: Effect of commercial handling methods on incidence of the disease”, Ann. Appl. Biol., 37: 29–37. Frank, A.B.,(1883), “Liber einige neueund weniger bekannte pflanzen krankheiten”, Ber. Dtsch. Bot. Ges., 1: 129–134; II: 58–63. Fraymouth, J. (1956), “Haustoria of the Peronosporales”, Trans. Brit. Mycol. Soc., 39: 79–107. Gaumann, E. (1950), Principles of Plant Infection, English edition, W.B. Brierly (Ed.), Crosby Lockwood, London, 1950. Goodman, R.N. (1977), “Physioligical and cytological aspects of the bacterial infection process”, in: Encyclo. of Pl. Physiol. (New series), R. Heitefuss and P.H. Williams (Eds.), Springer-Verlag, Berlin, 4: 172–193. Hahn, M.G. (1996), “Microbial Elicitors and their receptors in plants”, Ann. Rev. Phytopathol. 34: 387–412. Hasselbring, H. (1906), “The appressoria of the anthracnoses”, Bot. Gaz., 42: 135–160. Hepple, S. (1963), “Infection of pea plants by Fusarium oxysporum pisi in naturally infested soil”, Trans. Brit. Mycol. Soc., 46: 585–594. Hickman, C.J. and H.H. Ho (1966), “Behaviour of zoospores in plant pathogenic phycomycetes”, Ann. Rev. Phytopath., 4: 195–220. Hirata, K. (1937) Ann. Rev. Phytopath. Soc, Japan, 6: 319–326. Jhooty, J.S. and W.E. Mckeen (1965), “Studies on powdery mildew of strawberry caused by Sphaerotheca macularis”, Phytopath., 55: 281–285. Kerr, A. and N.T. Flentze (1957), “Host infection in Pellicularia filamentosa controlled by chemical stimuli”, Nature, 179: 204–205. Kosuge, T. and E.W. Nester (Eds.) (1984), “Plant Microbe Interactions. Molecular and Genetic Perspectives”, Vol. I, Macmillan, New York. McKeen, W.E. and S.R. Rimmer (1973), “Initial penetration process in powdery mildew infection of susceptible barley leaves”, Phytopath., 63: 1049–1053. Marsh, R.W. and M.M. Walker (1932), “The scab fungus (Venturia inaequalis) on apple shoots”, J. Pomol. Hori. Sci., 10: 71–90.

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Mehrotra, R.S. (1970), “Techniques for demonstrating accumulation of zoospores of Phytopthora species on roots in soil”, Can. J. Bot., 48(5): 879–882. Melander, L.W. and J.H. Craigie (1927), “The nature of resistance of Berberis spp. to Puccinia graminis”, Phytopath., 17: 45–114. Otieno N.C. (1962), “Ingress of Cephalosporium gramineum into winter wheat seedlings and subsequent histological relationship between the suscept and the pathogen”, Diss. Abstr., 22: 3804. Peries, O.S. (1962), “Studies on strawberry mildew, caused by Sphaerotheca macularis (Wallr. ex Fries) Jaczewski-II: Host parasite relationship on foliage of strawberry varieties”, Ann. Appl. Biol., 50: 225–233. Peyton, G.A. and C.C.Bowen (1963), “The host-parasite interface of Peronospora manschurica on Glycine max”, Am. J. Bot., 50: 787–797. Rice, M.A. (1927), “The haustoria of certain rusts and relation between host and pathogen”, Bull. Torrey Bot. Club, 54: 63–153. Schaffer, W. (1994), “Molecular Mechanisms of Fungal Pathogenicity to Plants”, Ann. Rev. Phytopathol., 32: 461–477. Smith, G. (1990), “The haustoria of the Erysiphae”, Bot. Gaz., 29: 153–183. Vidhyasekaran, P. (1998), “Molecular biology of Pathogenesis and induced systemic resistance”, Indian Phytopath. 51(2): I 20. Webster, J.M. (1969), “The host and parasite relationship of plant parasitic nematodes”, Adv. Parasitology, 7: 1– 4. Williams, P.H., J.R. Aist and P.K. Bhatacharya (1973), “Host–parasite relations in cabbage club root”, in: Fungal Pathogenicity and the Plants Response, R.J.W. Byrde and C.V. Cutting (Eds.), Academic Press, London and New York, 141–158. Wood, R.K.S. (1967), “Physiological Plant Pathology, Oxford and Edinburgh, Blackwell, p. 570. Yang, S.L. and A.H. Ellingboe (1972), “Cuticle layer as a determing factor for the formation of mature appressoria of Erysiphe graminis on wheat and barley”, Phytopath., 62: 705–714.

QUESTIONS 1. 2. 3. 4.

5. 6. 7. 8.

What is the latest concept of recognition by host of pathogens and nonpathogens? What are chemotaxis and electrotaxis? Distinguish between penetration and infection. Name at least two pathogens which enter their hosts through the following: i. Stomata ii. Lenticels iii. Wounds caused by natural agencies iv. Directly through cell walls v. Wounds caused by nematodes vi. Wounds caused by fungi vii. Root hairs viii. Through unfolding buds. Name some pathogens which grow superficially (i.e. ectotrophic), subcuticularly and endophytically in their hosts. Distinguish between appressoria and hyphopodia. What is the function of appressoria? What are the different types of appressoria produced by plant pathogens? Give examples of plant diseases in which insects play an important role in the entry of pathogen. How do the following pathogens enter their hosts: Heterobasidion (Fomes) annosus Gaeumannomyces (Ophiobolus) graminis Penicillium expansum?

4 Enzymes and Toxins in Plant Diseases

ENZYMES IN PLANT DISEASES

For successful infection by a pathogen, it should first enter the body of the host on which it feeds. According to Wilson (1973), hydrolytic enzymes are extremely important in pathogenesis because they provide the pathogen chemical means of entrance into the host and a process whereby nutrients can be digested. The plant cell is surrounded by a cellulose wall which is comparable to a closed box. If the parasite is to feed successfully, it should break this barrier which acts like a fortress wall. Decomposition and disintegration of host tissues are the constant components of plant disease syndromes. Enzymes and toxins are the main aspects under which the mechanism of disease has been studied in the last 80 years or so. The concept that cell wall degrading enzymes may be involved in pathological manifestations induced in plant tissues by biotic agents was introduced for the first time by de Bary (1886). He described the pathogen’s action on host tissues in two ways: one initiates the dissolution of certain constituents of the cell wall and subsequently the loss of tissue coherence, and the other kills the protoplast itself. The former effect was traced to some heat labile metabolites, possibly enzymes. The second effect could be due to the cell wall degrading enzymes themselves or it may be brought about by a second factor, possibly toxins. This view was substantiated by the researches of Ward (1888), Jones (1909), Brown (1915, 1917), and Hutchinson (1913) and they called these pectic enzymes. Pectolytic and cellulolytic enzymes are now implicated almost routinely as a feature of host-parasite interactions and their involvement in the degradation of the pectin and cellulolytic constituents of cell walls and of the middle lamella in plant tissues has been reported for such diverse types of diseases as soft rots, dry rots, charcoal rots, damping off, wilts, blights, and leaf spots, for such diverse pathogenic agents as fungi, bacteria, and nematodes; even virus-infected tissues have been reported to undergo alterations in their pectic constituents. This ability to elaborate pectic enzymes is widely distributed among organisms (Brown, 1965; Mahadevan,1970; Bateman and Basham, 1976; Cooper, 1983; Collmer and Keen, 1986). However, the ability to produce these enzymes does not indicate that an organism possesses pathogenic capabilities. In most plant diseases caused by microbial agents, cell walls are penetrated, tissues are colonized and permeability of host cells is altered. An understanding of the cell wall and membrane

Enzymes and Toxins in Plant Diseases

65

structure, together with a knowledge of the enzymes capable of degrading the components of these structures is essential for our understanding of disease physiology. COMPOSITION OF CELL WALL MATERIALS AND MIDDLE LAMELLA Cuticular wax

Plant waxes are formed as granular or rod-like projections or as a continuous layer outside or within the cuticle of many aerial plant parts. Wax formation is a continuous process and not a terminal phase in the development of a leaf. Recent indications are that minute pores exist beneath each wax projection and it is through these minute pores that wax is secreted. Cuticular waxes are made up of long-chain molecules of paraffin hydrocarbon, alcohols, ketones, esters and acids. They differ from fats in that glycerol is replaced by monohydric alcohols. Cutin

Cutin is the main component of the cuticular layer. The upper part of the layer is admixed with waxes, while in its lower part in the region where it merges with the outer walls of the epidermal cells, cutin is admixed with cellulose and pectin.

Fig. 4.1

Schematic representation of the structure and composition of cuticle and cell wall of foliar epidermal cells. (Redrawn from R.N. Goodman, Z. Kiraly and M. Zaitlin, The Biochemistry and Physiology of Infections Plant Disease, D. van Nostrand Company, Inc., 1967.)

Cutin is believed to be an insoluble polymer of hydroxylated octa-and hexadecanoic acids of which phloinolic and C17H32(OH)3COOH are characteristic. Each chain of the polyester consists of 16-18 carbon atoms and two to three are hydroxyl groups. The hydroxyl groups of one chain are linked to the carboxyl or hydroxyl groups of other chains which results in a complex three dimensional polymeric structure (Fig. 4.1 and 4.2). Cutler et al. (1982) have given a detailed account of the plant cuticle.

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Fig. 4.2

Distribution in the plant cell wall of the major wall constituents. [Adapted from D.F. Bateman and H.G. Basham, (1977), "Physiological Plant Pathology", in: Encyclo. of Pl. Physiol. (New Series), R. Heitefuss and P.H. Williams (Eds.), Springer-Verlag, Berlin, Heidelberg and New York.]

The cell wall

The cell wall of higher plants comprises a middle lamella and primary cell wall and in some cases, a secondary cell wall. Pectic substances are the basic constituents of wall layers and form almost the entire middle lamella, whereas in the other layers cellulose is also found in good amounts. Besides these two major group of compounds other components are hemicelluloses, lignins, proteins, and limited amounts of other materials. In the classical view, cell walls are a mesh of crystalline cellulose microfibrils embedded in an amorphous matrix of hemicellulose with the pectic compounds localized in the middle lamella serving to cement cells together. A serious challenge to this view came from Lamport (1967) who demonstrated that the cell wall contains protein rich in hydroxyproline. Functionally, the cell wall may be divided into three regions; middle lamella, primary wall, and secondary wall. The middle lamella acts as an “intercellular cement” that binds cells together in the tissue system. A schematic representation of the structure and composition of a typical cell wall of higher plants is given in Fig. 4.3.

Enzymes and Toxins in Plant Diseases

67

Cell lumen Plasmodesmata

Middle lamella (pectates) Primary cell wall (Cellulose, pectates) Secondary cell wall (almost entirely Cellulose) Intercellular spaces

Fig. 4.3

Schematic representation of structure and composition of plant cell walls. (From G.N. Agrios, Plant Pathology, Academic Press, London, New York, 1969)

Pectic Substances

Pectic substances are a group of complex, colloidal carbohydrate derivatives which occur in or are prepared from plants and the basic structure of which is a linear polymer of D-galacturonic acid units in a 1-4 glycosidic linkages, esterified with a methyl group or linked with other carboxyl groups in calcium or magnesium salt bridges. The esterification varies with the age, organ or species of the plant. The term protopectin is applied to substances which are insoluble in water and which, upon restricted hydrolysis, yield pectinic acid. Pectinic acid contains a variable quantity of methoxyl groups, but fewer than pectin which may have 75% or more of the uronic carboxyls methylated. Pectic substances essentially free from methyl ester groups are classified as pectic acids.

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COOH O HO H H H OH H

H OH

-O

COOH O H H

H OH

OH

H

H OH O

H OH

COOH O H H

OH

H

H

O

H OH

H H COOHO

a d-galacturonic acid

H

H

O

OH

Pectic acid

— —

O

H

OH

H

O

H O C—O—CH

H OH H

H OH

H H OH O

OH H

O

H

H O C—O—CH3

— —

H

COOH O H H

OH

— —

O

C—O—CH3 H O H H H OH OH H O H

O

O Pectinic acid

The proportion of carboxyl and methyl ester groups has profound effects on the properties of polymers. Calcium salts of pectin occur in the middle lamella of plant cells and represent a large portion of the pectic substances in plants. Cellulose

Cellulose is an aggregation of β -D-glucopyranose in which C1 is linked to C4. Cellulose is the major constituent of the cell walls of higher plants. Cellulose is an insoluble, crystalline substance in its native form (Fig. 4.4).

Structure of cellulose showing β -D-1, 4 linkages of glucopyranose form

Hemicelluloses

In addition to celluloses, these (hemicelloses) also occur in cell walls. Hemicelluloses include several polymers. Xyloglucan is an important hemicellulose in the primary wall region. It is made up of a β 1, 4 linked glucopyranose chain with terminal branches of 1,6 linked D-xylopyranose. Other hemicelluloses

Enzymes and Toxins in Plant Diseases

69

include glucomannan, a heteropolymer of D-glucopyranose and D-mannopyranose linked β-1, 4 and glactoglucomannan, a glucomannan with 1,6 linked D-gluctopyranose side branches.

Fig. 4.4

Schematic diagram of the gross structure of cellulose and microfibrils (a) and of the arrangement of cellulose molecules within a microfibril (b) MF = microfibril, GS = ground substance (pectin, hemicelluloses, or lignin), AR = amorphous region of cellulose, CR = crystalline region of cellulose, M = micelle, SCC = single cellulose chain (molecule). (From G.N. Agrios, Plant Pathology, Academic Press, London and New York, 1969.)

Lignin

This is a high molecular weight, structurally complex, amorphous polymer that is different from carbohydrates and proteins. Its structure is still not known but the basic unit which forms a polymer is phenylpropanoid.

Lignin is generated from the oxidation products of sinapyl, confiferyl and p-hydroxycinnamyl alcohols which condense by the free radical mechanism to form an amorphous, three dimensional, branched polymer. Lignin is found in the middle lamella, the cell walls of xylem vessels and the sclerenchyma fibres as a secondary thickening that strengthen plants. It may also be formed in other cells such as bundle sheath cells and occasionally epidermal cells. The lignin content of mature woody tissues may vary from 15 to 38% and is second only to cellulose. Lignin is formed by C—C and C—O bonds between the carbon and/or the hydroxyls of the aromatic ring and of the side chains of different phenyl propanoid or similar units. The polymer (3-dimensional) may consist of 103 or more phenylpropanoid units. Lignin can form covalent linkages with polysaccharides and confers upon them resistance to cell wall dissolving enzymes. Its structure, formation and role in resistance are considered by Ride (1983).

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Cell Wall Proteins

Proteins are also present in all plant cell walls in the region of the cuticle and possibly in the middle lamella. The structural wall proteins seem to be similar to other cell wall proteins in all aspects except that an amino acid hydroxyproline, is also present, which is not present in other plant proteins. Mucopolysaccharides are formed as a result of the covalent bonds between the protein molecules and cell wall polysaccharides and form a complex called extension, envisaged as playing a part in cell wall extension. Five classes of structural proteins have been found in cell walls: (i) extensions, which are glycoproteins. They are upto 0.5 percent of the cell wall mass in healthy tissues but may increase to 5 to 15 percent in infected tissues. (ii) proline-rich proteins (iii) glycine-rich proteins (iv) solanaceous lectins and (v) other structural proteins (Agrios, 1997). Lipids

Phospholipids and glycolipids, along with proteins, are the main constituents of all plant cell membranes. The common characteristic of all lipids is that they contain fatty acids that may be saturated or unsaturated. Oils and fats are formed in many cells specially as storage compounds especially in seeds. Wax lipids are formed on aerial epidermal cells. ENZYMES FOR WAXES AND CUTINS

It was earlier thought that no pathogens could produce enzymes that degraded waxes (Goodman et al., 1967). Wax layers are mainly penetrated by mechanical force alone. However, electron microscopic studies suggest that some pathogens such as Puccinia hordei produce enzymes that can degrade waxes (Agrios, 1997). The significance of cuticle-softening enzymes in plant diseases is uncertain and needs more investigation. The cuticle consists of a cutin framework with waxes embedded within it and extruded from its surface to give a water proof ‘bloom’. Until 1961, no enzymes were known which could degrade cutin. Heinen and Linskens (1961) demonstrated that the fungus Penicillium spinulosum from rotting leaves produced at least two types of cutinolytic enzymes. Cutin Esterase This catalyzes the hydrolysis of ester bonds occurring between free hydroxyl and carboxyl groups of cutin bonds.

Carboxycutin Peroxidase

This catalyzes the hydrolysis of the peroxide group of cutin.

Enzymes and Toxins in Plant Diseases

H2C(CH2)n—CH—(CH2)n—COOH

OH

OH

H2C(CH2)n—CH—(CH2)n—COOH

O

Carboxycutin peroxidase

O

H2O

O==C—(C H2)n—CH—CH—(CH2)n—CH2OH

71

OH

OH

H2C —(CH2)n—CH—CH—(CH2)n—COOH+ OH

1 O2 2

OH OH

Chaudhuri (1935) reported that the germinating spores of Colletotrichum gloeosporioides degrade the cuticle of orange leaves. Graniti (1962) reported the cuticular breakdown in olive leaves by Spilocaea oleagini. It is possible that in organisms such as Spilocaea oleagini, which live sub-or intracuticularly, the entire cuticle is also used as a food source. Roberts et al., (1960) noted a considerable reduction in the content of cutin in rose leaves infected by the fungus, Venturia inaequalis. However, some fungal pathogens may not produce quantities of cutin-dissolving enzymes sufficient to play an important role in the penetration of the cuticle. A localized secretion of cutin-dissolving enzymes is significant as it may aid in the penetration process. The work of Edwards and Allen (1970) is significant in this connection. They visualized a localized secretion of cuticle-degrading enzymes from the tip of the infection peg by the fungus Erysiphe graminis f.sp. hordei attacking barley leaves. Cutinolytic enzymes and pathogenesis have been reviewed by Van den Ende and Linskens (1974). Fungal penetration of the first line defensive barriers of plants has been critically discussed by Kolattukudy and Koller (1983). Enzymatic penetration of the plant cuticle by fungal pathogens has been reviewed by Kolattukudy (1985). It is believed that fungal pathogens constantly produce low levels of cutinase which, on contact with cutin release small amounts of cutin monomers (single molecules). These small monomers gain entry into the fungal pathogen which then trigger further expression of the cutinase gene and stimulate the fungus to produce large amounts of the cutinase enzyme. Fatty acids present in the wax can also stimulate the production of cutinase enzyme. Glucose on the other hand suppresses the production of cutinase enzyme. The involvement of cutinase in the penetration of host cuticle by plant pathogenic fungi is proved by several indirect evidences. It has been shown that the production of the enzyme is highest at the point of penetration of the germ tube of the pathogen. To prove the involvement of cutinase in penetration of pathogen into its host, secretion of cutinase by the penetrating fungus must be conclusively shown and conclusive evidence that cutinase is required for infection must be provided. Both of these have been accomplished for infection of pea by Fusarium solani f. pisi under controlled conditions. Ferritin-conjugated antibodies prepared against cutinase were used to determine whether the fungus penetrating the intact pea stem, secreted cutinase. Scanning electron microscopy showed that the spores placed in suspension on pea stem germinated and penetrated into stem in 18 hours. Treatment of the penetrating area with ferritin labelled anticutinase followed by electron microscopy demonstrated the presence of cutinase antibody complex, showing that the penetrating fungus (pathogen) secreted cutinase. Chemicals known to be inhibitors of cutinase such as diisopropyl fluorophosphate also prevented infection, although such chemicals did not appear to affect the growth of the pathogen. A similar approach was used to demonstrate that cutinase was essential for infection in case of papaya fruits infected with Colletotrichum gloeosporioides.

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PECTIC ENZYMES

Important reviews on the significance of pectic enzymes in plant diseases are by Bateman and Millar (1966), Sadasivan and Subramanian (1963), Wood (1960), Brown (1965), Demain and Phaff (1957) and Bateman and Basham (1976), Cooper (1983), Collmer and Keen (1986). The role of these enzymes in wilt diseases (Papavizas and Ayers, 1966; Hancock,1968 a,b ; Subramanian, 1970), storage rots (Vidhyasekaran et al., 1966), and fruit rots (Midha and Chohan, 1967) has been extensively studied. The importance of these enzymes in some anthracnose diseases (Hancock, 1966) and leaf spot diseases (Hancock and Millar, 1965) has also been demonstrated. Vidhyasekaran et al., (1973) emphasized the role of pectolytic enzymes in the pathogenesis of two obligate parasites (Puccinia purpurea and Sclerospora sorghi) and two facultative parasites (Helminthosporium turcicum and Cercospora sorghi). The capacity to produce pectic enzymes is widespread in fungi, bacteria, and nematodes. The pectic enzymes which are currently known are classified into two major groups. Pectinesterases (PE or PME)

Pectinesterases or pectin methyl esterases catalyze the hydrolysis of methyl ester groups of pectinic acids to methyl alcohol and pectinic acids of reduced methoxy content and eventually to pectic acid. PME is also found in most of the higher plants and in micro-organisms. The microbial PME and plant PME may, however, differ in their chemical and physical properties. It is of interest that the rate of cleavage of the α -1, 4 bond of the pectic substance is enhanced by the removal of the methoxyl groups. This is illustrated by the fact that endopolygalacturonase prefers demethylated pectin.

Polygalacturonases (PG)

Polygalacturonases or pectic glycosidases and lyases are chain-splitting enzymes which break the links between adjacent galacturonic acid units in pectic substances. This can occur by a hydrolytic mechanism (hydrolases) or an eliminative mechanism (lyases). There has been some confusion about the names of various chain splitting enzymes of pectinous materials. However, Wood (1960) followed the classification proposed by Demain and Phaff (1957) who distinguished between the polygalacturonses (PG) which prefer pectic acid of the de-esterified parts of pectinic acid as substrates, and pectin methyl galacturonases (PMG) which prefer the esterified chain parts. They further distinguished these polygalacturonases according to whether they caused cleavage in glycosidic linkages at random by an endo-type of polygalacuronase or polymethyl-galacturonase, or by an exo-type of action in which the end linkages are preferred.

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A significant advance was the discovery of the pectin transeliminases (PTE) by Albersheim et al. (1960) which split the pectin chain by a transeliminative mechanism. This enzyme breaks the glycosidic linkage of pectin at C4, accompanied by a simultaneous elimination of H+ from C5. Wood (1960) further classified these enzymes into those which preferably attacked esterified or nonesterified parts of the chain and determined whether enzymes attacked chains terminally, (exo-), or at random (endo-). MACERATING ENZYMES

Protopectinases (hypothetical enzymes) attack protopectin which is a native substance in cell walls. Although the name protopectinase is widely used for macerating acitivity, Demain and Phaff (1957) devised a new system of classification where the macerating principle was shown to be associated with the chain-splitting enzymes, polygalacturonases (PG and PMG). Protopectinases can bring insoluble substances into solution and can include the enzymes degrading the middle lamella pectates. Maceration of plant tissues has often been associated with hydrolases and lyases which degrade the α -1, 4 linkages in the galacturonic acid polymers of pectin substances in a random manner. Several workers have reported the “macerating enzymes” from the pectic enzymes. Byrde and Fielding (1962) separated the endopolygalacturonase from the macerating enzymes produced by Sclerotinia fructigena and subsequent studies have revealed that it is pectin methyl transeliminase. The work done by McClendon (1974), Zaitlin and Coltrin (1964) on purified pectic enzymes suggests that macerating enzymes are not of only one type. Looking at these discrepancies, Brown (1965), Bateman, and Millar (1966) emphasized that the non-specific term, macerating enzymes, should be retained. But all macerating enzymes which have been purified and identified to date have proven to be pectic enzymes. The capacity to produce pectic enzymes seems to be widespread among living organisms, specially higher plants, micro-organisms, and animals feeding on plants. Thus, the presence or secretion of these enzymes by any micro-organism is not indicative of its pathogenic capability. Pectin esterases occur in

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fruits, especially in ripening fruits. Bateman and Millar (1966) reported the occurrence of endo pectin transeliminase in healthy pea tissue. Many fungi and bacteria produce pectic enzymes, especially PME in culture and this may be stimulated by the presence of specific substrates. Production of pectic enzymes by a pathogen in culture does not necessarily mean that they are produced in the host plant or that they play a part in pathogenesis. Damle (1952) working on Pythium spp. was able to establish a correlation between the pathogenicity and pectolytic enzyme secretion. In addition to the correlation between pectinase secretion and pathogenicity, the degree of pectolytic action varied with the relative rapidity of symptom development. Subramanian (1960) compared the pectolytic enzyme secretion in vivo in wilting cotton (Gossypium arboreum-K2), pigeon pea (Cajanus cajan-NP24) and foot rot infected rice (Oryza sativa-MT09). There was a good correlation in the wilt caused by Fusarium oxysporum f. vasinfectum and F. oxysporum f. udum in the development of wilt-symptoms and pectolytic enzyme production. However, in the case of foot rot of rice caused by Fusarium moniliforme there was no such correlation. Pectolytic enzymes produced in culture by virulent and avirulent strains do not have any correlation with their pathogenic ability (Chan and Sackston, 1972; Goel and Mehrotra, 1974). Hancock et al. (1964), have demonstrated that different types of enzymes are produced by three Botrytis species pathogenic to onion, depending on whether the fungi are grown in culture on detached leaves or on leaves of infected plants. Hence, it is apparent that the determination of pectic enzymes produced by the pathogens in vitro may not be a reliable indication of the types of enzymes produced by the pathogen during pathogenesis. Vascular pathogens, such as Fusarium, Verticillium and some bacteria produce pectolytic enzymes. F. oxysporum f.sp. lycopersici produces a pectin depolymerase, pectin methyl esterase and cellulase (Gothoskar et al.1955; Husain and Dimond, 1960; Pierson et al., 1955; Waggoner and Dimond, 1955) and these appear to be involved in pathogenesis (Dimond, 1955; Dimond and Waggoner, 1953; Gothoskar et al., 1955). Juice extracted from stems of tomato plants infected with Verticillium alboatrum was found to contain higher amounts of PME and chain-splitting pectolytic activity than healthy plants (Deese and Stahman, 1962). Still, no direct evidence has been suggested for the involvement of pectins in reduced water transport. Pectic enzymes may partially hydrolyze pectic substances along with the walls of vessels. The action of depolymerase and pectin methyl esterase together might free pectic acids which probably cause plugging. In the presence of calcium ions, these materials could form calcium pectate gels. PME has been detected in a greater quantity in the tracheal fluid of infected plants than in that of healthy plants (Matta and Dimond, 1963; Waggoner and Dimond, 1955; Wood, 1961). As a result of the action of these enzymes in the vascular region, phenols are released into the xylem vessels and the phenol oxidase system of the plants aids in their conversion to melanins which are absorbed by the liquified xylem walls. The basis of the theory is that browning results from phenols produced in the xylem as a result of the action of the pectolytic enzyme (Gothoskar et al., 1955). Pectic enzymes are of great importance in soft rot diseases in which the parenchymatous tissue of the host parts is rapidly invaded. There is the dissolution of the middle lamella which results in a mass of soft, disorganized, water-soaked tissue. Although soft rot organisms produce pectic enzymes in culture, it is likely that other enzymes are also involved in the maceration of plant tissues. Some of the organisms responsible for soft rot in this manner are Pectobacterium carotovorum, Pseudomonas marginalis, Penicillium expansum, Botrytis cinerea, Choanephora cucurbitarum, Sclerotinia fructigena, Pythium spp., Phytophthora spp., Rhizopus spp., Rhizoctonia solani, and R. bataticola. The mechanism involved in the killing of plant cells by pectic enzymes and specifically the factors that regulate their activities in plant tissues are not currently understood. Pectic enzymes are relatively

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stable and are resistant to many classical enzyme inactivators or inhibitors and may act as a basis for the self-limited lesions in various plant diseases (Byrde, 1963). The cells of macerated tissues soon die although the reasons for the death of the cells are obscure. Brown (1965) distinguished a ‘macerating principle from a ‘lethal principle’. The death of protoplasts (often the action of the pectolytic enzymes) has been ascribed by Wood (1967) to the bursting of the protoplasts following their rapid expansion on absorbing water. This is supported by the results of the plasmolysis experiments performed by Tribe (1955) in which the cells in the discs of a potato tuber exposed to macerating enzymes of Erwinia aroideae and Botrytis cinerea remained alive much longer when plasmolyzed than when unplasmolyzed, although maceration was only slightly affected by plasmolysis. Microscopical examination failed, however, to support the bursting theory. Hall and Wood (1973) suggest that cell death caused by pectic enzymes is an osmotic effect which results in the rupture of plasma membrane. The role of pectic enzymes has now begun to be appreciated in certain specialized plant pathogens such as Puccinia graminis tritici, where it has been shown that the germinating uredospores of P. graminis tritici produce pectic enzymes which are thought to be involved in the growth of the hyphae between host cells and in the penetration of the epidermal cell wall. CELLULOLYTIC ENZYMES

These enzymes have been investigated in organisms which bring about the deterioration of textiles and wood but not much attention has been given to their significance in plant diseases. The enzymatic degradation of cellulose is imperfectly understood. Cellulose degrading enzymes are produced by fungi, bacteria, and nematodes and also by parasitic higher plants. They cause: 1. The break-up of the wall barrier for entry. 2. Provide food in the form of glucose from cellulose on hydrolysis. The enzyme cellulases degrade cellulose and its derivatives. Cellulases which degrade native cellulose by destroying its crystalline structure are designated as C1 enzymes. A second cellulase also attacks native cellulose and breaks it into shorter chains and exposes the glucan chains to a third group to enzymes β-1,4 endoglucanases, termed Cx enzymes, which degrade the glucan chains to cellobiose. The conversion of cellulose to glucose also requires a cellobiase or β-glucosidase. The presence of cellulolytic enzymes and the degradation of cellulose by bacteria and fungi have been reviewed by several workers. Host wall alterations by parasitic fungi have been reviewed by Cooper (1983) and Petrini and Quellete (1994). Wood (1967) proposed a scheme of cellulolytic enzymes when native cellulose is degraded. However, this has been slightly modified as follows: Native cellulose C1 attacks native cellulose cleaving cross-linkage between chains Native cellulose (modified without cross-linkage) C2 breaks native cellulose or modifies cellulose into short chains of cellulose Shorter chain of cellulose Cx (third group of cellulases which degrade shorter chains into disaccharide cellobiose) Cellobiose β-glucosidase Glucose

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Cellulases are generally adaptive in their formation because these enzymes are stimulated by the presence of cellulose. The capacity to degrade native cellulose is confined to a few organisms, while soluble cellulose is degraded by a large number of micro-organisms. It is generally believed that cellulases are not of much importance in the early stages of pathogenesis; perhaps cellulose is first protected by the enveloping matrix. Many soft rot organisms produce cellulases as well as pectolytic enzymes. Members belonging to the family Pythiaceae do not form cellulases in vitro, whereas others, such as Rhizoctonia solani, Fusarium moniliforme are reported to produce both cellulolytic and pectic enzymes. Sclerotium rolfsii produces cellulolytic enzymes and has the capacity to cause soft rot as well as degradation of hardened tissues (Husain and Kelman, 1957). In the root rot and foot rot of many plants and in leaf spot diseases, cellulases seem to play an important part in pathogenesis. Cellulolytic enzymes have been implicated in wilt diseases (Husain and Kelman, 1957, 1958; Talboys, 1958; Husain and Dimond. 1960) caused by Pseudomonas solanacearum, Verticillium alboatrum and Fusarium oxysporum f. sp. lycopersici. Wood (1960) suggested that the conditions in the trachea favoured the production of cellulases by vascular wilt pathogens and that local action of these enzymes might result in the formation of the compounds of high molecular weight which could impede the flow of sap in the transpiration stream. The production of cellulolytic enzymes in vitro is often reduced by sugars and by various inhibitors, such as polyphenols. Germinating uredospores of Puccinia graminis tritici are known to produce a cellulase but its role is uncertain. There is increasing evidence that a number of fungi, particularly the wood rotting fungi, produce several cellulolytic enzymes. Polyporus versicolor produces a multiple-component cellulase system. HEMICELLULASES

Many micro-organisms—saprophytic and parasitic—produce hemicellulases and can convert hemicelluloses into pentoses and uronides. Depending on the monomer released from the polymer on which they act the particular enzymes are called xylanase, galactanase, arabinase, mannase, and so on. Activated oxygen, hydroxyl and other radicals produced by attacking pathogens also bring about nonenzymatic break down of hemicelluloses (Agrios, 1997). Although it is well known that pathogens or non-pathogens produce hemicellulases and oxidative agents yet the exact manner in which these enzymes contribute to the breakdown of the cell walls or the ability to cause disease is not well understood. It is indeed surprising that so little attention has been given to cellulases and hemicellulases as compared with the pectolytic enzymes of plant pathogens. Hancock (1967), in the case of sunflower hypocotyls infected with Sclerotinia sclerotiorum, reported the production of xylanase and arabinase by the pathogen. Byrde and Fielding (1962, 1965) reported an arabino-furanosidase in culture filtrates of S. fructigina, while Strobel (1963) reported the production of at least two extracellular xylan-degrading enzymes by Diplodia viticola when grown on autoclaved grapes. Van Etten and Bateman (1969) found that the pathogen Sclerotium (Corticium) rolfsii produced exogalacturonase, endomannase, galactosidase and endoxylanase in culture and these enzymes have also been detected in bean tissues infected by the pathogen. Hemicellulases will no doubt be investigated in many other plant pathogens and their role elucidated in plant diseases.

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LIGNOLYTIC ENZYMES

Lignin is a resistant material and only about 500 species of basidiomycetous fungi are capable of degrading it. Bacteria are seemingly not important in the degrading of lignin. The nature and action of enzymes which decompose lignin have not been properly understood. It has been recognized for many years that the white rot fungi, together with carbohydrates, decompose lignin in plant tissues (Campbell, 1952). Cowling (1961) showed that Polyporus versicolor can destroy over 97% of the lignin in sweetgum wood, and many reports of the removal of most of the lignin from wood by white rot fungi is found in the literature. There are several hundred species belonging to a number of families of Hymenomycetes, namely, Agaricaceae, Corticiaceae, Hydnacece, Polyporaceae , and Thelephoraceae, that can bring about white rot. In addition, a few wood-inhabiting Ascomycetes in the order Sphaeriales cause a white rot type of decay (e.g. Hypoxylon deustum and Xylaria polymorpha). There is very little experimental data available on the role of lignolytic enzymes in disease syndrome. It appears that white rot fungi secrete an extracellular polyphenol oxidase, possible laccase, which enables them to use lignin. On the other hand, all types of wood-rotting fungi seem to produce transmethylase that removes methoxyl groups from lignin, thus “modifying” lignin and increasing the exposure of the embedded cellulose and other polysaccharides to the action of fungal enzymes. Then there are brownrot fungi. Like most white rot fungi, they are the members of Holobasidiomycetes and unlike white rot fungi, which decompose both carbohydrates and lignin in wood, brown rot fungi decompose the carbohydrates with only a slight decrease in lignin content. The chemistry and breakdown of lignins are described by Brauns (1952, 1962), Brauns and Brauns (1960), Kremers (1959), Neish (1960), and Pearl (1967). Husain and Kelman (1959) have pointed out that lignolytic enzymes might be playing an important role in many foot rot diseases of monocotyledonous plants which have a high lignin content in plant tissues. Certain species of Fusarium, such as F. lactis and F. nivale have been reported to degrade lignin (Fischer, 1953). Kirk (1971) has discussed the effect of micro-organisms on lignin. Delignification of wood by wood decay fungi has been reviewed by Blanchette (1991). PROTEOLYTIC ENZYMES

Cell walls contain a protein rich in hydroxyproline (Lamport, 1973). Pathogens are known to degrade proteins enzymatically, and since no particular enzyme responsible for the degradation of cell wall protein is known, it is presumed that its breakdown follows the same steps as the degradation of cytoplasmic proteins. Some pathogens are known to produce proteolytic enzymes in culture and in the infected plant. Some of the pathogens which are known to produce the cell wall dissolving enzymes have also been shown to produce proteolytic enzymes. Kuc’ (1962) believed that the proteases might be important in view of the proteinaceous gel existing in the cell wall. One of the well documented studies on the secretion of proteolytic enzymes was on Pyricularia oryzae by Otani (1959). Protease was detected in the mycelium of the pathogen and in culture fluids. The enzyme was also located inside the diseased tissues. Degradation of RNA by Phytophthora infestans, the late blight pathogen, was demonstrated by Page (1959). Hancock and Miller (1965) detected protease activity in alfalfa plant tissues infected with Stemphylium botryosum. Mahadevan (1970) reported the production of proteolytic enzymes and the existence of an RNAase in Pyricularia oryzae (now P. grisea). He also reported the production of proteolytic enzymes by the

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cotton wilt fungus (F. oxysporum f. vasinfectum) and Helminthosporium oryzae, responsible for the brown leaf spot of paddy. In the three organisms — Pyricularia grisea, F. oxysporum f. vasinfectum, and Helminthosporium oryzae—strong exocellular amino acid oxidases are present. Both amino acid oxidases and proteases help the pathogen to invade the tissues easily and effectively. LIPOLYTIC ENZYMES

Various types of lipids occur in plant cells, the most important of them being the neutral lipids functioning as energy sources, while phospholipids and glycolipids are the main components of cell membranes. Several fungi, bacteria and nematodes are capable of degrading lipids. Plant pathogens produce enzymes that attack major membrane constituents. Lipolytic enzymes, called lipases, phosholipidases, and so on, hydrolyze fatty acids from the lipid molecule by hydrolyzing the acyl ester and phosphate ester bonds of phospholipids. Phospholipase A is not known to be produced by plant pathogens. Phospholipase B is produced by Thielaviopsis basicola, S. rolfsii, and Botrytis cinerea. These enzymes and proteases might affect cell membranes since phospholipids and proteins are major constituents of plant membrane systems. According to Bateman and Basham (1976) more information is needed before any conclusions can be drawn about the importance of phospholipases and proteinases in the destruction of intact plant membranes. INACTIVATION OF ENZYMES

In some host-parasite combinations resistance may be related to inactivation or inhibited synthesis of enzymes by the parasite. In several instances of fungal enzyme inactivation, there is evidence that the inhibitors are of phenolic origin. Phenols and their oxidation products inhibit a wide range of enzymes and their effects on pectic enzymes have been studied by several workers. Byrde et al. (1960) suggested that in Sclerotinia attack on apple fruits, the hyphae grow through the tissue, macerating and killing the cells as they advance. From the time any cell is killed, the intermingling of phenolic oxidizable substances contained in the cell with the oxidizing enzymes supplied by both the host and fungus, leads to the formation of oxidized phenolic substances that inactivate the macerating enzyme. Natural inhibitors of fungal enzymes and toxins in disease resistance have been discussed by Byrde (1963). Deverall and Wood (1961) have suggested that the self-limited lesions of chocolate spot of broad bean are due to the inactivation of the enzymes of the pathogen Botrytis fabae. As Botrytis fabae, through its pectolysis of host cells, begins to macerate the latter products of pectin hydrolysis activate a latent phenolase in leaves. This phenolase then oxidizes polyphenols in host cells and the oxidation products inactivate the pectolytic enzymes. The varietal resistance of certain varieties of cider apples to brown rot has been attributed to their high content of polyphenols in parenchyma of the fruit. Patil et al. (1964) studied the relation of chlorogenic acid-free phenols in potato roots to infection by Verticillium alboatrum. They related the resistance of potatoes to Verticillium wilt to the content of chlorogenic acid in the superficial cells of roots. It is believed that this polyphenol inactivates hydrolytic enzymes during the early phases of host penetration.

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In the wilt of tomato, there is inhibition of pectolytic activity in hyphae invading parenchyma and the lack of inhibition of mycelium in vessels may account for mycelium being restricted primarily to vessels. Oxidized phenols, quinones, and related substances have been employed as chemotherapeutic agents in wilt diseases. Grossmann (1962 a, b and c) used rufianic acid (1,4-dioxy anthraquinone sulfone) which inactivates both polygalacturonases and pectinmethyl esterases. Patil and Dimond (1968b) have shown that polygalacturonase synthesis is inhibted by rufianic acid in Verticillium. Mahadevan et al. (1965) have demonstrated that the extracts of a number of plant species inhibit pectic enzymes in vitro and in some instances, occurrence of such inhibitors in vivo may be associated with resistance to pathogens. Turner (1961) made an interesting contribution to the field of molecular phytopathology while studying the take all disease of oats caused by Ophiobolus graminis. Oat leaves and roots contain a fluorescent glucoside (avenacin) which inhibits the growth of several fungi. Ophibolus graminis var. avenae is pathogenic because it produces a specific enzyme (glucosidase) which hydrolyzes avenacin. Thus, the pathogenic capability of O. graminis var. avenae is dependent on the production of an inhibitor. Albersheim et al. (1969), while discussing the biochemistry of the cell wall in relation to the infective process, state that the repressed synthesis of the polysaccharide-degrading enzymes, which enable some pathogens to attack plants, is the possible mechanism which brings about resistance in certain hostparasite combinations. Colletotrichum lindemuthianum is capable of producing a variety of polysaccharide-degrading enzymes and the virulence of the isolates has been correlated to their ability to produce α-galactosidase. When the alpha or gamma strain of the fungus is grown in a medium containing galactose as the sole carbon source, large quantities of α-galactosidase are secreted. In the presence of either glucose or xylose, α-galactosidase synthesis is suppressed. β-galactosidase appears to be produced constitutively in the alpha strain of C. lindemuthianum, while β-galactosidase is induced when either the alpha or gamma strain of the pathogen is grown in culture with xylose as the sole source of carbon. Patil and Dimond (1968a) have demonstrated that the addition of glucose to the cut stems of Fusarium oxysporum f. lycopersici infected tomato plants reduces both the rate of symptom development and the level of vascular system polygalacturonase activity. Glucose also represses the synthesis of polygalacturonase activity. Horton and Keen (1966) have studied the endo-polygalacturonase and cellulase formed in extracts of onion roots infected with Pyrenochaeta terrestris. The enzyme production in culture is repressed by the addition of glucose. Horton and Keen (1966) have shown that upon the removal of the cotyledons from young onion seedlings infected with P. terrestris the sugar content of the seedling is reduced to about one fourth. This is accompanied by an increase in both endo-polygalacturonase and cellulase activities and an acceleration in disease development. If plants are sprayed with glucose solution or maleic hydrazide, resulting in increased glucose levels in the tissues, there is reduced enzyme accumulation. Bateman (1964) has shown that in the case of bean seedlings infected by Rhizoctonia, infection causes accelerated respiration and this results in the accumulation of Ca++ and other cations in the infected region. The increased level of Ca++ activates pectin methylesterase which in turn, de-esterifies the carboxyl groups of polygalacturonide, converting them to pectic acid. Pectic acid then reacts with available Ca++ to form calcium pectate. The invaded tissue then becomes resistant to attack by polygalacturonases. This limits the size of the lesion. Another way in which plants are protected against cell wall-degrading enzymes secreted by pathogens is by a modification of the primary cell walls.

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The development of resistance in the hypocotyls of maturing red kidney beans to infection by Rhizoctonia solani is correlated with the conversion of pectin to calcium pectate in the hypocotyl cell walls. Calcium pectate is resistant to the polygalacturonase secreted by the fungus. Albersheim and Anderson (1975), while discussing the cell wall and wall degrading enzymes in the infection process, state that a pathogen is incapable of attacking a plant unless the former finds the proper environment to secrete sufficient amounts of endopolygalacturonase to overcome the amount of inhibitor present in the cell walls of the plant. Although there are several endopolygalacturonase inhibitors in the cell walls of dicotyledons, each of them appears to be able to inhibit the endopolygalacturonases of a variety of unrelated plant pathogens. The ability of plants to produce enzymes that are able to degrade the cell walls of pathogen may represent another general defence mechanism. Plants can synthesize large amounts of lysozyme, chitinase and endo-β-1, 3 glucanase. The chitinase of tomatoes appears to participate in the lysis of Verticillium albo-atrum mycelia that occurs during the infection of tomatoes by this pathogen. In some cases, pathogens have also evolved their own defence against the potentially lytic enzymes of their hosts. C. lindemuthianum which infects beans has been shown to secrete a protein that can specifically inhibit the action of such a wall-degrading enzyme present in beans. The inhibitor, an endo-β-1, 3, glucanase is produced in a large amount in bean tissues. TOXINS AND PLANT DISEASES

There is an enormous amount of literature on toxins and their role in plant diseases. Some of the important contributions in this area were made by Gaumann (1954), Ludwig (1960), Deverall (1964), Wheeler and Luke (1963), Dimond and Waggoner (1953) on vivotoxins; host specific plant toxins have been discussed by Pringle and Scheffer (1964); and toxins and plant diseases have been discussed by Kalyansundram and Charudattan (1966) and Husain and Janardhanan (1976). Toxins in plant diseases, their structure and mode of action have been ably discussed by Owens (1969). Wood et al., (1972) have compiled papers on phytotoxins and plant diseases. Host-specific toxins in relation to pathogenesis and disease resistance have been discussed by Scheffer (1976). Phytotoxins produced by plant parasites are reviewed by Strobel (1974). Toxins produced by phytopathogenic bacteria have been discussed by Patil (1974). Some recent reviews are those of Van Alfen (1989) “Reassessment of plant wilt toxins”, Walton and Panacione (1993) on “Host selective toxins and disease specificity”. There are some edited books such as those of Daly and Deverall (1983) Toxins in Plant Pathogenesis, Graniti et al. (1989) Phytotoxins and Plant Pathogenesis, and a review on “Bacterial phytotoxins: Mechanism of action” by Durbin (1991). Although the germ theory of disease became known earlier to the science of plant pathology (Prevost, 1807) than to human pathology, yet the role of toxins in infections and in diseases was first established in human medicine. The term toxin in plant pathology has actually been adopted from the conceptual sphere of human medicine. The idea that pathogenesis might be due to the production of poisons by the pathogen is by no means new, but it is only in recent years that it has been put on a scientific footing. De Bary (1886) showed that plant pathogens produced toxins but experimental evidence to support this hypothesis was available only in 1913 (Hutchinson, 1913). The credit for the discovery of toxin is generally given to Roux and Yersin (1888) who first visualized the presence of the toxin in the disease

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caused by Corynebacterium diptheriae. In the diseased animals he observed symptoms very far from the site of infection. They thought that this may not be due to the microbe itself but to a metabolic product which gets translocated and reveals symptoms at distant places from the actual site of infection. A toxin can be defined as a substance of microbial origin involved in host pathogenesis. Toxins are normally proteins which are antigenic in nature and have antitoxins. In addition, by introducing small doses of a toxin it is possible to develop immunity in the host against the toxin, and the organism producing it. However, this was not known when Roux first visualized the presence of a toxin in the disease caused by the bacterium, Corynebacterium diphtheriae. Plant pathologists have raised doubts about the existence of toxins in phytopathology. Actually, we do not have toxins in the real sense playing their part in the plant disease syndrome, as in human or animal pathology, because none of these satisfies the conditions of a toxin in mammalian pathology. Gaumann (1954) stated, “Micro-organisms are pathogenic only if they are toxigenic: in other words, the agents responsible for diseases can damage their hosts only if they form toxins-microbial poisons that penetrate into the host tissues”. The term toxin was used by Gaumann for all substances produced by the pathogens, including enzymes. The Concise Oxford Dictionary defines a toxin as, “a poison, especially one secreted by a microbe and causing some particular disease”, and a poison as, “a substance when introduced into or absorbed by a living organism destroys life or injures health, especially (popularly) one that destroys life by rapid action and when taken in small quantity”. Dimond and Waggoner (1953) regarded a toxin as a phytotoxic compound secreted by a microorganism. This excludes toxic substances secreted by plant parasites other than micro-organism. Such a restriction is unnecessary. They also recognized vivotoxin as a substance produced in the infected host by the pathogen and/or its host which functions in the generation of a disease, although it is not itself the initiating agent of the disease. Ludwig (1960), in his review, also regarded a toxin as a product of a micro-organism or of a microorganism host complex which acts on living host protoplasts to influence disease development or symptoms. Wheeler (1975) visualizes toxins as injurious substances produced by organisms. According to him, the term is generally restricted to substances active at physiologically low concentrations. Beyond these basic characteristics, there is little agreement on how toxins, with or without modifying prefixes, should be defined. Some follow the practice of excluding enzymes and growth regulators; others believe that one or both of these should be included in the toxin category. Still others restrict the term toxin to low molecular weight substances or to substances produced by micro-organisms. Toxins are different from enzymes in that they do not attack the structural integrity of the tissue but affect the metabolism in a subtle manner. It is the subtlety of action that differentiates toxins from enzymes. Toxins act directly on the protoplasts of cells and substances with the same end effect but an indirect method of action are not toxins. The toxin of Clostridium welchii, lecithinase, even though an enzyme, is a toxin because it disrupts cell membranes. Such enzymes are toxins. CLASSIFICATION OF TOXINS

Wheeler and Luke (1963) defined three broad classes of toxins, namely, phytotoxins, vivotoxins and pathotoxins according to their origin.

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Phytotoxins

Any compound produced by a micro-organism which is toxic to plants is a phytotoxin. Phytotoxins are nonspecific, incite few or none of the symptoms that are incited by the pathogen, and as happens in most cases, show no relation between toxin production and pathogenicity. Lycomarasmin and alternaric acid are examples of phytotoxins. Vivotoxins

Dimond and Waggoner (1953), who coined the term, defined vivotoxin “as a substance produced in the infected host by the pathogen and/or its hosts which functions in the production of the disease but is not itself the initial inciting agent of the disease”. They laid down certain criteria which a substance must fulfil before being called a vivotoxin. The requisites of a vivotoxin are: 1. It must be isolated from the diseased plant but should not be present in the healthy plant. 2. It must be characterized chemically. 3. When introduced in a pure form into a healthy host, it must produce the symptoms of the disease or a portion of the syndrome. Fusaric acid and piricularin are typical examples of vivotoxins. Pathotoxins

Pathotoxins play a causal role in disease production, and produce symptoms characteristic of the disease in susceptible plants (Wheeler and Luke, 1963). These toxins may be produced by the pathogen, host or interaction between them. Piricularin produced by Pyricularia grisea (oryzae) is a typical pathotoxin. Fusaric Acid Fusaric acid (5-n-butyl pyridine-2 carboxylic acid) is produced by several species of Fusarium, mainly belonging to the group Elegans Woolenweber [F. oxysporum (Schl.) Sny. et Hans.) and causing wilt diseases in tomato, cotton, pea, banana, and other plants. Some of these fungi also produce other toxic metabolites which may be closely related to fusaric acid, for example, dehydrofusaric acid with a double bond at the end of the side chains, or entirely different with a low or high molecular weight. Fusaric acid was first isolated as the metabolic product of Fusarium heterosporum Nees, a nonspecific parasite, by Yabuta et al. in 1934. In 1952, it was recognized as an important toxin of Fusarium oxysporum f.sp. lycopersici Sacc., Fusarium vasinfectum Atk., and Gibberella fujikuroi (Saw.) Wr. It is now known to play a pathogenic role in the wilt of tomato and cotton. Subsequently, there has been evidence to show that fusaric acid is involved in the Panama disease of banana caused by Fusarium oxysporum f. sp. cubense E.F. Smith. A possible correlation has been demonstrated between pathogenicity and the amount of fusaric acid production in vitro in respect of several strains of F. oxysoporum f. sp. niveum and F. oxysporum f.sp. pisi. Fusaric acid is called a wilt toxin as it is now known to play a more distinct role in the wilt of tomato and cotton plants than in any other disease. Even when the pathogen is restricted to the root, symptoms like vein clearing and epinasty are manifested on the leaves. This appears to be due to toxic metabolites translocated to distant parts of the plant from the locus of infection. Some of the toxic symptoms on diseased plants could be produced by pure toxin fusaric acid. Moreover, it has been firmly established

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that a concentration of 10– 4 M of fusaric acid is reached in the infected plants, a concentration lethal to the host tissues. The symptoms of fusaric acid injury appear first in the stem and soon after in the leaves in the form of intercostal necrosis of leaves. In the stem it is mainly the cortical tissue over the vascular bundles that is destroyed and light grey-green sunken furrows appear on the stems and gradually spread to the petioles. Next the petioles start to bend downwards. This is called epinasty and this injury is similar to that caused by ethylene. In contrast, the conducting strands in the stems and petioles are apparently uninjured and are not browned (Gaumann, 1957). At higher concentrations, the whole stem tissue is weakened and necrosis of the cortical tissues overlying the vascular bundles in the stem sets in In the leaves, necrosis extends to the interveinal areas, and subsequently, along the whole length of the veins. About 24 hours after toxin intake the leaves curl and become flaccid. Fusaric acid has been detected in various plants after inoculation with their wilt pathogens. Fusaric acid (5-n butylpyridine-2-carboxylic acid) has the empirical formula C10H13O2 N and molecular weight 179. It has the following structural formula:

Pure fusaric acid has a melting point of 98-100°C, but biogenic preparations often contain traces of dehydrofusaric acid, and therefore, possess a higher melting point (up to 109°C). The existence of dehydrofusaric acid in the prepration of fusaric acid has been demonstrated by Stoll (1954). It has the empirical formula C10H11O2 N and molecular weight 177, with melting point, 118–120 0C. It has the following structural formula:

Surprisingly, the unsaturated dehydrofusaric acid appears after the appearance of the saturated fusaric acid in culture filtrates of F. oxysporum f. sp. lycopersici. Fusaric acid is toxic to higher plants and micro-organisms, interfering with their metabolism in various ways. Before the appearance of the visible symptoms—necrotic spots on the leaf blades, the shrivelling and drying of leaves, and the shrinking and wilting of stem and petioles in tomato cuttings—the water permeability of protoplasts and the water balance of the whole plant are altered and the respiratory rate decreases. Fusaric acid inhibits polyphenol oxidase competitively at much lower concentrations than other enzymes and may also react with various phenol derivatives. A comparison of fusaric acid with other pyridine derivatives shows that two parts of the molecule are essential for toxicity. Inhibition of fungal spore germination, yeast respiration, and polyphenol oxidase depends on the presence of a carboxyl group in α-position to the nitrogen. The aliphatic side chain in the β-position determines primarily the effects on water permeability, the severity of the damage increasing with the length of the side chain, methyl to butyl.

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Plant Pathology

Fusaric acid forms stable chelate rings, catching a metal ion between the N atom of the pyridine ring and carboxyl radical in the α-position, forming a 5-membered coordinate link. Of the metals, the ferric ion is most strongly chelated with it. On the basis of the antagonism reported between ferrioxamine B and fusaric acid, it could be conjectured that fusaric acid specifically interferes wth the incorporation of Fe+++ in the porphyrin skeleton. The mode of action of fusaric acid has been discussed by Kalyansundram (1970). Sanwal (1961) described the physiological effects of this toxin, and suggested that it could cause a partial uncoupling of oxidative phosphorylation. According to Deverall (1964), it plays no part in the development of wilt symptoms, although it might act in conjunction with other metabolites. According to Nishimura (1962), fusaric acid is only one of the toxic substances which contribute to the fusarial wilt syndrome. He suggested that one of these – phytonivein—caused wilting; pectic enzymes caused stem flaccidity; and fusaric acid caused leaf necrosis. All these must be present to reproduce the wilt disease syndrome completely. Lycomarasmin Lycomarasmin is a wilt toxin of the tomato wilt pathogen F. oxysporum f.sp. lycopersici Sacc. Described by Clauson-Kass et al. in 1944, this toxin unlike fusaric acid, appears to be specific to F. oxysporum f.sp. lycopersici. Lycomarasmin, when administered to tomato plants, produces injury in the intercostal fields of the leaves and rolling of the leaf tips. The symptoms of tomato wilt simulate the toxic injury manifested by the synergistic action of lycomarasmin and fusaric acid. While fusaric acid has been demonstrated in infected tomato plants, no such direct evidence exists for lycomarasmin. The minimum dose of lycomarasmin that is needed to cause appreciable injury to tomato shoots is 150 mg per kg fresh weight. Lycomarasmin is a polypeptide with molecular weight 277.3 and empirical formula C9H15O7N3. The following structural formula has been tentatively assigned to this toxin (Miller, 1961):

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However, using the powerful technique of NMR spectroscopy in a comparative study of lycomarasmin with aspartic acid, N-acetyl aspartic acid and N (β carboxy-b-amino ethyl) aspartic acid, Hardegger et al. (1963) determined what is currently accepted as the correct formula for lycomarasmin.

— — — —

— — —

COOH

H

—

HOOC

C—NH—CH 2—C—H

CH

N—H

HOOC

CH 2

O== C—N H 2 Lycomarasmin

The toxicity of this substance seems to arise from fact that it can form metal chelates. Thus, the injury caused by lycomarasmin when administered to tomato plants causes a shock phase within the first few hours when there is a depressed rate of water intake as well as transpiration. Soon this is reversed, and the rate of transpiration far exceeds the normal rate, bringing about the wilt. The pathogenic effect of this toxin is partly due to the fact that its molecules, on their way through the tomato shoots, chelate the iron ions of the host which would not be able to migrate themselves, and transport them into the leaves in the form of a lycomarasmin-iron complex. Here, part of the complex is decomposed due to photochemical effects or due to exchange by more potent chelating agents in the cell itself. Thus, on the one hand, part of the lycomarasmin molecules are regenerated as chelating agents and cause appropriate local deficiencies, while on the other hand, iron ions are set free in the ‘wrong’ place lead to an iron plethora. Gaumann and Naef-Roth (1956) assume that the excessive transpiration which occurs due to lycomarasmin poisoning is mainly due to iron injuries. Lycomarasmin without iron has no toxic effect on transpiration. Another aspect of the study of this toxin has been its antagonism to the yeast growth factor, strepogenin, which is an oligopeptide containing glutamic acid. As against glutamic acid which is present in strepogenin, lycomarasmin has aspartic acid, and to this has been ascribed its antagonistic action. This growth factor, when supplied externally reverses some of the toxic symptoms of the lycomarasmin injury in tomato, leading to the conclusion that it may interfere with the strepogenin metabolism of the host. The role of toxin lycomarasmin in the development of disease symptoms has been disputed by a number of workers. Piricularin Pyricularia grisea (formerly P. oryzae) belonging to the group of Hyphomycetes, is the causal organism of the blast disease of rice and has a wide distribution in all rice-growing areas of the world. The symptoms of rice blast disease indicate that the causal fungus produces some very effective toxins.

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Plant Pathology

It appears that two of the metabolic products of the fungus are responsible for the characteristic symptoms of the disease. One of them is identified as α-picolinic acid and the other is piricularin. Piricularin is more active and semispecific in that it affects blast-susceptible rice plants more than resistant ones. Low concentrations of piricularin inhibit the germination of the conidia of P. grisea and the fungus has been shown to produce a piricularin binding protein, a copper oxidase, which binds piricularin and destroys its fungitoxicity but not its phytotoxicity. The ways in which piricularin affects plant tissues are not understood but it induces an increase of polyphenols and oxidases and its toxicity is counteracted by chlorogenic acid, one of the principal polyphenols of the rice plant. It increases respiration and growth at low concentrations but inhibits them at higher ones. Asparagine was found to be the best nitrogen source for the in vitro production of toxins by P. grisea followed by urea, ammonium sulphate, and sodium nitrate. P. grisea tends to lose toxin-producing ability after repeated serial cultures. Piricularin has been assigned a tentative empirical formula of C18H14N2O 3, melting point of 73.5°C and a molecular weight of 306. It is highly stable in water, especially at low pH. The toxic action of piricularin appears to be due to its inhibitory effect on the activity of several enzymes such as peroxidases, catalase, cytochrome oxidase and ascorbic acid oxidase, even at low dilutions. At still lower concentrations (1/1,600,000), the toxin stimulates the respiration and growth of rice plants. This stimulative effect of piricularin causes an increase in all the fractions of organic phosphorus, especially nucleic acid phosphorus and protein phosphorus. The data on RNA content clearly shows a definite increase, especially in the ribosomal RNA. The toxic effect of piricularin is removed by chlorogenic acid or ferulic acid, each of them natural phenolic constituents of the rice plants. It is presumed that the combination of piricularin with these substances is a natural detoxifying mechanism. The inhibitory activity of the toxin to a test organism, Bacillus subtilis, was considerably reduced by the presence of catechol, chlorogenic acid, and caffeic acid, in the toxin extract. Toxin produced in vitro inhibited plumule elongation and oxygen uptake in varieties susceptible to blast but not in those resistant to it. A third substance, pyriculol (C14H 16O4) was isolated from cultures of P. grisea, which causes natural blast lesions and also inhibit the growth of rice seedlings. The compound is a heptaketide (Turner, 1971). Rao and Suryanarayanan (1971), who earlier failed to detect piricularin and α- picolinic acid, were able to identify pyriculol in the culture filtrates of six isolates of P. grisea. In addition to the above three toxins, two more toxic substances have been recorded by Iwasaki et al. (1972), namely, 3,4 dihydro 3,4,8 trihydroxy-1 (2H) naphthelenone and tenuazonic acid. The latter substance has been isolated from infected tissues also, and thus, is a vivotoxin. Alternaric Acid Alternaric acid occurs in the early blight of tomato and potato and is caused by Alternaria solani. Some of the lesions appear to be devoid of the fungus under certain conditions, suggesting that a mobile toxin is involved. Cultures of A. solani produced a dibasic acid—alternaric acid which caused necrotic lesions similar to those caused by the fungus. The toxin also inhibits or severely restricts growth of the fungal germs tubes without actually preventing germination. Alternaric acid is thought to be fairly nonspecific and its significance in early blight is uncertain. Thus, some highly pathogenic strains of the fungus failed to produce it in culture and it is clear that other factors and possibly other toxins are involved. Alternaric acid produced by A.solani has antifungal but not antibacterial activities. In addition, it is highly fungitoxic. This substance is an optically inactive, unsaturated diabasic acid with an empirical

Enzymes and Toxins in Plant Diseases

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formula of C21H30O8. The effect of alternaric acid on the water economy of host plants is similar to that observed with the toxin lycomarasmine but it is highly potent. This substance, or a substance closely related to it, has been detected in the naturally-infected plants and there is considerable evidence to show that it is involved in the disease caused by A. solani. Tabtoxin (or Wild Fire Toxin)

Pseudomonas syringae pv. tabaci produces a pathotoxin called tabtoxin which is host non-specific. The pathogen causes a disease known as wild fire disease of tobacco. This toxin is also produced by other strains of pv. tabaci attacking other hosts such as bean and soybean and other pathovars (subspecies) of P. syringae such as those occuring on maize, oats and coffee. The pathogen causes necrotic spots on the leaves with a yellow halo surrounding the necrotic spots. Sterile culture filtrates of the organism as well as purified toxin, produce symptoms which are similar to those characterstic of wild fire disease of tobacco and other hosts. It is interesting to note that mutants that have lost the ability to produce the toxin (Tox– ) show reduced virulence and cause necrotic spots without a yellow halo. Tox – strains can not be distinguished from P. angulata which causes angular leaf spot of tobacco. P. angulata is now believed to be a nontoxigenic form of P. syringae pv. tabaci. Tabtoxin is a dipeptide composed of a common amino acid threonine, and the amino acid tabtoxinine (not known previously). Tabtoxin itself is not toxic but in the host cells the toxin is hydrolyzed with the release of the toxic moiety tabtoxinine (which is the active toxin) and threonine. Tabtoxinine is toxic to cells because it inactivates the enzyme glutamine synthetase which leads to depleted levels of glutamine with subsequent accumulation of ammonia which reaches toxic levels. Ammonia accumulation leads to the uncoupling of photosynthesis and photorespiration. The thylakoid membranes of the chloroplasts are destroyed which result in the chlorosis and necrosis of the tissues in the leaves.

Phaseolotoxin

Phaseolotoxin (host non-specific toxin) is produced by the bacterium Pseudomonas syringae pv. phaseolicola which causes halo blight of beans and some other legumes. The localized and systemic chlorotic symptoms produced in infected plants are identical to those produced on plants treated with the toxin alone. Plants treated with toxin alone or those infected with the bacterial pathogen show reduced growth of newly-formed and expanding leaves, disruption of apical dominance and the accumulation of the amino acid ornithine.

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Plant Pathology

Phaseolotoxin is a modified ornithine-alanine-arginine tripeptide carrying a phosphosulfinyl group. The toxin affects cells by binding to the active site of and inactivating enzyme ornithine carbamoyl transferase which normally converts ornithine to citrulline, a precursor of arginine. Thus the toxin causes accumulation of ornithine and depleted quantities of citrulline and arginine. Phasaolotoxin also inhibits the synthesis of pyrimidine nucleotide biosynthesis, reduces the activity of ribosomes, interferes with lipid synthesis, changes the permeability of the membranes and results in the accumulation of large starch grains in the chloroplasts. It is believed that the toxin breaks the disease resistance of the host. Tentoxin

Tentoxin (host non-specific toxin) is produced by the fungus Alternaria alternata (previously called Alternaria tenuis). The pathogen causes chlorosis in the seedlings of many plant species. Tentoxin is a cyclic tetrapeptide that binds to and inactivates a protein (chloroplast-coupling factor) involved in energy transfer into chloroplasts, and also interferes or inhibits the light dependent phosphorylation of ADP to ATP. An additional but apparently unrelated effect of tentoxin is that it inhibits the activity of polyphenol oxidases, enzymes involved in resistance mechanisms of plants. There are several areas such as the molecular site of action of tentoxin and the exact mechanism by which it brings about these effects are still not known. There are several other non-host-specfic toxins such as fumaric acid produced by Rhizopus spp. in the almond hull disease, oxalic acid produced by Sclerotium and Sclerotinia spp. in various host plants, and by Cryphonectria parasitica the cause of chestnut blight disease, ceratoulmin produced by Ceratocystis ulmi in Dutch elm disease, fusicoccin produced by Fusicoccum amygdali in twig blight disease of almond and peaches, cercosporin produced by Cercospora spp. and several others which are involved in host pathogenesis but are not host-specific. Host-Specific or Host-Selective Toxins

A host-specific or host-selective toxin is one which only affects plants susceptible to the pathogen producing it. There are several host-specific toxins such as victorin (HV toxin) on the victoria variety of oats, T-toxin produced by H. maydis, now called Cochliobolus heterostrophus, on maize lines having Texas male-sterile (Tms) cytoplasm, HC-toxin produced by Helminthosporium (Cochliobolus) carbonum on specific maize lines, AM-toxin produced by apple pathotype of Alternaria alternata (previously called A mali), HS-toxin produced by H. (Cochliobolus) sacchari affecing sugarcane, ACLtoxin produced by Alternaria citri (lemon race) affecting rough lemons, ACT-toxin produced by A. citri (tangerine race) affecting Dancy tangerine, AK-toxin on Japanese pear, AT-toxin on tobacco. PC-toxin produced up Periconia circinata in sorghum, PM-toxin in corn that has Texas male cytoplasm, and CC-toxin produced by Corynespora cassicola in tomato. Evidence that a particular host specific toxin is involved in a disease can be obtained from the following: 1. Purified toxin reproduces all disease symptoms in the host.

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2. Toxin affects susceptible but not resistant genotypes. 3. Tox– strains are non pathogenic and 4. In crops between Tox+ and Tox– strains there is co-segregation among progency for toxin production and pathogenicity Some host-specific toxins will be discussed here in some detail. Victorin or HV Toxin In the late 1940s, oat crops in the USA were affected by a seedling blight caused by Cochliobolus (Helminthosporium) victoriae in the variety victoria or its derivatives. Although the pathogen was typically localized in the basal portion of infected plants, symptoms appeared on the leaves which often collapsed. Litzenberger (1949) demonstrated that the culture filtrates of the fungus contained a toxin that could reproduce accurately the visible symptoms of the disease. What was particularly interesting about this toxin was that resistant oat cultivars were not affected by it. The name “victorin” was given to this toxin by Wheeler and Luke (1954). The toxin is highly specific in its activity and is claimed to produce the symptoms only in the pathogen-susceptible variety of oats. Thus, victorin was the first host-specific toxin to be described. Since then a number of other toxins with host specificity and an ability to incite all of the symptoms associated with a particular disease have been identified. These are known as host-specific toxins to distinguish them from those which are non-specific. Victorin is by far the most potent and selective pathotoxin known on a dry weight basis; refined preparations produce toxic effects on oat plants susceptible to Cochliobolus (H.) victoriae at concentrations below 2 × 10–4 µg/ml. Its chemical structure has been determined. It is a complex chlorinated, partially cyclic pentapeptide (tricyclic secondary amine), with molecular weight of around 2000. The earliest effect detected was in permeability. This led to the suggestion that the disruption of cell permeability could be an initial event which triggered subsequent pathological changes in many diseased plants.Victorin causes a decrease in C6/C1 ratio of susceptible oat leaves. This provides additional evidence that victorin induces metabolic alterations similar to those found in other diseased plants. Victorin is able to incite toxic symptoms only in the blight-susceptible variety of oats. Perhaps this could be attributed to the presence of a specific enzyme on the surface of the cells of the susceptible and not the resistant variety (Sadasivan and Kalyansundaram, 1966). Cell wall modifications and increased secretory activity were the earliest ultrastructural changes found in victorin-treated tissues. The primary target of the toxin seems to be cell membranes (plasma membranes) where the toxin is believed to be binding to several proteins. But the exact mechanism of its action is still not clear. The widespread use of new crop varieties often results in new disease problems. Thus, the black spot disease of Japanese pears (Pyrus serotina) appeared after the new natural mutant variety, Nijisseiki was introduced. The causal organism was identified in 1917 as a species of Alternaria, later named A. kikuchiana (Tanaka, 1933). Tanaka, in 1933, reported a host labile-a host selective factor produced by the causal fungus in culture. Convincing evidence for a host-specific toxin produced by A. kikuchiana was supplied by Hiroe et al. (1958) and confirmed by Mori (1962). The host-specific toxin was extracted from narurally infected leaves (Ohkawa and Torikata, 1967). The effect of AK-toxin on pear appears to be similar to the effect of HV toxin on oats. AK-toxin caused an increase in electrolyte loss from susceptible but not resistant leaves within 20 minutes of initial exposure.

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Plant Pathology

T-Toxin T-toxin is produced by race T of Helminthosporium (Cochliobolus) heterostrophus, the cause of southern corn leaf blight in the USA. It attacks corn that have Texas male-sterile (Tms) cytoplasm. Corn with normal cytoplasm is resistant to the pathogen. T-toxin is a mixture of long (35-45 carbon) polyketols. The toxin acts specifically on mitochondria of susceptible cells and inhibits the synthesis of ATP. The toxin reacts specifically with a specific receptor protein molecule (URF 13) that is located on the inner mitochondrial membrane of sensitive mitochondria (of Texas male sterile cytoplasm).When T-toxin is present it forms pores in the inner mitochondrial membrane of maize lines with cytoplasmic male sterility. The pores cause loss of mitochondrial integrity which results in selective permeability of the mitochondrial membrane and disease. There are several other toxins which have been reported during the last few decades. Some of these are host-specific while others are non-specific. A list of various toxins with the effects they produce on the host plants or tissues, and the possible mechanism of their action is given in Table 4.1. TABLE 4.1 Fusaric acid

Fusarium oxysporum 5 n butylpyridine f.sp. vasinfectum, carboxylic acid F. heterosporum, F. cubense, F. moniliforme, Nectria cinnabarina, F. oxysporum lycopersici (host: cotton, tomato, banana, rice)

Non-specific vivo-toxin

Lycomarasmin

F. oxysporum lycopersici (host: tomato)

Amino acid derivative

Non-specific

Piricularin

Pyricularia grisea (formerly P. oryzae) (host: rice)

Empirical formula Non-specific activity C18H14N2O3

α-Picolinic acid

P. grisea (formerly P. α-Picolinic acid oryzae) (host: rice)

Pyriculol

P. grisea (host: rice)

C14H16O4

Affects permeability of plant membranes, water intake, depresses respiration and polyphenoloxidase, disturbs oxidative phosphorylation, inhibits Fe-porphyrin oxidases, disturbs balance of inorgainc ions in diseased plants Causes strepogenin deficiency, chelates Fe+++ free and bound, injures permeability of leaf cells. Increases respiration and growth at low concentration and inhibits them at high doses, inhibits the enzyme peroxidase, catalase, cytochrome oxidase and ascorbic acid oxidase Affects catalase, chelates iron in respiratory enzymes. Unknown (Contd.)

Enzymes and Toxins in Plant Diseases

Victorin

Helminthosporium victoriae (host: oat)

Polypeptide linked Host-specific to a tricyclictoxin, not yet secondary amine detected in the host tissues

Periconin or PC toxin

Periconia circinata (which causes milo disease of Sorghum vulgare var. subglabrescens)

Alternaric acid

Alternaria solani (host: potato, tomato) Colletotrichum fuscum (anthracnose of Digitalis) Endothia parasitica (host: chestnut) Fusicoccum amygdali (host: almond, peach)

Probably a polypeptide of fairly low molecular weight. Two toxins-toxin A: peptide + base, B: peptide Dibasic acid Alternaric acid

Colletotin

Diaporthin

Fusicoccin

Tentoxin

Alternaria tenuis

Host-specific toxin, although it remains to be shown that the toxin is a vivotxin Non-specific phytotoxin

Similar to alternaric Non-specific acid, polysaccharide phytotoxin and peptide characteristics Structure similar Phytotoxin to isocoumarins A terpenoid Phytotoxin (a glucoside of carbotricyclic terpene) Cyclo-n-methyl Non-selective dehydrophenyl phytotoxin alanyl-L-n-methyl alanyl

91

1. Primary effect on plasma membranedamages permeability 2. Increases respiration and has secondary effects on several other metabolic systems. Loss of electrolytes from host cells, disruption of chloroplasts, disruption of internal membrane systems,effects on permeability of the cell membranes, leading to the loss of cellular contents, inhibition of root growth, transpiration and auxin induced cell elongation, victorin acts by uncoupling oxidative phosphorylation Increased respiration, loss of cellular materials, decreased ability to incorporate amino acids, interference with membrane functions. Role in disease doubtful, wilting, necrosis and chlorosis Disrupts cell permeability or affects pectic enzymes Necrosis of the conducting vessels. Possesses growth regulatory properties, causes stomata to open Reduction in chlorophyll content, inhibits photophosphoylation, causes stomatal closure (Contd.)

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Plant Pathology

Altenuene

A.tenuis (brown spot of tobacco)

Structually related Selective to alternariol

Induces chlorosis on the leaves

Wildfire toxin (Tabtoxin)

Pseudomonas syringae pv.tabaci, P. angulata, P. coronofaciens (host: tobacco)

Non-specific pathotoxin

Formely thought to interfere with methionine metabolism but recent studies indicate that it may act by inhibiting glutamine synthetase activity

Rhizobitoxin

Rhizobium japonicum (host: tobacco)

Phytotoxin not host specific

Sole cause of chlorosis in plants infected, inhibits βcrystathionase

Alternariol monomethyl ether Tenuazonic acid

A. tenuis

Wooley et al. (1952, 1955) proposed that the wildfire toxin is composed of an amino acid tabtoxinine linked to lactic acid by a lactone ring, Sinden and Durbin (1970) failed to detect lactic acid but did find that threonine and serine were present Basic sulphur containing amino acid presumably composed of two molecules of an ether derivative of homoserine linked through thioester bridge Alternariol monomethyl ether Tenuazonic acid

-

Inhibits phenol metabolism of the diseased plants Chlorotic haloes, inhibits the release of newly formed proteins from the ribosomes and thus inhibits further synthesis.

Amylovorin

Alternaria longipes (leaf spot of tobacco), A. alternata (leaf blight of Datura innoxia), Pyricularia grisea (oryzae) (blast of rice) Erwinia amylovora

A refined polypeptide antibiotic

Vivotoxin

Non-specific

Broad spectrum, antibiotic, disrupting cellular permeability, induced shoot cankers and necrotic leaf spots (Contd.)

Enzymes and Toxins in Plant Diseases

HC toxin

HS toxin

Helminthosporium carbonum (host: corn) Helminthosporium sacchari (host: sugar cane)

Cyclic polypeptide

93

Host-specific phytotoxin

Effect on nitrate uptake, reduction of corn tissues

Glycoside Host-specific helminthoselective sporiside (2 hydroxy cyclopropyl-D galactopyranoside) Chemical structure Hostnot yet established specific (appears to be a ninhydrin positive low molecular weight substance)

Disruption of chloroplast lamellae. Plasmalemma disruption

HM-T toxin

Helminthosporium maydis race T (host: corn)

AK toxin (phytoalternarin)

Alternaria kikuchiana (host: pear) Phyllosticta maydis (host: corn) Phyllosticta maydis (host: corn)

Has not been identified chemically

Helminthosporium sativum (host: wheat, barley) Helminthosporium oryzae (host: rice) ,

Sesquiterpenoid

Phytotoxin, non-specific

Effects similar to T-toxin, plants with Tms cytoplasm are more susceptible, mitochondria are affected Inhibits respiration in root tissues of barley and wheat

Oxalic acid

Phytotoxin, non-specific

Phenol metabolism is affected

Phytotoxin

Necrosis

Phytotoxin

Symptoms develop in winter when snow and ice might prevent the gas from escaping

PM toxin

Helminthosporal Ophiobolin A, formerly known as cochliobolin Oxalic acid

Hydrogen cyanide

Aspergillus niger (crown rot of groundnut seedlings), Sclerotinia sclerotiorum Unidentified psychrophilic basidiomycetous fungus responsible for white crown rot (snow mould of alfalfa)

Hostspecific

Hostspecific

HCN

Swelling of mitochondria and loss of respiratory control, initially affects respiratory centres, alters membrane permeability. HM-T toxin stops light induced K +uptake by guard cells, thus preventing opening of stomatas. Perhaps causes damage to cellular membrane

(Contd.)

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Plant Pathology

Fumaric acid

Pseudomonas phaseolicola toxin Pseudomonas solanacearum toxin

Rhizopus sp. (in the hull rot of almonds associated with the rotting of the fruit mesocarp of almond) Pseudomonas phaseolicola (host: bean) Pseudomonas solanacearum (host: potato)

Either fumaric acid or its derivative

Non-selective pathotoxin

The toxin is translocated from the affected fruit and causes blighting of adjacent leaves and twigs

Polysaccharide

Pathotoxin

Polysaccharide

Pathotoxin

Inhibits ornithine carbamoyl transferase activity Blocks water flow in the vascular system

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Tribe H.T. (1955), “Studies on the physiology of parasitism-XIX: On the killing of plant cells by enzymes from Botrytis cinerea and Bacterium aroideae”, Ann. Bot., 19: 351–368. Turner, E.M.C. (1961), “An enzymic basis for pathogenic specificity in Ophiobolus graminis”, J. Exp. Bot., 12: 169–175. Van Etten, H.D. and D.F. Bateman (1969), “Proteolytic activity in extracts of Rhizoctonia infected bean hypocotyl”, Phytopath., 59: 968–972. Van den Ende, G. and H.F. Linskens (1974), “Cutinolytic enzyme in relation to pathogenesis”, Ann. Rev. Phytopath., 12: 247–258. Vidhyasekaran, P., C. Paramkaramani and C.V. Govindaswamy (1973), “Role of pectolytic enzymes in pathogenesis of obligate and facultative parasites causing sorghum diseases”, Indian Phytopath., 26: 197–204. Vidhyasekaran, P., G. Mithuswamy and C.L. Subramanian (1966), “Role of seed-borne microflora in paddy seed spoilage-1: Production of hydrolytic enzymes”, Indian Phytopath., 19: 333–341. Waggoner P. E. and A. E. Dimond (1955), “Production and role of extracellular pectic enzymes of Fusarium oxysporum f. lycopersici”, Phytopath., 45: 79–87. Ward, H.M. (1888), “A lily disease”, Ann. Bot., London, 2: 319–382 Wilson, C.L. (1973), “A lysosomal concept for plant pathology”, Ann. Rev. Phytopath., 11: 247–272. Wood, R.K.S. (1961), “Verticillium wilt of tomatoes—the role of pectic and cellulolytic enzymes”, Ann. Appl. Biol., 49: 120–139. Wood, R.K.S. (1960), “Pectic and cellulolytic enzymes in plant disease”, Ann. Rev. Pl. Physiol., 11: 299–322. Wood, R.K.S. (1967), Physiological Plant Pathology, Blackwell Scientific Publ., Oxford. pp. 570. Zaitlin, M. and D. Coltrin (1964), “Use of pectic enzymes in a study of the nature of the intercellular cement of tobacco leaf cells”, Pl. Physiol., 39: 91–95. For Toxins Clauson-Kass, N., P.A. Plattner and E. Gaumann (1944), “Uber ein welkeerzeugendes stoffwechselprodukt von Fusarium lycopersici”, Sacc. Ber. Schweiz Botan. Ges., 54: 523–528. De Bary, A. (1886), “Uber eingie Sclerotinien und Sclerotien Krankheiten”, Bot. Z., 44: 377–474. Deverall, B.J. (1964), Quoted in Microbial Behaviour in vivo and in vitro, H. Smith, and J.Taylor (Eds.) Cambridge University Press, London, pp.165–186. Dimond, A.E. and P.E. Waggoner (1953), “On the nature and role of vivotoxins in plant disease”, Phytopath., 43: 229–235. Durbin, R.D. (1991), “Bacterial phytotoxin: Mechanism of action”, Experientia, 47: 776–783. Gaumann, E. (1954), “Toxins and plant diseases”, Endeavour, 13: 198–204. Gaumann, E. (1957), “Fusaric acid as a wilt toxin”, Phytopath., 47: 342–357. Gaumann, E. and S. Naef-Roth (1956), “Uber die chelierende wirkung einiger welke toxine IV. Die Verschiebungen der Toxizital durch steigende Alssa Higung mitverschiedenen Schwermetallionen”, Phytopath. Z., 16: 257–288. Graineti, A. et al. (Eds.) (1989), Phytotoxins and Plant Pathogenesis, Springer-Verlag, Berlin. Hardegger, E., P. Liechti, L.M. Jackman, M. Boller and P.A. Platner (1963), “Welkstoffe aud Antibiotica Die Konstitution des Lycomarasmins”, Helv. Chem. Acta., 46: 60–74. Husain, A. and K.K. Janardhanan (1976), “Role of toxins in plant diseases”, in: Glimpses in Plant Research, P.K.K. Nair (Ed.), Vikas, New Delhi, pp. 148–186. Hutchinson, C.M. (1913), “Rangpur tobacco wilt”, Deptt. Agr. India, Bact. Ser., 1: 67–83. Iwasaki, S., H. Muro, S. Nozoe, S. Okuda and Z. Sato (1972), “Isolation of 3,4-dihydro-3,4,8-trihydroxy-1(2H)naphthalenone and tenuazoic acid from Pyricularia oryzae”, Tetrahedron Letters, 1: 13–16. Kalyansundaram, R. (1970), “Mode of action of Fusaric acid”, In: Plant Disease Problems, Proc. First Int. symp. Pl. Pathol., IARI, New Delhi, pp. 135–142.

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QUESTIONS 1. What do you understand by cell wall degrading enzymes? How are these involved in the production of disease syndromes? Explain with reference to soft rots in vegetables or fruits. 2. How will you classify cell wall degrading enzymes on the basis of their substrate utilization and mode of action? 3. What do you understand by the mechanism of disease development in soft rots? 4. What are “macerating principles”? Explain their role in disease development. 5. What are toxins? How are these involved in the development of vascular wilt syndrome? 6. Explain the following: i. Host-specific toxins ii. Vivotoxins, phytotoxins and pathotoxins iii. Role of proteolytic enzymes in pathogenesis iv. Role of cutinolytic enzymes in pathogenesis v. Role of lignolytic enzymes in pathogenesis. 7. What is the role of toxins in host-specificity? 8. What do you understand by mycotoxin, phytotoxin, pathotoxin and vivotoxin? Explain by giving suitable examples. 9. Write in brief about the chemical ‘weapons’ utilized by the pathogens in disease development. 10. What do you know of the detoxification of enzymes and toxins? 11. What is the mechanism of action of the following host-specific (selective) toxins(i) Victorin (ii) T-toxin (iii) Ak-toxin 12. The discovery of host-specific toxins raised hopes that the biochemical basis of pathogenicity, and conversely host resistance, might turn out to be simple, determined by a right compound of major effect. Overall these hopes have not been fully realized. Why? 13. What can be the evidences for involvement of a hosts specific toxin in plant disease?

5 Alteration in Plant Physiological Function due to Plant-Pathogen Interaction

PERMEABILITY CHANGES IN DISEASED PLANTS

Permeability phenomena in plant diseases have been reviewed by Wheeler and Hanchey (1968). The opening paragraph of their review article is of great relevance: “Ignorance rarely goes unmasked. Thirty years ago, our ignorance of the fundamental nature of plant disease wore a physiological mask. Today, permeability is in fashion tomorrow nucleic acids, masked or unmasked, will probably be the vogue.” There is also a review by Wheeler (1976). In addition, a number of reviews have dealt completely or in part with the effects of phytotoxins on permeability (Wheeler and Luke, 1963; Owens, 1969). Dube and Srivastava (1975) have discussed altered permeability as a trigger for pathogenesis. Membranes are composed primarily of lipids and proteins, with small amounts of carbohydrate. These are arranged as a bilayer of phospholipid molecules with the ionic groups facing outwards and the nonpolar ends of the phospholipids facing inwards with embedded protein molecules protruding on either side. These proteins may move within the fluid bilayer. It is generally accepted that all water relations are controlled by the plasma membrane although in plant cells the rigid cell wall is also important in maintaining turgor. Due to its semipermeable nature, the plasma membrane is of central importance in regulating the passage of ions and organic molecules into and out of the cell. Thus the plasma membrane is a very complex and dynamic structure which maintains a suitable intracellular environment for various activities of the cell. Similar membranes also occur in cell cytoplasm as endoplasmic reticulum and delimiting various organelles. Membranes are responsible for ion transport. There are two methods of ion transport. One is by passive diffusion in response to concentration gradients. The other method is against a concentration gradient and against electrochemical gradients by active transport in which ions are pumped across membranes using metabolic energy, in the form of ATP, and mediated by ATPases. Membrane permeability plays a vital role in this inward and outward movement of substances. Among the several disturbances that a sick plant shows, some are primary while others are secondary. Regardless of the disease type or the nature of the pathogenic agent, changes in permeability are

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present in all diseased tissues which have been examined. Changes in permeability have been detected before any other symptom of the disease appeared or in advance of the pathogen. This, plus the fact that changes in permeability can be caused by a variety of biological, chemical or physical agents, led to the suggestion that a change in cell permeability might be the initial event in pathogenesis. The first case of altered permeability was reported by Hutchinson (1913) for Rangpur tobacco wilt caused by Pseudomonas solanacearum. Thatcher (1939, 1942, 1943), however, made the first comprehensive study of the extent of permeability changes in plant diseases, that is rusts, powdery mildews, soft rots, dry rots, and wilts and concluded that: 1. Cells of the fungus pathogens have higher osmotic pressure than those of their hosts. 2. Altered permeability of the host cells is consistently associated with disease. 3. For rusts, which were most extensively studied, an increase in host cell permeability characterizes susceptibility, while decreased permeability results in resistance. The biotrophic fungus which forms haustoria establishes a very specific relationship with the host cell membranes. These fungi do not cause massive membrane damage since this would kill their host cells. The plasma membrane of the host and the pathogen remain in very close contact near the point at which the cell wall is penetrated. As the fungal haustorium grows the host membrane becomes invaginated (extra haustorial membrane) to accommodate the fungus and seals form at this point. The extra haustorial membrane lacks ATPase, membrane bound enzymes which control pumping of H+ ions out and K+ ions in, across the membrane. The haustorium of the pathogen can deplete solutes from the matrix which surrounds it and maintains a high concentration gradient. The fungus thus maintains a tight control over membrane permeability. Thus biotrophic pathogens also increase the permeability of host tissues. Cells penetrated by haustoria can still be plasmolyzed so the integrity of the membrane is maintained but there is a definite change in the semipermeable properties of the plasma membrane which is related to the uptake of nutrients by the pathogen. The extra haustorial membrane loses control of nutrient transport so that sugars pass freely to the pathogen. Ionic imbalance has been reported in Verticillium wilt of cotton and blast disease of rice by Dube, (1971, 1973). Bateman and Millar (1966), Beckman (1964) and Kuc (1966) related calcium to resistance in plants infected by Rhizoctonia solani. Membrane damage has been observed in the electronphotomicrographs of infected tissues. Hall and Wood (1970) and Mount and Bateman (1970) have reported that endo-PGTE, a pectic enzyme, damages plasma membranes and causes the leakage of ions. The rupturing of plasma membrane occurs at plasmodesmata as a result of excessive influx of water from the hypotonic solution (formed by the enzymic dissolution of middle lamella) into the host cells. Spalding and Abdul-Baki (1973) have shown that maceration and cell death were closely correlated when potato discs were treated with a purified pectinmethyl trans-eliminase produced by Penicillium expansum. Tissues exposed to pectic enzymes undergo changes in permeability before cells are separated or killed; the relationship among these phenomena is not clear at all. If tissues are plasmolyzed in hypertonic solutions, cells death and permeability changes can be greatly delayed during exposure to pectic enzymes but such conditions have little effect on the maceration. Attempts to correlate tissue permeability with disease resistance have yielded variable results. It had been shown that when the leaves of Minden wheat were exposed to chloroform vapour, there was an increase in the permeability of the tissue and there was definitely an increase in the susceptibility to rusts. However, Johnson and Johnson (1934) found no correlation between osmotic pressure and resist-

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ance to rusts. The susceptibility of several varieties of wheat to rust and cotton of Fusarium wilt has been correlated to the quantities of organic substances secreted. In an interesting work done by Stall and Cook (1979), they recorded the levels of conductivity, measuring leakage of electrolytes released from leaves of pepper plants inoculated with three races of the bacterium, Xanthomonas campestris pv. vesicatoria. Disruption of membranes and electrolyte leakage occurred much earlier and at a much greater rate when the leaves were inoculated with bacterial races carrying virulent genes while release of electrolytes occurred later and at a slower rate with a virulent race of the pathogen. The substitution of pathogens by toxins has greatly helped in understanding the various steps in pathogenesis. Linskens (1955) treated tomato tissues with fusaric acid and lycomarasmin. Ions of Na+, K+, Ca++, as well as free amino acids were detectable in leaf exudates after treatment with fusaric acid and lycomarasmin. This gave support to Gaumann’s theory that damage of plasma membranes was the primary cause of pathological wilting of plants. Victorin, a host-specific toxin (pathotoxin), produced by Helminthosporium victoriae, which causes victoria blight of oats, provided a lot of information (Wheeler and Luke, 1963). Wheeeler and Black (1963) noted the passive leakage of large quantities of electrolytes and increased oxygen uptake in victorin-treated leaf tissues. However, victorin has no effect on isolated mitochondria. Thus, increased respiration of tissues under toxemia was not the result of direct action of victorin on mitochondria (Black and Wheeler, 1966; Wheeler and Hanchey, 1968). Apparently some site other than the Krebs cycle is affected which brings about increased oxidation rates in an unknown way. The electrolyte leakage from a victorin-treated tissue was very quick (within two minutes) and it was almost an instant response (Samaddar and Scheffer, 1968). That permeability damage is a critical event which ensues much before the loss of respiratory control is proved beyond doubt in the experiment with victorin. Respiratory changes occur as the secondary effects of toxin action. Mechanisms responsible for membrane permeability alterations under pathogenesis still remain unknown. According to one hypothesis these toxins combine with some unknown component or receptor in the susceptible cell and thus bring about its disorganization. Phosphatidase, tyrosinase and also pectic enzymes have been implicated in membrane damage. The rupture of the plasma membrane at the site of plasmodesmata has been suggested by Hall and Wood (1970) to explain the leakage of electrolytes from endo-PGTE treated tissues. It has been suggested that in the bacterial-hypersensitive reaction, an inducer disrupts the tertiary structure of the membrane protein by breaking the disulfide bond. SH-containing compounds, such as mercaptans, bring about similar splitting. Thus, membrane stability and self-repair may be crucial determinative factors in susceptibility and resistance. Some phytoalexins have been shown to cause membrane damage as well as other effects on the host plant. The phytoalexin pisatin, influences stomatal opening by affecting membrane function. Leakage of electrolytes has also been reported in virus-infected tissues, especially roots, but this does not seem to be a common symptom in other diseases . EFFECT OF PLANT PATHOGENS ON TRANSLOCATION OF WATER AND NUTRIENTS IN THE HOST PLANT

Many plant pathogens interfere in one or more ways with the absorption of water and uptake of nutrients and their translocation in plants. In many diseases such as the damping off fungi, the root rotting fungi, bacteria, nematodes and viruses where the pathogens bring about extensive damage or destruction of the

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roots, absorption of water is considerably reduced. Root injury directly affects the number of functioning roots and decreases proportionately the amount of water absorbed by the roots. In vascular wilt diseases there is resistance to water flow which may be due to physical blockages which obstruct the vascular system by the fungal biomass (as in Verticillium spp. Fusarium oxysporum and F. solani), or action of fungal toxins and enzymes. There may be host plant responses which may lead to xylem vessel blockage. The formation of tyloses is often reported, particularly as host response to vascular wilt infections. Xylem parenchyma cells baloon through pit membranes and very effectively clog or close xylem vessel lumen. Gels and polysaccharide gums are also produced by the host plant, probably in response to the release of cell wall degrading enzymes by the invading pathogens. These may also obstruct xylem vessels, often accumulating at perforation pits. Dysfunction of the water system has been discussed by Talboys (1978) and Isaac (1992) and dysfunction in the flow of foods by Zimmerman and McDonough (1978). Distrubed mineral nutrition has been discussed by Huber (1978). Ion transport is also interfered with as a result of infection. Ions diffuse differently through solution than through pure water. Their diffusion coefficients are greatly reduced in soil. PO3-4 diffuses through soil very slowly and a depletion zone may occur around the active absorbing roots. It is therefore, very essential that new root growth occur in growing plants. Ca+2 moves through plant tissues with water and enters the symplast at the casparian strip. Suberization blocks the uptake of Ca+2, Mg+2 and Fe+2ions. K+ and PO43– ions are taken up by the symplast in the root cortex, so that increased suberization of maturing roots does not affect the uptake of these ions. Increased transpiration rates leads to increased ion uptake and such as K+ which accumulate at the infection sites. In take all disease of wheat caused by Gaeumannomyces graminis var. tritici, infected plants are seen to lead to the reduction of K+, P and Ca+2. As far as organic nutrients are concerned these are produced in the leaf cells through photosynthesis and move through plasmodesmata into adjoining phloem elements. From there they move down the phloem sieve tubes and eventually again through plasmodesmata they move into the protoplasm of living photosynthetic cells, where these are either utilized or stored into storage tissues. Plant pathogens may interfere with the movement of organic nutrients from the leaf cells to the phloem, with their translocation through the phloem elements, or possibly, with their movement from the phloem into cells that will utilize them. Biotrophs such as powdery mildew fungi and rusts cause an accumulation of photosynthetic products as well as inorganic nutrients in the areas invaded by the pathogens. As indicated earlier, in these diseases the infected areas are characterized by reduced photosynthesis and increased respiration. However, it has been seen that the synthesis of starch and other compounds as well as dry weight are temporarily increased in the infected areas. In some viral diseases such as leaf curling type and some yellows diseases, starch accumulation in the leaves is mainly the result of degeneration of the phloem of infected areas. There is another possibility in the interference with translocation of starch in that the virus inhibits the enzymes involved in the breaking down of starch into smaller translocable molecules. PHOTOSYNTHESIS

Photosynthesis is a basic function of green plants that enables them to transform light energy into the energy of chemical bonds, literally, photosynthesis means “synthesis with the help of light”. This covers a variety of processes in organic and inorganic chemistry. However, the term is usually applied to one

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reaction only—the synthesis of organic matter by plants in light—a process also called carbon assimilation. In the basic reaction of photosynthesis, carbon dioxide from the atmosphere and water from the soil are brought together in the chloroplasts in the presence of light. They react to form glucose with the concurrent release of oxygen. Light

6CO2 + 12H2O ¾¾¾® C6H12O6 + 6O2 + 6H2O chlorophyll

D Go = 686 kcal Carbon dioxide is reduced to the oxidation-reduction level of a carbohydrate, as symbolized by CH2O. Each molecule of CO2 fixed into a carbohydrate requires a net input of 1,18,000 calories from light. As an inherent feature of this process, light energy is also converted to chemical energy by a process known as photosynthetic phosphorylation, where light energy is stored in the useful high energy organic phosphate compound – adenosine triphosphate (ATP). The details of the perturbations in the phenomenon of photosynthesis by plant pathogens have received little attention and there is scanty information available on this subject. The carbon balance of diseased plants had been reviewed by Daly (1976) and bioenergetic and metabolic disturbances in diseased plants by Hutcheson and Buchanan (1983). Principal attention has been paid to the effect of the pathogen on the overall rate of the photosynthesis of plants because the photosynthetic apparatus—the chloroplast—is destroyed or disturbed by viral, fungal or bacterial pathogens. Our understanding of the perturbations in photosynthesis as a result of infection will not be complete unless we study the mechanism in a healthy plant. Our description of the process of photosynthesis in healthy plants will be very brief. The pigments, enzymes and cofactors of photosynthesis are all located in the chloroplast. The chloroplast is an ellipsoidal structure with an axis 3.5 mm long. It is a highly organized structure in which the pigments are found in a series of concentrated lamellae in the regions termed grana. A smaller number of lamellae are also found between the grana. The lamellae consist of an ordered array of chlorophyll, lipid, and accessory pigment molecules (such as carotene) oriented so as to allow the transfer of light from the pigment molecules which absorb the photons of light to the pigment molecules in which the energy is ultimately utilized in photosynthesis. We know that photosynthesis is a two-phase process. In the first phase which is completed in the presence of light, water is broken down (photolysis) and an energy-rich bond or ATP is generated, together with a molecule of NADP which is reduced to NADPH2. ATP and NADPH together form the assimilatory power. In most plants, two light reaction systems—Photo system I and Photo system II—are responsible for the trapping of light energy (Fig. 5.1). The mechanism by which cyclic and noncyclic photophosphorylation are generally considered to be effected by the thylakoid membrane of chloroplasts is shown in Figure 5.1. The heart of the system is a light-driven transport of electrons from two different active chlorophylls (P680, P700) to each of the two acceptors AII and AI . In the non-cyclic reactions, electrons from water flow through two photochemical reactions (Photo system II and I) to ferredoxin and then the NADP. In the cyclic reactions, electrons flow through a single photochemical reaction (Photo system I) and cyclic in a closed system without net oxidation or reduction.

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ADP + Pi

- 0.6

Redox Potential Em (volts)

- 0.2

AII

ADP + Pi

+ 0.4

e- hn

+ 0.6

O2 H2O

Fd

Fp

NADP+

e- hn

PQ

+ 0.2

AI

cyt b6

0

+ 0.8

Fd

ATP

- 0.4

P680

cyt f

ATP

PC P700

Photon

Photosystem I

Photon

Photosystem II

Fig. 5.1

Mechanisms of non-cyclic and cyclic photophosphorylation in chloroplosts. A11, primary electron acceptor of Photosystem II; PQ, plastoquinone, cyt. f, cytochrome f, PC, plastocyanin; P 700, reaction centre chlorophyll of photosystem I, A1, primary electron acceptor of photosystem 1; Fd, ferredoxin; Fp, Favoprotein (Ferredoxin-NADP reductase) cytb6, cytochrome b6

Carbon-Reduction Reactions

The second phase of photosynthesis is completed in the absence of light. CO2 is reduced to form a carbohydrate molecule through a series of reactions by the utilization of assimilatory power generated in light period. The dark reactions are called the Calvin-Benson cycle. Alternative Paths of Carbon Fixation

The Calvin-Benson cycle of CO2 fixation into carbohydrates is not the only means by which plants can fix CO2. In 1966, Hatch and Slack discovered that in sugarcane the first stable intermediate is not a 3carbon atom compound such as the one which occurs in the Calvin-Benson cycle but a 4-carbon atom compound, oxaloacetate, another intermediate in plant and animal glucose metabolism. The key feature of this C4 cycle is that the primary carboxylation reaction is catalyzed by PEP carboxylase so that the resulting product is oxaloacetate. This 4-carbon acid is formed by a completely different pathway in which a CO2 molecule is connected with a 3-carbon atom precursor to form the 4-carbon acid. This discovery of Hatch and Slack has added a fascinating page to the history of photosynthesis research, in as much as it is apparent that plants using the 4-carbon atom cycle have a very fundamental difference in physiology as compared to plant using the 3-carbon atom pathway as their main carbon fixation device. The 4-carbon atom pathway plants (sugarcane, corn and certain grasses) have been termed efficient plants by some recent workers for the reason that unlike that 3-carbon plants (or nonefficient plants) they are able to grow fast at high temperatures and light intensities. Furthermore, there is a phenomenon common to many non-efficient plants whereby as much as 30% of CO2 initially fixed into carbohydrates compounds is released as CO2 through a light-dependent process called photorespiration. The efficient plants also have a photorespiration pathway that can degrade carbohydrates back to CO2 and water, but it is compensated for by a very efficient system that traps any CO2 released by photorespiration and incorporates it into the 4-carbon oxaloacetate. Therefore, all the

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CO2 fixed into carbohydrate remains and can be stored as starch or used for plant growth. Many of the tropical plants growing in the region of high light intensity and temperature have been found to employ the efficient 4-carbon pathway which has obvious evolutionary advantages. It is interesting to find that the efficient plants have certain cells with chloroplasts that exhibit different membrane structures as compared to chloroplasts in the cells of non-efficient plants. The efficient plants have the Calvin-Benson 3-carbon pathway along with the 4-carbon path, but the latter predominates. The most diagnostic and easily determined feature of all plants with the C4 pathway photosynthesis is a Kranz or wreath-like leaf anatomy. The anatomy of the typical C3 dicot is characterized by a distinct dorsiventrality of the internal leaf structure as a result of a well defined palisade and spongy mesophyll cell layers, both of which contain numerous chloroplasts. In marked contrast to the wreathlike anatomy of C4 grasses such as maize, in which the vascular tissue is surrounded by a concentric layer of large bundle sheath cells containing numerous starch-filled chloroplasts, this layer is surrounded by one or more rings of mesophyll cells which also contain numerous chloroplasts. The growth of the host is dependent on its ability to synthesize food material from CO2 and water in the presence of light. Many plant pathogens affect this photosynthesis. Some do so because they kill the tissues, such as Phytophthora infestans on potato, while other leaf infecting pathogens, such as viruses, the powdery mildews, and rusts, affect this in a more subtle manner. Kosuge (1978) had reviewed the work on the capture and use of energy by diseased plants. Photosynthesis, as influnced by viral, bacterial, and fungal pathogens is discussed below. Symptoms of viral infections in the leaves of a plant often include characteristic symptoms of mosaics of green and yellow areas in which chloroplasts are reduced in number or show ultrastructural abnormalities such as swelling and fewer lamellae which are the actual sites of photochemical reactions. PHOTOSYNTHESIS AS INFLUENCED BY VIRAL INFECTIONS

Viruses affect the photosynthesis of their host plants considerably, and in advanced infections, photosynthetic rates might be 75–80% of those of uninfected plants. The characteristic symptom of a viral disease often is chlorosis. The intracellular manifestation of chlorosis is either loss of chlorophyll or the breakdown of the chloroplast or both. Peterson and McKinney (1938) reported less chlorophyll in diseased than in healthy tissues as a result of the influence of four mosaic diseases. Roberts et al. (1952) reported a reduction in photosynthetic rates in the case of those virus diseases in which the symptoms were mild or absent as well as the induction of chlorosis, mosaic or local lesion formation. Roberts and Corbett (1965) reported reduced photosynthesis in tobacco plants infected with Tobacco Ring Spot Virus. They demonstrated that a symptomless tissue of low virus content showed reduced photosynthesis (O2 evolution) per unit of chlorophyll. This suggests that in this case, the perturbation of photosynthesis does not result from chlorophyll loss; in fact, this type of tissue has a slightly increased chlorophyll content per unit leaf area and appears greener than the uninfected tissue. Apparently, other components of the phytosynthetic apparatus are affected before the conversion of chlorophyll to the chlorine ring containing magnesium (the chlorophyllide) and chain alcohol (the phytol).

chlorophyllase ¾ ¾¾¾¾¾¾® Chlorophyllide + phytol ¬¾¾¾¾¾¾¾ Chlorophyllase enzyme located in the chloroplast is involved in what is probably the fist step in the destruction of chlorophyll in vivo. Chlorophyll is removed from the lamellar structure of the chloroplast and chlorophyllide crystallizes within the cell. Chlorophyll

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Owen (1957b) reported an increase in the photosynthetic rate of tobacco leaves infected with mosaic virus which was confirmed by Zaitlin and Hesketh (1965). However, Doke and Hirai (1969) found no difference until the development of mosaic symptoms. A reduction in photosynthesis in tobacco carrying tobacco etch virus was reported by Owen (1957a) who maintained that decline began two days after inoculation. This can be explained by a combination of the reduction in chloroplast number and the very low efficiency of fixation by chloroplasts. Tu et al. (1968) reported an 18% decrease in chloroplast size and number of leaves of corn systemically infected by maize dwarf mosaic and a decrease of 24% in total chlorophyll. Reduction in photosynthesis was 31%. Jensen (1968) found in barley plants infected with barley dwarf virus, a reduction of 50% photosynthesis per unit of chlorophyll as compared to that of healthy leaves. This discrepancy may be partly explained by the reported increase in dark respiration (Tu et al., 1968), while the data by Jensen (1969) cannot be explained in this manner. When expressed on a comparable dry weight basis, dark respiration, in contrast to photosynthesis, is unafffected by disease. Stimulated photorespiration during the infection of this C3 plant may be the reason for this phenomenon. According to Hutcheson and Buchanan (1983) viral infections can alter photosynthesis in either of two ways depending on the disease: (1) As exemplified by squash plants (Cucurbita pepo) systemically infected with squash mosaic virus in which there is a shift in the product from sugars to amino acids (particularly alanine), an increase in cytoplasmic ribosomes, and fewer chloroplasts. There was no difference between the healthy and diseased chloroplasts with respect to most parameters tested: sensitivity of CO2 assimilation to DCMU and Antimycin A; ultra centrifugation profile; products of photosynthetic CO2 assimilation; activity of the enzymes, ribulose 1, 5 – biphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase; chloroplast ultrastructure. Despite the presence of fewer chloroplants, Hutcheson group found, on chlorophyll basis, a slight increase in the rate of CO2 assimilation in infected plants. (2) As exemplified by turnip yellow mosaic virus on photosynthesis, the research groups of Bove (Goffeau and Bove, 1965) and of Matthew (Bedbrook and Matthews, 1972, 1973) used Brassica pekinensis in their studies and it was concluded that the choroplasts were structurally and biochemically altered after infection. Bove’s group reported an increase of both cyclic and non-cyclic photophosphorylation in chloroplasts from infected plants. Matthew’s group observed that infection led to a shift in the products of photosynthesis from sugars to organic acids and amino acids. Some other investigations have also reported such a shift with other viruses. It is interesting to note that with turnip yellow mosaic virus, the viral nucleic acid is formed in the vesicles of the chloroplast envelop and the virus is thus a parasite of the choroplast. Deranged starch metabolism, with either an excessive accumulation or paucity of starch, is a common biochemical symptom of viral diseases. Starch accumulates in the chloroplasts which are affected by viral diseases. Holmes in 1931 observed that tobacco plants infected with TMV failed both to form and to metabolize starch at a normal rate. There was more starch in virus-infected areas. This phenomenon was always consistent; leaves taken at the end of the period of active photosynthesis often contained less starch in virus-infected plants than those taken from the greenhouse and placed in the dark under conditions favourable for the starch to be metabolized. Holmes (1931) found that the infected leaves had a reduced capacity for synthesizing and degrading starch. Robbins (1921) found an accumulation of starch in mosaic leaves of sugar beet, while Narayanaswamy and Ramakrishnan (1965) reported a reduced level of starch in pigeon pea mosaic infected leaves. Vidhyasekaran and Kandaswamy (1972) also observed that the mosaic disease incidence of Phaseolus aureus did not alter the starch metabolism. Starch accumulation is also characteristic of a number of other viral diseases, among them

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potato leaf roll, sugar beet yellows and barley yellow dwarf. It is not known where the virus affects these processes, although it can influence the loss of some enzyme activity from the chloroplast. The inability of some virus-infected plants to degrade starch is also of interest. How starch is degraded within tissues, still requires further investigation. Photosynthesis as Influenced by Bacterial Pathogens

An attack by bacterial pathogens frequently involves chlorotic symptoms, for example, a number of leaf spotting xanthomonads where there is a direct effect on chloroplasts. This is perhaps by the toxic metabolites of the pathogen. Wildfire-toxin or tabtoxin produced by Pseudomonas syringae pv. tabaci suggests that the toxin affects photosynthesis. Tabtoxin as such is not toxic but in the host cell the toxin is hydrolyzed and releases tabtoxinine which is the active toxic molecule. Tabtoxin inactivates the enzyme glutamine synthetase, which leads to depleted levels of glutamine and as a consequence accumulation of ammonia to toxic levels. The latter uncouples photosynthesis and photorespiration and destroys the thylakoid membrane of the chloroplasts, thereby causing chlorosis and eventually necrosis. Photosynthesis may also be affected indirectly by the occlusion of the vascular system by a bacterial pathogen leading to water stress and wilting. This results in the closure of stomata, and consequently, there is inhibition of CO2 absorption. Beckman, Brun and Buddenhagen (1962) ascribed the symptom of wilting in banana (Moko disease) induced by Pseudomonas solanacearum to one of the two causes: (1) vascular occlusion or, (2) a toxic effect which would influence water retention by host cells. Photosynthesis as Influenced by Fungi

Fungal pathogens seem to affect the photosynthetic process by affecting the chloroplast. Marcoscopic observations of many fungal diseased tissues also suggest that the chloroplasts are affected. Rusted wheat leaves and leaves of bean infected with Uromyces phaseoli retained chlorophyll in regions termed green islands at the edge of the infection. Wang (1961) has shown in the case of bean rust, that these regions are photosynthetically active and starch tends to accumulate in them to a greater extent than in the surrounding areas infected by Uromyces phaseoli, while Schippper and Mirocha (1969 a, b) showed the depletion of starch in the same infected tissues. Certain effects on the photosynthetic system may be observed anatomically. There may be chlorosis of the whole leaf. This is due to a reduction in the amount of chlorophyll per chloroplast rather than to a reduction in chloroplast number, at least in Puccinia recondita on wheat. Green islands may appear around a pustule when the remainder of the leaf is yellow. Bushnell and Allen (1962) found that Erysiphe graminis on barley caused the tissues around a pustule first to become chlorotic and then to redevelop chorophyll. Green islands might also be induced by extracts of conidia of E. graminis or urediniospores of Puccinia graminis. Auxins or kinetins may also induce green island formation. It has been shown that in barley kinetin induced island contain more starch but less nitrogen than mildew-induced islands. Electron micrographs show that grana and intergranar lamellae of chloroplasts in leaves infected with E. graminis become progressively more disorganized with the progress of the diseases. Figure 5.2 shows the net photosynthetic rate of oak leaves infected by Microsphaera alphitoides (powery mildew pathogen). There is a slight initial stimulation of photosynthesis in inoculated leaves but this is followed by a gradual decline.

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Fig 5.2

Changes in photosynthesis of oak leaves following infection by the powdery midew fungus Microsphera alphitoides. (From C.H. Dickinson and J.A. Lucas, Plant Pathology and Plant Pathogens, Blackwell Scientific Publications, Osney Mead, Oxford, 1977, p. 94.)

According to Magyarosy et al. (1976), the rate of 14CO2 uptake by leaf discs from sugar beet infected with powdery mildew (E. polygoni) is also reduced as compared to healthy tissues (Fig. 5.3). Chloroplasts isolated from mildewed beet leaves show a reduced capacity to form ATP by non-cyclic photophosphorylation. In powdery mildewed-infected barley leaves it has been shown that there is also a progressive reduction is the activity of several key enzymes of the Calvin cycle. This reduced capacity to form ATP by non-cyclic photophophorylation may be linked to a change in the concentration of soluble carbohydrates in infected tissues, which in turn influences the rate of photosynthetic CO2 fixation. In Arabidopsis leaves infected by Albugo candida, the reduction in the rate of photosynthesis is paralleled by a decrease in the amounts of Rubisco (ribulose biphosphate carboxylase) protein present the host.

Fig 5.3

Effect of infection by Erysiphe polygoni on the rate of photosynthetic 14CO2 assimilation by sugar beet leaf discs. (From Dickinson and Lucas, Plant Pathology and Plant Pathogens, Blackwell Scientific Publications, Osney Mead, Oxford, 1977, p. 95)

Much of the reduced rate of photosynthesis has been attributed to reduction in the photosynthetic machinery of leaf cells. Chloroplasts show ultrastructural changes after infection; sometimes concentration has been shown to be reduced in mildewed barley plants and in barley with brown rust infection. One very interesting observation in case of barley plants with powdery mildew infections and for rusted bean plants is that uninfected leaves exhibit stimulated or enhanced CO2 fixation. This suggests that

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uninfected leaves are able to compensate for the reduced capacity of infected leaves by increasing photosynthesis (Fig. 5.4).

Fig. 5.4

Comparison of 14CO2 uptake by rust-free leaves from healthy (H) and rust-infected (I) bean plants at different times after inoculation. (From Dickinson and Lucas. Plant Pathology and Plant Pathogens, Blackwell Scientific Publications. Osney Mead, Oxford, 1977, p. 95)

In the case of facultative parasites, studies have shown a significant inhibition of photosynthesis within a day after the inoculation of wheat with Septoria nodorum and of ragi with Helminthosporium nodulosum (Vidhyasekaran, 1974). This early inhibiting effect suggests that there are other factors involved in the decline of photosynthesis for apparently healthy leaves with normal chlorophyll content. Changes in starch content following infection have been observed in many foliar diseases. The general pattern is an initial decrease followed by a marked increase with heavy accumulations around the margins of lesions. Still later, the starch content again declines. Starch accumulates in wheat leaves infected with stripe rust. Work with wheat leaves infected with Puccinia striiformis led to the suggestion that changes in the activity of ADP-glucose photophosphorylase during disease development might account for the pattern of changes in starch content (MacDonald and Strobel, 1970). MacDonald and Strobel (1970) assayed these enzymes in the presence of activators (sugar phosphates) and inhibitors (inorganic phosphates) at concentrations found in diseased leaves at various times after inoculation. They reported that the pattern of changes in the activity of this enzyme was similar to that of changes in starch content. Changes in starch in leaves infected with P. striiformis were very similar to those observed in other diseases. In contrast, photosynthesis in infected leaves increases sharply, reaches a peak of twice the normal as symptoms appear, and remains above normal for at least six days after sporulation. These results, which differed markedly from those obtained from other diseases, indicate that P. striiformis infected plants may represent a special case. According to Vidhyasekaran and Kandaswamy (1972), the powdery mildewed (Oidium sp.) infected tissue of Phaseolus aureus showed a severe reduction in both starch and sugar content. The phenomenon of depletion of starch in mildew-infected tissues has already been observed (Mirocha and Zakai, 1966) and the metabolism of starch seems to play an active role in pathogenesis, possibly supplying metabolites to support the growth of the fungus immediately after the penetration of the host as well as during spore formation (Schipper and Mirocha, 1969a). According to Schipper and Mirocha (1969a), this may be due to the activation of starch-hydrolyzing amylase in the infected tissues. The facultative parasites, such as Cercospora, also cause a depletion of starch. Kolandaisamy (1964) reported a reduction in starch content of banana leaves and an initial increase in sugar content of the groundnut leaves infected by Cercosporidium personatum. However, there was a sharp decline in sugar

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content. Vidhyasekaran and Kandaswamy (1972) observed in the case of Cercospora cruenta infecting Phaseolus aureus that even in the initial stages of infection, there was a sharp fall in the sugar content. Jayapal and Mahadevan (1968) also found a quick decline in the sugar content of banana leaves due to Cercospora infection. Although increased respiration reported in the tissues infected with facultative parasites may be a factor in the depletion of sugars, it is also possible that the sugars may form the basic material for the growth and sporulation of the pathogen. The most important exception to the usual sequence of chlorosis and reduced photosynthetic activity is also seen in diseases caused by the rust and powdery mildew fungi. In rust infections colonised leaves become chlorotic but immediately around the infection sites chlorophyll is retained, appearing as a green halo known as green islands. In powdery mildew fungal infections, chlorophyll is initially lost from the leaf but later resynthesized around infection sites (regreening). There has been much discussion of the significance of these green islands. The selective retention of chlorophyll around infection sites suggests that the pathogen exerts some degree of control over host physiology. Photosynthesis in the green island areas occurs at a rate comparable with that in uninfected tissues or even slightly greater rate which it is believed, serves to support the invading fungus through sporulation. RESPIRATION

Respiration is the sine qua non for life. In normal healthy plant cells the function of respiration is twofold—to provide (1) energy and (2) carbon skeletons. Respiration is the oxidation (aerobic or anaerobic breakdown) of organic material, primarily carbohydrates, to simple compounds, H2O and CO2, with the production of utilizable energy for the cell in the form of ATP. In aerobic respiration this takes place with the uptake of oxygen from the air. In fermentation the degradation of organic material is anaerobic. Energy is liberated through the process of respiration. Actually, the oxidation of carbohydrates and related compounds is the main source of energy but energy derived from photosynthetic phosphorylation is also a characteristic of plant metabolism. In the process of respiration energy is not “liberated” as such, it is first redistributed in molecules formed by oxidative reactions, creating bond energy. The energy-rich group generated by oxidation is capable of initiating reactions requiring energy. Hence the coupling of the chemically bound energy to other systems is the key reaction in the biosynthetic processes of plants. One of the simplest substrates for oxidation is glucose. The overall reaction involved in the respiration of glucose is as follows: C6H12O6 + 6O2 = 6CO2 + 6H2O + DG° = 686 kcal. The respiratory process, however, can be divided into two portions : 1. The initial phase known as the Embden-Meyerhof -Paranas pathway or glycolysis, in which each glucose molecule is converted into two molecules of pyruvic acid. 2. The terminal phase (citric acid cycle or Krebs cycle or tricarboxlic acid cycle), in which pyruvate is oxidized to CO2 + H2O, utilizing oxygen as the final hydrogen acceptor. The glycolytic phase can take place in the absence of O2 (anaerobically) and is common both to aerobic respiration and to alcoholic or lactic acid fermentation. The terminal phase is strictly aerobic. If an adequate amount of oxygen is lacking and if anaerobic pathways are favoured, this scheme is altered. Glycolysis occurs but intermediates of glycolysis acting as the final hydrogen acceptors are reduced to other end products. A typical overall reaction for anaerobic respiration is C6H12O6 = 2CH3CH2OH + 2CO2 + 2ATP (ethyl alcohol)

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Note that the energy yield of fermentation is less because the end product is incompletely oxidized, and still stores energy. The discussion of respiration is divided into (1) glycolysis, (2) fermentation end products, (3) CA-cycle. Glycolysis

Glycolysis is the conversion of glucose to pyruvic acid by any biochemical pathway. The most common glycolytic pathway is the Embden-Meyerhof-Paranas pathway, although the hexose monophosphate pathway (HMP) and the Entner-Doudoroff (ED) pathway may provide alternative mechanisms. The interrelationships between the EMP, HMP, and ED glycolytic pathways are given in Fig. 5.5. The EMP and HMP pathways occur in animals, plants, bacteria and fungi, while the ED pathway is restricted to bacteria and fungi. The EMP pathway in fungi is the principal glycolytic pathway as in most of the other organisms and usually accounts for at least 50% of the glucose dissimilated; in some fungi it accounts for all the glucose dissimilated. However, the EMP pathway may be secondary in importance to either the HMP or ED pathway in a few fungi or totally lacking, as in the case of Cladariomyces fumago. The EMP pathway becomes increasingly important under anaerobic conditions as the HMP pathway accounts for all the glucose dissimilated. The HMP pathway is common in plants and animals. The enzymes required to carry out the HMP pathway are widely distributed in fungi and may be universal. The presence of the enzymes, however, is not a proof that the HMP pathway occurs. It is found in a number of fungi, usually accounting for the less than 40% of the glucose dissimilated. The relatively low level of the HMP reactions is perhaps due to limiting amounts of NADP. The HMP pathway is the major route of glycolysis in 5 of the 15 fungi surveyed (Blumenthal, 1965). Glucose ATP ADP Glucose 6-phosphate ATP

NADP NADPH2

ADP 6 Phosphogluconic acid Glyceraldehyde-3 phosphate

Pentoses

2 ADP 2 ATP

2 NAD

2-keto 3 deoxy-6 phosphogluconic acid

2 NADH2 2 ADP 2 ATP Fermentation products ( EM

Fig. 5.5

Pyruvic acid

Citric acid cycle HMP

ED)

The interrelationships between EM, HMP and ED glycolytic pathways

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Respiration in Plants Under Pathogenesis

Plant respiration as an index of disease has been the subject of investigation for a number of years. Increased respiration in tissues generally accompanies a variety of pathological conditions except in plants systemically infected with viruses. Merrett and Bayley (1969) have criticallly examined the special topic of viral infections. A study of the altered respiration of infected plants is also of interest to plant physiologists and biochemists investigating the nature of the respiratory rate. There is some evidence that genotypically-controlled resistance to disease depends on differences in metabolism and respiration, and respiratory rates as indices of such differences seem to open up new lines of interest both to the pathologist and the plant breeder. Prior to 1953, studies on respiratory stimulation in infected plants were confined mainly to the quantitative aspect of the increase. It was Allen (1953,1954) who first pointed out that disease was the result of disturbances in host metabolism induced by the diffusible metabolites (toxins) of the parasite. He postulated that increased respiration in mildew disease was the result of a toxin produced by the fungus which uncouples phosphorylation from electron transfer in host cells. Attempts have been made by several workers to isolate toxic agents inducing stimulation of respiration. Figure 5.6 shows the rate of oxygen uptake in cabbage cotyledons during colonization by Peronospora parasitica as shown by Thorton and Cooke (1974).

Fig. 5.6

Oxygen uptake by healthy cabbage cotyledons and those infected by Peronospora. (From Dickinson and Lucas, Plant Pathology and Plant pathogens, Blackwell Scientific Publications, Osney Mead, Oxford, 1977 p. 90)

Mechanism of Respiratory Increase

In a diseased plant there are a number of changes which accompany increased respiration, including changes to pathways used, increases in the concentration of enzymes involved and changes to the uncoupling of respiration to other related cellular processes. There are a number of theories which seek to explain the enhanced respiration rate in diseased plants (Fig. 5.7). These are: 1. Uncoupling of oxidative phosphorylation 2. Augmented synthetic processes

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3. Changes in respiratory pathways 4. Increased activity of terminal oxidases.

Fig. 5.7

Possible mechanisms of stimulated respiration in diseased plants due to infection

The direct contribution of the parasite in the increased respiration rate is negligible. Uncoupling of Oxidative Phosphorylation

It has been suggested that the pathogens may uncouple host respiration (oxidative phosphorylation). This uncoupling of electron transfer from phosphorylation can be brought about by chemical inhibitors such as 2-4-dinitrophenol (DNP). The synthesis of ATP that accumulates via the electron transfer chain is prevented and ADP accumulates, stimulating respiration and increasing the rate of oxygen uptake. The hypothesis that pathological increases in respiration result from an uncoupling of phosphorylation from electron transport was put forward by Allen (1953). The effect has often been attributed to diffusible substances or toxins produced by invading pathogens e.g. Helminthosporium maydis, H. victoriae, and Gibberella saubinetti. The evidence that the pathogen or their toxins uncouple oxidative

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phosphorylation is proved by the fact respiration in diseased plants in not increased or stimulated even after treatment with DNP as the pathogen or its toxin has already done so. In the presence of oxygen (aerobic condition) glucose is used more efficiently through normal respiration and fermentation is suppressed. This is known as the Pasteur effect. Under anaerobic conditions, however (i.e. in the absence of oxygen) pyruvate cannot be oxidized, instead it undergoes fermentation and yields lactic acid or alcohol. In diseased plants, aerobic carbohydrate breakdown becomes less efficient but at the same time energy requirements are high and ADP levels rise. This results in an increase in respiration rate. The Pasteur effect is lost in a similar way as treatment with DNP. An important gain to the plant by the abolition of the Pasteur effect is the accumulation of intermediates of carbohydrate breakdown which are utilized in the formation of phenolic compounds. Phenolic synthesis is triggered in the diseased plant. Augmented Synthetic Processes

Another cause for the mechanism of respiratory increase is ascribed to the active metabolism accelerating the ATP-utilizing reactions of host tissues. The synthetic processes requiring energy, accelerate ATP breakdown to ADP which in turn causes respiration to rise. In support of the above mechanism, many characteristic features indicating the activation of the metabolism of the host have been reported, such as : 1. Accumulation, mobilization and synthesis of phosphorus and carbon compounds 2. The growth of the host tissue 3. Synthesis of proteins, including activation of enzymes and enzyme systems 4. Increase in protoplasmic work, such as protoplasmic streaming (cyclosis) According to the results of Daly and Sayre (1957), safflower infected with Puccinia carthami exhibited a pathological-stimulated cellular growth and a concomitant increase in tissue respiration. The rise was at its maximum during the sporulation of the parasite. In supporting this abnormal growth of diseased host cells ATP is consumed; thus the breakdown of ATP is accelerated and eventually enhances the respiratory rate of the host. Stimulated growth, accumulation and mobilization of metabolites occurs in rust and bean plants, synthesis of proteins in rotted sweet potato and late blight of potato and increased protoplasmic streaming in late blight of potato. All these demand energy that is ATP and thus respiration is automatically regulated through the control of the level of ADP (phosphate acceptor). Besides the respiratory rate, the temperature of diseased tissues is also higher than that of healthy ones. The increase in temperature is from 0.3 to 0.7oC for mildew and rust diseases (Yarwood,1953). It is not yet known whether respiration of infected plants is less efficient than that of healthy plants. It is possible that the high rate of respiration of infected plants is in part at least, a wasteful one. Changes in Respiratory Pathways

The EMP pathway is the most important pathway for carbohydrate breakdown but this respiratory pathway becomes altered in higher plant tissues as a result of biochemical transformations during differentiation and maturation and it is not surprising that infection with viruses, bacteria, and fungi also brings

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about such changes. The HMP pathway is of minor importance, except in special conditions. The work done in this sphere on healthy and infected tissues strongly suggests that the HMP pathway (pentose phosphate pathway), becomes quite important during infections. The work of Farkas and Kiraly (1955) was the first in this area. Daly et al. (1957) have put forward three arguments for a change in the nature of respiratory pathways in rust diseases. 1. It was observed that at sporulation the ratio of anaerobic to aerobic carbon dioxide production fell below the limit of 0.33 which was expected if only glycolysis and the TCA cycle were operating. 2. Sodium fluoride inhibits the reaction in the EMP pathway by which phosphoenol pyruvate is formed from phosphoglycerate through the activity of enolase. Inaction to sodium fluoride showed that the EMP pathway is not operative. The tracer methodology has been utilized for this purpose by which radioactive carbon is incorporated into either the C6 or the C1 position of the glucose molecule. The ratio of labelled CO2 released from each source during respiration is measured which indicates the predominating pathway. Activation of the pentose phosphate pathway leads to an increased contribution from the C1 position, and hence, the C6/C1 ratio is lower. Infected plants, the C6/C1 ratio is typically lower than the value obtained from healthy tissues, suggesting an increased participation of the pentose route. A shift from the glycolytic to the HMP pathway has been detected in several diseases caused by fungi, bacteria, and viruses (only in the local lesion type). Some of the examples are as follows : 1. Infection of Datura stramonium by TMV 2. Tomato tumour caused by Agrobacterium tumefaciens 3. Halo blight of tobacco caused by Pseudomonas tabaci 4. Rusted and mildewed wheat 5. Potato tubers infected with Phytophthora infestans 6. Safflower hypocotyl infected with Puccinia carthami 7. Tobacco leaves infected with several strains of potato virus 8. Bean hypocotyls infected with Rhizoctonia solani The actual significance of the switch to the pentose pathway is linked to its role in the biosynthesis of various compounds in addition to providing pentoses for the biosynthesis of nucleic acid. Pentose phosphate intermediates are involved in the production of numerous aromatic compounds, particularly phenols and their derivatives. Changes in the operation of the Krebs cycle have been recorded in several plants by studying the effect of malonic acid which inhibits an enzyme system (succinic dehydrogenase) in the cycle of oxygen uptake. Some of the examples are as follows : 1. Rusted and mildewed wheat 2. Potato tuber infected with Gibberella saubinetii 3. In Cercospora infection on groundnut, particularly in later stages of infection. Increased Activity of Terminal Oxidases

In addition to changes in respiratory pathways, alternative terminal oxidation systems may operate in diseased tissues. Apart from the usual cytochrome system terminating with cytochrome oxidase, systems involving phenol oxidases and ascorbic acid oxidases have been detected in plants.

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The reduced coenzyme NADPH produced via PP pathway may be oxidized by a non-cytochrome oxidative system in diseased plants. With the increase in oxygen uptake in diseased plants there may be an increase in the activity of polyphenol oxidases e.g. in diseases caused by Phytophthora, Ceratocystis and Fusarium species. Ascorbic acid oxidation has also been reported to increase as oxygen uptake increases, for example, in cabbage infected by Fusarium oxysporum and rusted wheat plants or oat plants infected with Helminthosporium victoriae. The precise mechanisms and significance of such oxidation systems are not clear but phenol oxidases play an importance part in the production of phenolics which have a bearing on the resistance of the plant. These alternative pathway oxidases are unable to participate in the formation of ATP during oxygen uptake. Relationship between Resistance and Susceptibilty

Resistant and susceptible cultivars have been shown to react differently to infection by fungal pathogens and it has been suggested (Smedegaard-Peterson, 1984) that different mechanisms may operate. In infected resistant cultivars (incompatible reaction) oxygen uptake has been shown to rise sharply quite early in infection, although at a later stage the level is reduced to normal. In infected susceptible cultivars (compatible reaction), there is much less change initially and a rise in oxygen uptake occurs a few days after infection. Many workers have studied the relationship between respiration and resistance to plant pathogenic organisms. Samborski and Shaw (1956) observed that in wheat rust, the resistant variety recorded a steep rise followed by an equally steep fall in respiration while the susceptible variety had a continuous rise in respiration. Some interesting correlations between the respiratory rate and resistance to parasitic attack have been observed at Madras laboratory. Andal (1959) studied the respiratory rate of three varieties of rice differing in their degree of susceptibility to Fusarium moniliforme. It was found that the respiratory rates of both root and shoot tissues were directly proportional to the degree of susceptibility. Similar correlations were obtained by Lakshmanan (1956, 1959) in cotton varieties resistant and susceptible to Fusarium oxysporum f. sp. vasinfectum. Lakshmanan (1956) had further shown that there is a correlation between respiratory activity and induced resistance. Studies by Rubin and his co-workers in the USSR (Farkas and Kiraly, 1958) on potato-Phytophthora, cabbage-Botrytis, citrus-Penicillium and other diseases led them to conclude that resistant varieties respire more intensively than susceptible ones. Balasubramanian et al. (1971) studied the respiratory pattern of Botrytis fabae and B. cinerea on a common host. In the case of B. fabae, where lesions were spreading, the respiratory rate was three times after 24 hours than in the limited lesions caused by B. cinerea. CHANGES IN NITROGEN METABOLISM OF INFECTED PLANTS

In many diseased tissues, especially in those characterised by overgrowths, galls or other growth abnormalities, increased metabolic activity is accompanied by marked increases in nucleic acid and protein

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synthesis. Cytochemica1 evidence for major changes in nuclei, nucleoli, and changes in ribosomes is available. Nucleic Acid Metabolism

No field of biological science has developed and progressed to such an extent during the last few decades as that of nucleic acids because they are involved not only in the transfer of genetic information but also in the regulation of gene activity. Nucleic acid metabolism in infections caused by obligate parasites had been reviewed by Heitefuss (1966). Nucleic acids in host-parasite interactions had been reviewed by Heitefuss and Wolf (1977). Changes in the nucleic acid level have been reported in diseases caused by all kinds of pathogens. The host-RNA content in TMV-infected tobacco leaves increases shortly (8 hours after inoculation), but afterwards (70 hours) the viral RNA increases together with the total increase of RNA. Total RNA increases in infected tissues, while it decreases in the uninoculated control tissues. In various virus-host plant combinations, transitory increases, moderate declines, or no significant changes in DNA have been reported. Such variations probably reflect differences among the plants and viruses studied. In the case of tobacco leaves infected with TMV, one investigator reported that t-RNA increased fourfold after infection but another worker while using similar methods, found no such increase. Thus, the explanation given earlier does not account for inconsistent data in this case. Similarly, a rapid breakdown in ribosomes following TMV infection has not been confirmed. Marked increases in ribosomes have been observed around necrotic areas on local-lesion hosts inoculated with TMV. Gall cells induced by various fungi, bacteria, and nematodes have been shown to contain a very large quantity of nucleic acids. In the case of Plasmodiophora brassicae, causing club root disease of crucifers, the infected cells each contain an enlarged nucleolus which may be as much as 30-or more fold in volume over nucleoli in non-parasitized cells and the DNA content may be sixteenfold greater than in the non-infected cells. Hypertophy of host cells together with increases in the sizes of nuclei have been reported for various other host-parasite combinations. In the same disease, the nucleolus of root hair cells begins to enlarge within two hours after penetration and after twenty hours this volume has increased sixfold. The increase in nucleolar volume has accompanied by an increased nuclear-RNA and non-histone proteins. The lysine-rich portion of histones decreases. Since histones have a regulatory role, any change in their quantity is related to a change in the regulation of gene action. It seems that the normal transcriptional processes of invaded cells are altered by parasites (Williams et al., 1973). A higher level of RNA in the rust-infected enlargement of the nucleolus has been reported by a number of workers. The higher level of RNA in rust-infected wheat is indicated also by an increased incorporation of P32 into RNA in four to five days after inoculation (Fig. 5.8). An increase in the RNA content of the nuclei of the cells of diseased plants probably indicates an induced synthesis of proteins in the host. Experiments of Millerd and Scott (1963) and Quick and Shaw (1964) have shown a correlation between increased respiration and RNA synthesis in barley, powdery mildew disease and rust infections of wheat. There is an initial swelling and later degradation of the infected host nuclei in many fungal diseases of the plants. This is accompanied in every case by an increase in RNA and protein synthesis. Eventually the collapse of the nuclei is caused by proteolysis in the host. A decrease in the RNA and DNA contents of soybean leaves infected with Peronospora manschurica has been demonstrated by Millikan et al. (1965). Callow (1973) showed that an increase in the total RNA content of cucumber leaves during the

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period of leaf expansion is retarded by inoculation with Erysiphe chicoracearum. But when the expansion of the leaves went up to 75% of their final area, a very slight increrase in cytopasmic RNA was observed.

Fig. 5.8

Amount of P32 incorporated into RNA of wheat leaves CV Little Club (compatible) and CV Khapli (incompatible) inoculated with Puccinia graminis. H = healthy and I = infected leaves. (From Dickinson and Lucas, Plant Pathology and Plant Pathogens, Blackwell Scientific Publications, Osney, Mead, Oxford, 1977, p. 109)

Haustoria of powdery mildew fungi only penetrate epidermal cells, which do not contain chloroplasts. Loss of nucleic acids from chloroplasts must therefore be caused by diffusible metabolites released by the pathogen. In incompatible reactions of soybean plants infected by Phytophthora magasperma f. sp. glycinea, increases in m-RNA levels may precede the formation of phytoalexins. CHANGES IN PROTEIN METABOLISM

Uritani (1971) had surveyed the changes in the proteins of diseased tissues and compared them to those induced by mechanical injury. Changes in protein metabolism in plant tissue during the initiation of pathogenic infection and course of disease development had been reviewed by Uritani (1976). The total content of the protein (that of the plant and pathogen) usually increases during the early stages of infection. When the contribution of the pathogen is seen, little change, or in some cases, a decrease in plant protein has been found in most diseased tissues. In the case of crown gall tumours, there is much more protein in them than could be expected from synthetic activities of the pathogen. In late degenerative stages of disease, large decreases in protein, sometimes accompanied by increases in free amino acids, are often found. It is not possible to generalize the influence of viral infections on the host’s protein metabolism. A few viruses such as TMV and potato virus X, in connection with which this problem has been most thoroughly studied, may be atypical of plant viruses as a whole; these viruses multiply to such an extent that the viral protein contributes significantly to the total protein of the leaf. The synthesis of viral protein in turn, must exert a considerable direct stress on host-protein metabolism. Most viruses however, contribute very little to the total plant protein. Levels of protein synthesis measured in infected plants at the infection site in susceptible plants are usually increased over levels in healthy or resistant plants. Much of this increase in protein level can be attributed to synthesizing activities of the invading pathogen.

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It is generally agreed that there are marked differences between the nitrogenous constituents of normal resting cells and actively dividing tumour cells incited to division by the crown gall bacterium, Agrobacterium tumefaciens (Braun, 1962). Neish and Hibbert (1943) have reported that while both normal and tumour tissues contain about the same amount of non-protein nitrogen, tumours have three times the protein of normal tissues and the difference in water-soluble protein is 6:1 in favour of tumour tissues. This was interpreted as a greater tendency and capacity on the part of tumour tissues to synthesize protein. According to studies by Klein (1952), the greatest increases in proteins and soluble nitrogen became apparent in tumour tissues late in their development—at the time of cell-volume increase. He suggested that increased protein synthesis registered in the tumour tissues at this time did not occur entirely at the expense of free amino acids and amides in close proximity to the tumour. On the basis of observations of the stunted and nitrogen-deficient appearance of tumorous plants, Klein stated that the nitrogen of the whole plant was being mobilized into the tumour. Patel and Walker (1963) studied the amino acids and amide pool of the bean plant, susceptible and resistant to the haloblight bacterium Pseudomonas phaseolicola. The amino acids and amide content were not appreciably altered in resistant varieties as a result of infection. Chromatographic analysis of a healthy and inoculated leaf of a susceptible variety revealed 22 ninhydrin-positive compounds, and of these, 15 were identified as amino acids and amides. Changes in amino compounds were determined in inoculated and chlorotic tissues and again there was a large accumulation of ornithine, as well as histidine and methionine in the inoculated leaf tissue. More than five times as much ornithine accumulated in the uninoculated chlorotic leaves. Increases in the protein synthesizing activities of green island tissue are quite considerable whereas in the chlorotic tissues between these sites, protein synthesis is depressed to low levels. Some proteins novel to the host plant may be synthesized in resistant plants and are usually associated with hypersensitive response and resistant mechanisms (Chapter VI). These novel proteins are termed pathogenesis-related proteins (PR proteins). Such polypeptides are usually of low molecular weight. These have been detected in infected plants and also in tissues which have been treated with elicitor molecules from microbes or abiotic factors. Detailed aspects of pathogenesis-related proteins will be discussed in Chapter VI. PHENOLS

Phenols are compounds which contain one or more aromatic (benzene) rings with one or more phenolic hydroxyl groups. The simplest compound is phenol. Compounds having more than one hydroxyl group on the benzene ring are called polyphenols. Phenolic compounds are widely distributed in higher plants and fungi. Anthocyanins, leucoanthocyanins and anthoxanthins, hydroxy benzoic acids, glycosides, sugar esters of quinones and shikimic acids, esters or hydroxycinnamic acids and coumarine derivatives make up a vast class of compounds known as phenolics. Formerly, the term tannin, and more recently, the term polyphenols, have been used to refer to these compounds. In their discussion on the pathological darkening of plant tissue, Rubin and Artsikhovskaya (1964) stressed the diversity of physiological roles played by phenolic compounds which characteristically accumulate in diseased plants. The extensive literature on the role of phenolic compounds in disease development has been covered in proceedings and reviews by Pridham (1960), Wood (1967) and Kosuge (1969). Phenolic compounds function as hydrogen donors or acceptors in oxidation reduction reactions and they play an essential role in

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lignification. Some phenolics show antiauxin activities and are potent growth inhibitors. Others act synergistically with auxins in growth stimulation. Phenolic compounds have been shown to play key roles in the resistance of plants to disease, causing micro-organisms (Farkas and Kiraly, 1962; Cruickshank, 1963; Rohringer and Samborski, 1967; Kuc’, 1972). Phenolic compounds and their role in disease resistance has been reviewed by Nicholson and Hammerschmidt (1992). In addition, phenolics may also serve as protectants against leaf-eating insects, for example, they may influence the availability or digestibility of proteins which insects obtain from leaves. Phenolics may also interfere with growth and other energy-dependent activities by uncoupling oxidative phosphorylation. The oxidation of phenols yields highly reactive quinones which inhibit enzymes by complexing with metal ions, reacting with sulfahydryl groups or binding nonspecifically to proteins. It is obvious that even small changes in phenol metabolism may severely disrupt many processes which are essential for normal plant growth and development. Evidences from 14C-tracer elements indicate that the biosynthesis of phenolic compounds in plants may proceed via one or more of three different pathways: 1. Shikimic acid pathway 2. Acetate malonate pathway 3. Acetate-mevalonate pathway The most important of these appears to be the shikimic acid pathway (Fig. 5.9) in which the initial reaction involves the condensation of phosphoenol pyruvate and erythrose-4-phosphate to yield a sevencarbon intermediate which is converted by a series of reactions into shikimic acid. Glucose Glucose-6-P —— 6-Phospho gluconate —— Pentose-P-Cycle Fructose-6-P

Erythrose-P CHO

P-Enol Pyruvate H COH Pyruvate

H COH

KREBS CYCLE

H

Dehydroxyquinic acid

CH2OH H

OH OH

COOH Shikimic acid

OH H

Fig. 5.9 The shikimic acid pathway

The Shikimic Acid Pathway

The shikimic acid pathway, elucidated first in bacteria, appears to function in higher plants for the synthesis of phenolic compounds. The initial reaction of this pathway involves the condensation of phosphoenol pyruvate and erythrose-4-phosphate.

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Phosphoenol pyruvate is supplied by glycolysis, whereas erythrose-4-phosphate is derived from the pentose-phosphate pathway. One reaction, the reduction of 5-dehydroshikimic to shikimic, requires NADPH which is also a product of the pentose-phosphate pathway. Transaminations convert shikimic acid to phenylalanine or tyrosine. In dicotyledons, phenylalanine ammonia lyase (PAL) converts phenylalanine to trans-cinnamic acid and thus provides phenyl propane skeletons which can serve as building blocks for lignin or be utilized in the synthesis of flavonoids and various other phenolic derivatives. In cereal grasses, tyrosine ammonia lyase converts tyrosine to p-coumaric acid (4-hydroxycinnamic) and this, in addition to the PAL system, provides phenylpropane skeletons which serve the same functions as those provided by PAL. In several plant tissues, ammonia lyase activity has been found to be closely related to the biosynthesis of phenolic compounds (Kosuge, 1969). PAL activity has been shown to increase in response to a number of stimuli, including light and mechanical injury as well as infections by viruses, bacteria, and fungi. The level of PAL activity increases to a peak within 24 hours of infection by Ceratocystis fimbriata and this increase is followed by a rise in the concentration of polyphenols (Fig. 5.10).

Fig. 5.10

Activity of PAL and polyphenol accumulation in discs of sweet potato tissue following inoculation with Ceratocysis fimbriata. (From Dickinson and Lucas, Plant Pathology and Plant Pathogens, Blackwell Scientific Publications, Osney Mead, Oxford 1977, p. 115)

Acetate Malonate Pathway

Some phenolic compounds are synthesized by head to tail condensation of acetate units. This pathway, known as the acetate malonate pathway, is thought to involve acetyl Co-A and malonyl Co-A as intermediates and resembles fatty acid synthesis. Isocoumarin (3-methyl, 6-methoxy, 8-hydroxy, 3-4 dihydro isocoumarin) which accumulates in carrot slices is apparently synthesized via the acetate malonate pathway. Acetate-Mevalonate Pathway

In a third pathway, the acetate-mevalonate pathway, acetyl Co-A units are condensed to form mevalonic acid which is then converted into cyclic terpenes or steroids. The synthesis of compounds with two or more aromatic rings often involves interactions among different pathways. For example, both the acetate malonate and shikimic acid pathways contribute to the synthesis of the isoflavone and pisatin.

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The current interest in phenol metabolism stems from the identification of a large number of compounds synthesized via the shikimic acid or acetate pathways which accumulate after infection or injury. Many of these compounds, called phytoalexins, have antibiotic properties and are thought to function in disease resistance. These have been discussed in Chapter VI. Phenol Metabolism

Phenol oxidizing enzymes—laccase and tyrosinase—are the two most improtant phenol oxidases. These occur both in higher plants as well as in fungi. The enzyme laccase catalyzes the aerobic oxidation of p-hydroquinones or p-phenylenediamines to p-quinones or p-quinonediamines. The other enzyme, tyrosinase, is actually composed of two enzymes. One of the enzymes possesses only catecholase activity, whereas the other shows cresolase activity. This enzyme (tyrosinase) complex is also known as phenolase, phenoloxidase, polyphenoloxidase or DoP A-oxidase. Tyrosinase is also an aerobic oxidase which is able to oxidize many phenol compounds by oxygen. However, neither para nor metadihydroxyphenols are suitable substrates for this enzyme since it is able to oxidize only orthodihydroxyphenols. The enzymatic activity which makes O-diphenol from monophenol oxidatively (monophenol + ½ O2 = O-diphenol) is frequently referred to as cresolase activity. On the other hand, catecholase activity oxidizes the produced diphenol to quinone (O-diphenol + ½ O2 = O-quinone + H2O). These activities are given in Fig. 5.11. O-Quinone produces “melanin”, which is dark-coloured pigment produced in the course of further enzymatic or non-enzymatic reactions. Melanin is considered to be an end product but the details of the formation of this complex are not yet known. OH

OH OH ◊ 1 O2 2

Monophenol

Orthodiphenol O

OH OH

O

◊ 1 O2 2

◊ H2O

Orthodiphenol

Orthoquinone

Fig. 5.11 Catechol activity of the phenolase enzyme

Changes in the activity of phenol-oxidizing enzymes, peroxidase and phenolase may also pay a role in the regulation of metabolic pathways in diseased or injured tissues. Phenolase is a copper-containing enzyme which catalyses the oxidation of mono- and O-dihydroxyphenols to phenols. The resistance to several fungal pathogens has been ascribed to a higher concentration of fungitoxic phenolic substances and their oxidation products, to increased phenolic substances and their oxidation products, to increased polyphenol oxidase acitivity which generally, but not invariably results from infection. More than 65 years ago Dufrenoy (1936) observed that when the progress of some pathogens is checked, phenolic

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compounds, mostly tannins of the gallic group, develop in abundance within the vacuoles of adjacent cells, whereas in specialized parasites, tending to become systemic, tannin is formed very slowly in small amounts and only in vacuoles close to the parasite. Subsequently, it was suggested that the establishment of Puccinia coronata in oat leaves depended on the supply of phosphorus from the host tissue; on the depletion of this element, relatively innocuous host phenolic compounds were dehydrogenated by tyrosinase into toxic quinones, leading to the localized necrosis of the tissue. Importance of Phenol Oxidation in Plant Diseases

The phenol oxidase system plays a role in respiration by transferring electrons from respiratory substrates to other hydrogen or electron acceptors. Oxidation products of phenols (quinones) may be reduced to their original phenol form by respiratory carriers. NaDH2 and NADPH2, generated by the oxidation of respiratory substrates, may reduce quinones in the presence of quinone reductase enzyme. Polyphenolases oxidize monophenol to polyphenols by means of cresolase activity in which the hydroxylation of monophenol leads to the formation of complicated polyphenols, such as flavonoids, tannins, lignins, and others, whereas the catechol activity has a role in the browning reaction of plant tissues. Phenolase may influence the amino acid metabolism of plants. It has been seen in many cases that a close correlation exists between the enhanced activity of phenolase and peroxidase and the concentration of phenolic substances. Phenolic substances in turn, have been correlated with resistance to pathogens. The role of phenolases in host resistance has been summarized in several reviews. The increased phenolase activity in diseased or wounded areas of plant tissues is generally accompanied by an increased concentration of phenolic substances. Polyphenols, like chlorogenic and caffeic acids, are known to play a role in the inhibition of the activity of indole acetic acid oxidase. Phenolics and their oxidation products seem to play a role in the auxin metabolism of plants. Polyphenols, such as chlorogenic and caffeic acid, known to be powerful inhibitors of IAA-oxidase, may in turn be able to reverse the enzyme activity and confer resistance via an auxin-spring action on plants that produce them in response to infection . The phenols synthesized by plant tissues after infection appear to have a broad antifungal spectrum. Oxidized polyphenols inhibit the pectinolytic enzyme produced by various fungi and they have early been associated with the resistance of apples to brown rot. It has been shown in many experiments that a correlation may exist between the degree of resistance and the phenol level in healthy plants. Chandramohan et al. (1967) showed that varieties of Amaranthus tricolor resistant of Alternaria sp. accumulate O-D phenols and the amount of the latter was many a times that of the susceptible varieties. Walker and Stahmann (1955) showed that onion varieties resistant to Colletotrichum circinans accumulated flavones, anthocyanine and simple phenolics, such as protocatechuic acid and catechol in the dried bulbs scales which are red pigmented. It has been shown that oxidized phenols as well as oxidized phenolic glucosides are even more effective. In the case of apple leaves, resistance to Venturia inaequalis has been attributed to phloretin and that of pea shoots to Erwinia amylovora is attributed to arbutin. This is due to the action of bglucosidases, polyphenol oxidases and peroxidases. Lee and Le Tourneau (1958) reported the accumulation of higher amounts of chlorogenic acid in the potato roots of varieties resistant to Verticillium wilt disease. Mahadevan (1965) showed that phenols

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and their oxidation products showed inhibitory activity on the pectolytic and cellulolytic enzymes of the pathogen, Cladosporium cucumerinum. He suggested that the defence mechanism in resistant plants should involve the importance of phenolic constituents and their oxidation products on the cell walldegrading enzymes of the parasite. The inactivation of extracellular microbial enzymes by the phenols or quinones present in plants after penetration is certainly a defence mechanism. According to Johnson and Schall (1957), the periderm of tubers from a variety resistant to scab (Streptomyces scabies) showed much more chlorogenic and caffeic acid and were largely confined to the outermost tissues where the scab organisms proliferate. In the Verticillium wilt of potato, the resistance is ascribed to a greater quantity of chlorogenic acid in root tips, sprout tips and the vascular system of resistant varieties. In the case of Phytophthotra infestans infection, the leaves of resistant potato varieties show a more intense accumulation of toxic oxidative derivatives of polyphenols than do the leaves of susceptible varieties. According to Tomiyama et al. (1958, 1963), the resistance to Phytophthora is reversed by copper enzyme inhibitors that interfere with the formation of toxic quinones via polyphenol oxidases (PPO). Following inoculation with Phytophthora infestans, both polyphenol and PPO increased in resistant potato varieties but not in susceptible varieties which suggests a resistance mechanism to the enzyme. Voros et al. (1957) have reported that when streptomycin is absorbed through the root, it gives resistance to potato against Phytophthora. This is explained by the fact that antibiotic activity generally enhances PPO activity, and hence, the resistance in plants. The activity of PPO would seem to be important in that it can oxidize phenolics to quinones which may be more fungitoxic. Polymerisation of quinones to dark coloured tannins may occur. In tomato wilt (F. oxysporum f. sp. lycopersici) conjugated phenols such as glucosides, tannins and lignins may be hydrolyzed by enzymes to toxic quinones which then polymerize to cause characteristic vascular discolouration as suggested by Davis and Dimond (1954) and Davis et al. (1953), Waggoner and Dimond (1953). PPO produced by the pathogen might oxidize host polyphenols to more highly fungitoxic substance which prevent further development of the fungal pathogen for example, a PPO inhibitor reduces the toxicity of catechol to Cochliobolus miyabeanus (leaf blight to rice) and prevents the development of melanin, presumably by preventing enzymatic conversion of catechol into fungitoxic quinones (Oku, 1967). The melanin is apparently fungitoxic although in other cases these insoluble pigments are relatively non-toxic to fungi. Such inhibition can thus result from the production of PPO by the pathogens. The germinating uredospores of Puccinia graminis release phenols and PPO and the resistance of barely plants to Erysiphe graminis appears to be associated partly with an accompanying release of a phenolic substance which accumulates around the haustoria and inhibits further development of the fungus. Aromatic substances, such as polyphenols, phenolic glucosides, flavonoids, anthocyannis, aromatic amino acids, and coumarin derivatives tend to accumulate in and around infected plant tissue and also in tissue adjacent to wounds, where presumably they might exert a fungitoxic effect. Kuc’ (1963) described two types of reactions in apple varieties resistant to Venturia inaequalis. Phloridzin, a polyphenolic glucoside present in the bark and leaves of apple, was hydrolyzed to glucose and phloretin by a b-glycosidase and the phloretin was oxidized by PPO to a fungitoxic intermediate compound and later to melanins. These intermediate compounds, possibly quinones, may have been responsible for resistance to the fungus. They are produced in susceptible and resistant apple leaves but their formation is much quicker in the latter, thus leading to rapid inhibition of the invading pathogen.

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Kuc’ (1963) suggested that a great variety of phenolic compounds occurring in plants might provide a partial explanation for the specificity of the host-parasite interaction. For example, certain plant species (resistant) might contain or produce phenolic substances toxic to a particular pathogen, whereas others (susceptible) might lack them. Other workers, however, are sceptical and point to the paucity of adequately documented cases. Cruickshank (1963) notes that there is little direct evidence that the brown pigments (broadly described as polyphenol oxidation products) often associated with infection are of any great significance in pathogenesis—they may be a diffuse secondary attendant phenomenon of defence reaction which has no causal relationship to resistance per se. The significance of phenolic compounds in host-parasite interaction undoubtedly is complex and needs more critical investigations GROWTH REGULATORS IN PLANT DISEASES

Symptoms such as galls, overgrowth or malformation of host tissues, epinasty, elongation, witches broom effects, etc. indicate abnormal growth activities in many plant diseases. The extracts of tissues which exhibit such abnormal growth patterns are often ten times more active in the tests for growthregulating substances than are the extracts of healthy plants. Even in the absence of obvious growth abnormalities; diseased tissues often contain elevated levels of growth regulators. Four classes of compounds are known to be involved in plant diseases. All the major types of plant growth regulators (auxins, gibberellins, cytokinins and volatile hormone ethylene) are produced by viral, bacterial, and fungal pathogens. An evaluation of the role of individual growth regulators in pathogenesis is complex because their physiological effects in many cases overlap. Growth regulators in plant diseases have been reviewed by Sequeira (1963). Endogenous cytokinins in healthy and diseased plants have been discussed by Dekhuijzen (1976). Endogenous auxins, gibberellins, and ethylene in healthy and diseased plants have been discussed by Pegg (1976). Mahadevan (1984) has comprehensively discussed the role of growth regulators, microorganisms, and diseased plants. The role of auxins in plant disease development has been recently reviewed by Yamada (1993). Auxins

The auxin naturally occurring in plants is indole-3-acetic acid (IAA). It is continually produced in growing plant tissues in which it occurs in concentrations of about 5mg per kg of green material. Perhaps, no other single compound in plants has received as much attention from plant biologists as has b3-indole acetic acid (IAA or auxin). One might also add that so many diverse primary functions have never been ascribed to a compound present in the plant in such a comparatively small amount. Of all the endogenous plant growth regulators, IAA is perhaps the nearest to being ubiquitous. Auxin is an organic substance that promotes growth along the longitudinal axis when applied in low concentrations to the shoots of plants which are freed, as far as possible, from their own inherent growth-promoting substance. Went (1926) first showed the effect of IAA on Avena, following which it was demonstrated that IAA had a widespread distribution in higher plants and especially in diseased tissues. Auxin is also produced freely by many species of bacteria and fungi. The synthesis of IAA in extremely small quantities takes place presumably in young leaves and root tips. IAA is required for cell elongation and differentiation.

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Cell elongation is thought to be brought about by the removal of Ca++ and Mg++ cation bridges from the pectate network which leads to the formation of straight chain pectic acids or pectinic acids and this by forming a complex with, and thus inactivating the enzyme pectin methyl esteras (PME) on the cell membrane and so further inhibiting the formation of sites suitable for the attachment of divalent cations. The absorption of IAA to the cell membrane also affects the permeability of the membrane. Many primary functions have been attributed to IAA. It has been linked with cell metabolism, phenol metabolism, increase in respiration, enhancement of the pentose phosphate pathway activity, and membrane permeability. Evidence has also accumulated of the primary site of action of IAA being on the nuclear control of RNA synthesis or on RNA itself, thus affecting the level of certain key enzymes in cell growth.

R

N The indole nucleus

CH2COOH N H Indole-3 acetic acid (IAA)

Changed level of auxin in diseased tissues The study of plant growth substances in diseased plants is comparatively recent. This is somewhat surprising, since in many diseases, symptoms typically illustrate the effect of enhanced growth regulatory substances. There are some good review articles on this subject, such as the ones by Sequeira (1963,1973), Braun (1959,1962),Veldstra (1968) and Van Andel and Fuchs (1972), Pegg (1977,1981,1984) and Yamada(1993). Viral Diseases Grieve (1943) attributed the reduction in the growth of tomato plants infected with tomato spotted wilt virus to a decrease in auxin content and to reduced rates of translocation through the stem. Surprisingly, Jones (1956) did not find a decreased auxin content in the tomato tissues infected with spotted wilt virus. This can be explained by the observation made by Best (1936,1944) that such infected plants contain a high level of scopoletin which is a known inhibitor of IAA oxidase (Sequeira,1969). Symptoms of dwarfing in many viral infections are believed to be due to the lower auxin content of the tissues. Lower auxin content has been associated with TMV and potato leaf roll virus. Lower endogenous levels of auxins have also been found in Lycopersicon esculentum, Phaseolus vulgaris and Beta vulgaris plants infected with curly top virus rather than in healthy plants. Bacterial Diseases The production and role of auxins in plant disease has been studied more extensively in some bacterial diseases of plants such as Agrobacterium tumefaciens, Pseudomonas savastoni and Pseudomonas solanacearum. One of the best known plant diseases, displaying overgrowth symptoms, is crown gall caused by Agrobacterium tumefaciens. There is ample evidence that the abnormal amounts of auxins found in crown gall tumour are produced by the host. This disease has been studied by many investigators as a model system in cancer and tumour genesis. In crown gall, a disease caused by the bacterium on more than a hundred plant species, galls or tumours develop on roots and stems. Tumours develop when the crown gall bacteria enter fresh wounds on a susceptible host. The cells around the wound produce various phenolic compounds specially (acetosyringone) and are activated to divide. The pathogenic bacteria do not invade or enter cells of the host but simply attach themselves to the cell walls. In response to the phenolic compounds and other signals they begin processing the DNA in their

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Ti-plasmid (tumour inducing plasmid). Intense cell divisions begins to take place after wounding and two or three days after wounding, the plant cells are somehow conditioned and made receptive to a piece of bacterial plasmid DNA (T-DNA, for tumour DNA). A part of the bacterial plasmid DNA is cut out from the Ti-plasmid and is transferred into the host plant cells as T-DNA-protein complex. This T-DNA then becomes incorporated or integrated into the nuclear plant DNA and transforms normal plant cells into tumour cells (Agrios,1997). Tumour cells subsequently grow and divide independently of the bacteria, and their organization rate of growth, and rate of division can no longer be controlled by the host plants. The transferred (or T) region of plasmid DNA carries several genes which, when expressed in the host cell, cause the characteristic tumour. The one (as in oncogenic-cancer causing) gene specify the synthesis of auxins, and cytokinins leading to uncontrolled cell division, while other gene code for the production of unusual amino acids known as opines. These unusual amino acids can not be utilized by the plant, but are instead catabolized by the pathogen. In another hyperplastic disease, the knot disease of olive and oleander, the pathogen Pseudomonas savastoni produces IAA which induces infected plants to produce galls. Pseudomonas solanacearum the cause of bacterial wilt of solanaceous plants, induces a 100-fold increase in the IAA level of diseased plants compared to healthy plants. Increased plasticity of the cell walls as a result of higher IAA content may facilitate their degradation by cell wall renders the pectin, cellulose and protein components of the wall more accessible to degradation by the enzymes of the pathogen. Fungal Diseases There are numerous reports about the production of IAA in quantity from culture media supplemented with tryptophan or other IAA precursors. This has been used as more than circumstantial evidence for the production of IAA in vivo by the particular pathogen. Critical studies have not been made to find out the relative roles of the fungus and host in the hyperauxiny syndromes. Many fungal diseases of plants cause an increase in the levels of auxin in tissues (hyperauxiny) often to very high concentrations. The best known example of increased levels in plant trees infected by fungal pathogens is club root disease of crucifers caused by Plasmodiophora brassicae. The pathogen on entry to the roots through motile zoospores upsets the plants normal regulatory control of IAA synthesis which leads to root distortion in infected individuals. Increased level of IAA has also been found in rustinfected tissues of safflower with Puccinia carthami. A ten-fold increase has been measured in susceptible potato tubes tissues infected by Phytophthora infestans and in corn infected by Ustilago zeae. Higher levels of IAA may also be due to the fact that in infected tissues IAA oxidase has been shown to be lower than in healthy tissues of Euphorbia cyparissias infected by Uromyces pisi. Thus there was a correlation between increase in IAA and the decrease in IAA oxidase in healthy plants. Auxins are also involved in wilt diseases. Beckman et al. (1953) showed that cultures of the fungus produced a substance similar to IAA. It was claimed that the production of this substance in vivo increased the plasticity of xylem parenchyma walls allowing a pectic enzyme weakening of pit membranes and resulting finally in tylosis. IAA solution induced the formation of tyloses in tomato shoots. This supported the view that auxin is involved in the disease syndrome of the wilt. Increased levels of IAA have been reported in infected stems. Auxins have also been implicated in resistance to vascular wilting. Gibberellins

Gibberellins are a group of chemically similar growth-regulating substances. They can be considered to be normal constituents of green plants. The existence of gibberellin in plants might still have been

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unknown if the “bakanae” or “foolish seedling” disease (which had a devastating effect on the rice economy of Japan) had not broken out. Japanese farmers noted that plants affected with this disease outgrew normal plants but were thinner and paler than their normal counterparts and were sometimes devoid of fruits (Phinney and West, 1960). In the early part of the 20th century Japanese pathologists first demonstrated the connection between the bakanae disease and the fungus called Gibberella fuzikuroi (the teleomorph of Fusarium moniliforme). Sawada (1912) postulated that disease may be caused by some secretion of the fungus. Yabuta and Hayshi (1939) were able to isolate the crystalline gibberellin from the infected rice plants. Since that time, gibberellins and gibberellin-like substances have been found in higher plants and implicated in disease symptoms. About 60 more are known and form a group of compounds which possess the same general carbon skeleton and all are able to promote either stem elongation or cell division or both in plants. They have a gibbane skeleton.

Numerous functions have been attributed to gibberellins. Thus, gibberellin is the chemical signal, that is, the hormone secreted by the embryo in the seed that activates the cells of the aleurone layer into secreting hydrolytic enzymes, such as amylase, which is responsible for liquifying the reserve starch. It also activates the enzymes that promote cell wall degradation, thereby aiding in the digestion of the endosperm cells and in weakening the seed coats which subsequently allows the seeds to germinate. Besides inducing amylase activity in the aleurone layer of seeds, gibberellins exert a potent stimulus on terminal meristems, and thus in some resects, mimic the effects of auxins on plant growth. Gibberellin also activates or induces the synthesis of other enzymes, including proteinases. Proteinases break down the reserve proteins of the endosperm and release various amino acids, including tryptophan. Tryptophan is then activated to form auxin (IAA). Gibberellins generally have striking growth-promoting effects; they speed the elongation of dwarf varieties to normal size, promote flowering, elongate the stem and root and encourage the growth of fruit. Auxins and gibberellins may also act synergistically. The point of action of the gibberellins in the cells seems to be close to the nucleic acid system, somewhere between DNA and m-RNA. The involvement and role of gibberellins in plant disease has been studied in relatively few diseases. In the “bakanae” disease of rice, it is not known whether the infected plants contain more gibberellin than the healthy plants, or in what manner the fungal gibberellin causes the symptoms observed. Following the classical discovery of the bakanae disease, the first truly experimental investigations on endogenous gibberellins and infected plants were made by Radley (1961). It was shown by her that pea nodules infected with Rhizobium contain an increased level of growth like substances (GLS) as compared to healthy roots. Gibberellic acid has effects in infection of cassava (Manihot esculenta) caused by Sphaceloma manihotica, which initially causes the formation of necrotic leaf spots and stem canker, but subsequently gives rise to massive elongation of internodes as a characteristic disease symptom. Gibberellins have been studied more extensively in relation to viral diseases. The spraying of diseased plants with gibberellin has been shown to overcome some of the diseases caused by viruses. Thus, the stunting of corn plants infected with corn stunt virus and tobacco plants infected with severe etch virus was reversed after treatment with gibberellin. Axillary bud suppression caused by sour cherry yellows virus (SCYV) on cherry and leaf curl virus on tobacco was also overcome by gibberellin sprays.

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The fact that stunting caused by some viruses, can be reversed by applying exogenous gibberellins suggests that the reduced growth of these virus-infected plants might be due to decreased gibberellin content. Cytokinins

Cytokinins are potent growth factors necessary for cell growth and differentiation. The first cytokinin discovered was kinetin by Skoog and Miller in the early 1950s. The main action of cytokinin, an exogenously applied kinetin, is on cell division. It also seems to be capable of orienting plant growth induced by auxins. According to Osborne (1965), cytokinins stimulate protein synthesis at the level of DNA-dependent RNA synthesis. Cytokinins also delay senescence in plant tissues. The action of cytokinins is dependent on auxins. They are derivatives of adenine. Kinetin (6 furfurylaminopurine), has been isolated from yeast DNA by Miller et al. (1956). Kinetin was, however, isolated from herring sperm DNA and has not yet been shown to occur naturally in green plants. Several other adenine derivatives with cytokinin-like activities have been synthesized and recently, similar compounds, that is, zeatin and N6 (2-iso pentenyl) adenosin (IPA) have been isolated from young corn seeds and from spinach and peas, respectively. Thrower (1965), Scott (1972), Sequeira (1963, 1973) and Dekhuijzen (1976) have reviewed the role of cytokinins in plant diseases. Cells in cultures show no response when they are supplied with kinetin alone, and they increase in size but do not divide when supplied with only IAA. When both substances are supplied together, the cells enlarge and divide. The relative concentrations of kinetin seem to determine the organs that will differentiate, low concentrations inducing root formation, higher concentrations inducing bud formation. The mechanism of action of cytokinins is not yet known but the available evidence indicates that it is similar to that of gibberellins. Cytokinins act at the stage of m-RNA synthesis, possibly by preventing genes from being turned off and by activating genes that have been previously turned off. The role of cytokinins in plant disease has only now become a subject of study. A six-fold increase in cytokinin activity was detected in extracts from the galled roots of root knot nematode-infected tobacco plants compared with extracts from healthy plants; the cytokinin, however, appear late in the infection since they can be detected in galled roots 75 days, and not 35 days, after inoculation. Root cells near a nematode’s head display hypertrophy with repeated nuclear divisions together with the incorporation of neighbouring cells, and this results in large, thick-walled multinucleate syncytia or giant cells. The endogenous levels of several hormones are reported to increase in several hosts infected by nematodes. Cytokinin activity also increases in rust-infected beans and broad bean leaves and seems to be related to both, the juvenile features of the green islands around the infection centres and the senescence outside the green island. In the Helminthosporium blight disease of oats, cytokinins do not increase the toxicity of toxin victorin but they increase the quantity of toxin absorbed. It appears that cytokinins may enhance the effectiveness of the pathogens that produce pathotoxins. On the other hand, cucumber leaf discs treated with kinetin become resistant to the powdery mildew fungus, and tobacco leaves infected with the wild-fire toxin and treated with kinetin fail to develop the typical toxin-induced chlorosis. A cytokinin has been implicated in the initiation and development of the fasciation of peas caused by Rhodococcus (Corynebacterium) fasciens (Sequeira, 1973). Culture extracts of the pathogens and of infected tissues contained materials with cytokinin-like properties. The major active component obtained from the bacterium is 6-(3 methyl-2-butanylamino) purine, the riboside of which is found in tRNA of many bacteria. Plants treated with a high concentration of kinetin develop symptoms similar to

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those infected by the bacterium. Although these findings suggest that cytokinins play a role in this disease, a low amount of cytokinin produced by the bacterium in culture indicates that other factors are probably involved. It has been suggested that cytokinins may be involved in the cell proliferation stage of tumour development in oleander infected with Pseudomonas savastanoi. Studies on kinetin and plant virus multiplication have been stimulated by the work of Richmond and Lang (1957) and later by Osborne (1962) and Mothes et al. (1961) concerning the effect of kinetin on protein and nucleic acid metabolism and on amino acid accumulation in leaves. It will be recalled that kinetin tends to arrest the breakdown of proteins and nucleic acids which occurs when a leaf is removed from a plant and it tends to mobilize the accumulation of amino acids at the point of its application to a leaf. Osborne has suggested that the new RNA and proteins are even synthesized under these conditions which, without kinetin, greatly favour catabolism. Kiraly and Szirmai (1964) observed a decrease in TMV production in the leaves and leaf discs of a local lesion host Nicotiana glutinosa treated with kinetin (40-50mg/ litre). The kinetin treatment to be effective, has to be applied before or shortly after the infection. Pozsar and Kiraly (1964) showed that kinetin reduced virus multiplication in a systemic host. There are conflicting reports of the role of cytokinin in the green island formation in infected leaves. It has been pointed out that the green island formation in mildew infected wheat leaf does not merely represent retention of chlorophyll; they actually are areas in which chlorophyll was destroyed and formed subsequently. On the other hand it had been suggested that the green island formation is the result of chlorophyll retention. The green island formation in Pinto bean leaves infected with Uromyces phaseoli depends on light conditions after the inoculation of the plants. The total green area which finally becomes visible is the result of a retardation in chlorophyll breakdown and the resynthesis of chlorophyll. Brian (1967) speculated that the green island formation may be due to a release of cytokinin by the pathogen. This is, however, doubtful. Most probably the increased cytokinin level originates from host cells and not from the pathogen. Atkin and Neilands (1972) have speculated about the possible role of siderochromes in the formation of green islands. An increase in cytokinin content has also been noticed in tumours caused by Ustilago zeae and Synchytrium endobioticum, club root disease of cabbage caused by Plasmodiophora brassicae and peach leaves infected with Taphrina deformans. However, peach leaf curl symptoms cannot be entirely attributed to changes in cytokinins, as the level of IAA also increases in infected leaves. Ethylene

Ethylene (C2H4) is a plant growth regulator which is biologically highly active. It has an important role in regulation of plant growth and is implicated in a number of disease symptoms such as tissue swelling, stimulation of root formation, leaf abscission, epinasty and fruit ripening. It is produced by plants and some species of pathogenic organisms, is effective in very low concentrations and is synthesized from methionine in the presence of light. Ethylene stimulates the production of a number of enzymes. In healthy plants ethylene is reported to influence the levels of hydroxyproline-rich proteins in cell walls which are important to wall structure. It has been seen that the ethylene treatment increases the levels of cellulase and b-1, 3-glucanase which are implicated in the degradation of cell wall polymers. According to Hadwiger et al. (1970) the role of ethylene in disease resistance may be due to non-specific induction of phytoalexins synthesis and the stimulation of enyzmes which are active in the synthesis of phytoalexins for instance, PAL, peroxidase, polyphenol oxidase and pectin esterase. These enzymes oxidize phenolic compounds and are involved in

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resistant reactions of the host plants. Ethylene is ubiquitously present in plant tissues and is produced in increased amounts in damaged or diseased plants. Applied exogenously, it is effective in very low concentrations (below 0.1 ppm) and produces a variety of plant responses. Some of these responses— epinasty, leaf abscission, premature senescence, fruit ripening and shedding of leaves—are similar to the symptoms of certain plant diseases. A number of bacterial or fungal pathogens produce ethylene and in the case of fusarial wilt of tomato caused by F. oxysporum f. sp. lycopersici, ethylene production by the pathogen is sufficient to account for the epinastic symptoms of a disease. The defoliating symptoms caused by Verticillium wilt of hop plants is attributed to increased levels of ethylene in infected hop plants. In Verticillium wilt disease ethylene has been attributed a dual role; acting directly as a toxin on plant tissues covering leaf drop, epinasty and premature senescence and also inducing disease resistance in infected plants. The origin and possible role of ethylene in diseased plants has been discussed by Abeles (1973), Hislop et al. (1973), Sequeira (1973), Pegg (1976) and Mahadevn (1984). In a number of viral diseases, leaves with necrotic local lesions produce more ethylene than those of systemically infected plants without local lesions. Necrosis induced by toxic chemicals also results in increased ethylene evolution. These results suggest that ethylene is a product rather than the cause of necrosis. Erwinia amylovora does not produce ethylene in culture but when cauliflower florets are inoculated with this bacterium, it stimulates ethylene production. This indicates that the evolution in diseased tissue most probably is of host origin arising as a result of cellular damage in a still-living tissue. A pectate lyase in the culture filtrate seems to be responsible for the increased production by the plant. Ethylene undoubtedly interacts with other growth-regulating substances, and Van Andel and Fuchs (1972) have pointed out that changes in the balance of growth regulators as a result of such constantly changing interactions may be more important in symptom development than the quantity of any single component. It must be emphasized that it is still not clear whether ethylene is a primary factor in triggering metabolic charges or it is merely produced as a result of these changes. TRANSCRIPTION AND TRANSLATION IN DISEASED PLANTS

This subject has been dealt in two review articles by Samborski et al. (1978) and van Loon and Callow (1983). All living cells hold genetic information in DNA molecules which can be transcribed into RNA and translated into polypeptides according to the code of a particular gene. Mechanisms involved in transcription and translation are now fairly well understood, at least in prokaryotes. Transcription is the process by which genetic information encoded in DNA is copied during the process of mRNA synthesis. Translation is the process by which the information is used to specify certain amino acids sequences during peptide synthesis. Several pathogens, particularly viruses and fungal biotrophs such as rusts and powdery mildews, affect the transcription process in infected cells. In diseases caused by viruses, the pathogen through its own enzyme or by modifying the host enzyme (RNA polymerase) that makes RNA, utilizes the host cell nucleotides and machinery to make its own RNA. Effect on Transcription

In some cases the pathogens affect transcription by changing the composition, structure or function of the chromatin associated with the cell DNA. Ribonucleases have been investigated during obligate

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biotrophic infections such as rusts and powdery mildews. Ribonucleases in infected plants have been shown to exihibit different substrate preferances from those in infected plant tissues (host RNase) and from those of invading fungal mycelium (fungal RNase). Additionally, this type of novel activity was not detected in resistant hosts which therefore suggests that the changes in RNases may be related to the expression of resistance in the host or to the modification of host RNase by the pathogen. It is generally believed that the greater RNA levels and therefore increased transcription in cells indicates increased synthesis of substances involved in the defence mechanism of plants. Effect on Translation

Infected plant tissues often show increased activity in several enzymes, particularly those associated with respiration or with the production of various phenolic compounds, some of which may be involved in the defence mechanism of the plants. Although certain amounts of these enzymes (proteins) may be present in the cell at the time of infection of plants by pathogens, several new enzymes or protiens are synthesized de novo. Thus there is an increased level of transcription and translation activity in the infected tissues. It has been commonly seen that the increased level of proteins in hosts are those which are resistant to the pathogens. If the resistant tissues are treated with actinomycin-D an inhibitor of protein synthesis, their resistance to the pathogens is reduced. Thus it is very clear that the increased activity of the protein synthesis in plants attacked by pathogens is related to their defence mechanism.

REFERENCES Permeability Changes Bateman, D.F. and R.L. Millar (1966), “Pectic enzymes in tissue degradation”, Ann. Rev. Phytopath., 4: 119– 146. Beckman, C.A. (1964), “Host responses to vascular infection”, Ann. Rev. Phytopath., 2: 231–252. Black, H.S. and H. Wheeler (1966), “Biochemical effects of victorin on oat tissues and mitochondria”, Am. J. Bot., 57: 1008–1012. Dube, H.C. (1971), “ Studies on permeability changes in cotton (Gossypium hirsutum) caused by Verticillium albo-atrum”, Proc. Indian Acad. Sci, 50(B): 185–193. Dube, H.C. (1973), “Electrolyte changes in blasted rice”, Curr. Sci., 42(7): 249–250. Dube, H.C. and H.P. Srivastava (1975), “Altered permeability as a trigger for pathogensis”, in: Advances in Mycology and Plant Pathology, Published. by Prof. R.N. Tandon Birthday Celebration Committee, New Delhi, pp. 271–275. Hall, J.A. and R.K.S. Wood (1970), “Plant cells killed by soft rot parasites”, Nature, 277: 266. Hutchinson, C.M. (1913), “Rangpur tobacco wilt”, Mem. Dept. Agr. India, Bact., 1: 67 – 83. Johnson, T. And O. Johnson (1934), “Studies on the nature of disease resistance in cereals – II: The relationship between sugar content and reaction of stem rust of mature and immature tissues of wheat plant”, Can. J. Res., 11: 582 – 588. Kuc¢, J. (1966), “Resistance of plants to infectious agents”, Ann. Rev. Microbial., 20: 337–370. Linskens, H.F. (1955), “Der Einflus der toxigenen Welke auf die Blattausscheidungen der Tomatenpflanze”, Phytopath. Z., 23: 89–106. Mount, M.S. and D.F. Bateman (1970), “Cellular injury caused by an endopolygalacturonate trans-eliminase from Erwinia amylovora”, Phytopath., 60: 577.

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QUESTIONS 1. What do you know about the changes in the permeability of membranes as a result of infection by a pathogen? Suggest various methods by which you can measure the altered permeability. 2. Define photosynthesis. How is this process affected as a result of infection by a pathogen? 3. Define respiration. How is this process affected due to infection by a pathogen? 4. What are the various mechanisms responsible for augmented respiration in diseased plant? 5. Explain the differences in respiration between resistant and susceptible varieties. 6. What are laccases and tyrosinases? How are these involved in the resistance mechanism? 7. What is transcription and translation? How is the disturbance in any of these leads to drastic changes in structure and function of the affected cell?

6 How Plants Defend Themselves Against Infection

A great variety of plant and animal life exists in a common environment but parasitism of one form by another is rare. All plants are resistant to some pathogens under certain conditions. Host–parasite relationships may be inhibited or completely hindered if the host is able to ward off the pathogen by passive or active processes. Disease resistance appears to be the rule rather than the exception in nature. Its genetic constitution and the environmental conditions under which the genes operate govern the capacity or ability of a plant to defend itself against a pathogen. The attributes of the hosts that reduce the chances of infection or the development of the pathogen are the defence mechanisms. Different plants defend themselves against pathogens in different ways. The defence mechanisms of plants against pathogens fall into two categories. 1. Morphological or structural defence mechanism 2. Biochemical defence mechanism MORPHOLOGICAL OR STRUCTURAL DEFENCE MECHANISMS

The histology of defence in plants has been reviewed by Akai (1959), Akai and Fukutomi (1980), Dickinson (1960) and Royle (1976). The first line of defence against pathogens is the surface barrier, which a pathogen must penetrate before it can cause infection. The entry of the pathogens may either be through the epidermal cell walls directly or through the natural openings in the epidermis, such as stomata, lenticels, and hydathodes, or through injured areas caused by living or non-living agencies. Once a pathogen has gained entry into the host, its movement and spread is further checked between or through succulent parenchymatous cells, and in the vascular diseases through the lumen of the xylem vessels. Certain structural features of the epidermis or its interior may greatly affect the ability of the pathogen to penetrate or to invade a host plant. Such morphological defence structures may be present before penetration or infection or may be produced afterwards as a result of the interaction of the host and the pathogen.

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Defence Structures Existing Before Infection Waxes and Cuticles The cuticle, which consists of cutin and waxes, forms the outermost covering of the epidermal cells and appears as a non-cellular, membranous layer. Waxes play a defensive role by forming a hydrophobic surface which acts as a water repellent and thereby prevents the retention of water on sloping plant surfaces on which pathogens might be deposited and subsequently invade the host. The cuticle acts as a physical or chemical barrier to infection. In Berberis spp. infected with Puccinia graminis, Melander and Craigie (1927) attributed disease resistance to the outer epidermal cell wall with a thicker cuticle. There are several examples where cutin acts as a physical barrier and enables a greater degree of resistance, for example, Linum usitatissimum (linseed) attacked by Melampsora lini and coffee attacked by Colletotrichum coffeanum. Wilson and Jarvis (1963) attributed the greater infection of white blister, Albugo candida on the leaves of mutant forms of Brussels sprout to the absence in the mutant of surface wax. The resistance of strawberry varieties to Sphaerotheca macularis was shown by Peries (1962) and Jhooty and McKeen (1965) to vary with the cuticle thickness. Martin (1964) discussed the role of the cuticle in the defence of plants against diseases. Structure of Epidermal Cell Wall The thick and tough outer wall of epidermal cells forms an important barrier for certain pathogens. Lignification or the presence of silicic acid in the epidermis of some plants acts as an important structural defence mechanism. In the rice blast fungus Pyricularia grisea (oryzae), the outer walls of most of the epidermal cells of the leaves are lignifed and the entry of the pathogen is generally through the thin-walled motor cells having pectin rather than lignin. Suberin deposition in potato tuber cells enables better resistance to penetration by some pathogens. Structure of Natural Openings Most pathogens enter plants through natural openings. In the case of the stem rust of wheat caused by Puccinia graminis tritici, the varieties in which stomata open late in the morning are resistant because the germ tubes of the spores germinating in the night dew desiccate. This is due to the evaporation of the dew before the stomata begin to open. This is sometimes called functional resistance. The kind of structure of the stomata of the host gives resistance to penetration in certain pathogenic bacteria. Xanthomonas campestris pv. citri is responsible for Citrus canker disease and affects grape and Mandarin oranges when artificially introduced into them. The Mandarin variety, Szinkum, is resistant to the citrus canker bacterium because it possesses a broad cuticular ridge projecting over the stomata and a narrow slit leading to the stomatal cavity so that rain drops containing fungal spores and bacteria rarely reach the mesophyll of the leaf (Fig. 6.1). Internal Structural Barriers to Pathogen Invasion In certain varieties of wheat, the presence of bundles or increased areas of sclerenchyma cells prevent infection in cereal rusts. Defence Structures Formed after Infection

Defence structures are produced inside the host plant to block or prevent the spread of the pathogen. These include the formation of cork layers, tyloses and abscission layers; deposition of gums, resins and tannin-like substances; swelling of the cell walls; sheathing of hyphae, and so on.

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Fig. 6.1 Stomata of Szinkum Mandarin: (a) resistant to the citrus canker bacterium ( Xanthomonas campestris pv. citri) and of a variety (b) susceptible to the pathogen. SP = stomatal pore.

Formation of Cork Layers In the Rhizoctonia diseases of potato tubers, following infection, cork layers are produced just below the areas of infection. This layer prevents further invasion by the pathogen (Fig. 6.2). It blocks the spread of toxic substances produced by the pathogen and checks the flow of nutrients and water from the healthy to the infected area. This results in the starvation of the pathogen. The speed of the formation of the defence barrier is also important. Cork layers are also formed between healthy and diseased areas on Prunus domestica leaves attacked by Coccomyces prunophorae (Fig. 6.3).

How Plants Defend Themselves Against Infection 145

Fig. 6.2

Formation of cork layer on potato tubers following infection with Rhizoctonia.

H

CL

P

I

Fig. 6.3 Formation of cork layer between infected and healthy areas of leaf. CL = cork layer, H = healthy tissue, I = infected, P = phellogen.

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Formation of Tyloses Tyloses are overgrowths of the protoplast of adjacent living parenchymatous cells, which protrude into xylem vessels through half-bordered pits. These effectively clog xylem vessels. Tyloses are usually considered to be one of the factors, which cause wilt. The time and rapidity of the formation of tyloses determine whether their role will be defensive or whether they themselves will be one of the factors for the causation of the disease (Fig. 6.4). In sweet potato wilt caused by the fungus Fusarium oxysporum f. sp. batatas, tyloses in some varieties are formed abundantly and quickly before the pathogen. It results in a greater resistance as it prevents the further spread of the pathogen. Evidence for the importance of vessel tylosis as a defence mechanism is available for vascular wilt of tomatoes. Blackhurst and Wood (1963a) reported that the percentage of vessels occluded by tyloses–nineteen days after root inoculation with Verticillium albo-atrum–was higher in the wilt resistant variety, Loran Blood (36%), than in the susceptible variety, Ailsa Craig (23%).

Fig. 6.4 Development of tyloses in xylem vessels: (a) longitudinal and (b) cross-section view of healthy vessels (c) longitudinal and (d) cross-section of vessels with tyloses (e) longitudinal and (f) cross-section of vessels completely clogged with tyloses. PP = perforation plate, V = xylem vessel, XP = Xylem parenchyma cell, T = tyloses

Formation of Abscission Layers As a result of infection by several fungi, bacteria or viruses abscission layers are formed in the young active leaves of stone fruits. Due to the formation of an abscission layer (upon infections) in peach leaves by the bacterium Xanthomonas arboricola pv. pruni or the fungus Clasterosporium carpophilum and in sour cherry trees due to infection with necrotic ring spot and other viruses, there is a swelling in the two layers of cells surrounding the infected spot. They become thin-walled while the pectic materials of the middle lamellae get dissolved which results in the formation of abscission layers (Fig. 6.5). A shot hole effect results from the development of an abscission layer which extends from the upper to the lower epidermis around the infected tissue and causes the latter to drop out, as in the shot holes of peach caused by Clasterosporium carpophilum.

How Plants Defend Themselves Against Infection 147

Fig. 6.5

Abscission layer formation around a diseased spot of a Prunus leaf.

Gum Deposition Gum deposition along the borders of diseased lesions often serves as a protective demarcation and constitutes a type of mechanical resistance. In the silver leaf disease in plums caused by Stereum purpureum, the wood of the host resistant variety, Pershore, produces so much gum in advance of the fungus in a relatively short time that the fungus becomes completely enclosed by an impenetrable gum barrier in comparison to the susceptible Victoria variety. Hesler (1916) has found a similar gum barrier in the diseased part of the apple twig infected with Physalospora cydoniae (Fig. 6.6). In varieties of rice resistant to blast disease or Helminthosporium leaf spot, gum deposits are formed in the intercellular spaces that aid in restricting the fungus to the area of primary invasion. These deposits are found to be highly developed in Shoemed rice (resistant to helminthosporiose).

Fig. 6.6 Gum barrier in the apple twig infected by Physalospora cydoniae. A = wood parenchyma, B = xylem vessel, C = wood fibre

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Sheathing of Hyphae It has been seen that the hyphae penetrating the cell wall are often enveloped in a sheath formed by the extension of the cell walls and prevent the pathogen from spreading (Fig. 6.7). The nature of the sheath is not clear. It consists of cellulose, callose substances or other materials, and in some cases, a sheath may be the deposition product of the cytoplasm rather than the cell wall. Lignitubers may develop as protuberances on the inner side, opposite the site of penetration. Sometimes, the cell wall being penetrated by the infection peg swells and becomes suberized or lignified.

Fig. 6.7 Formation of sheath around hypha penetrating cell wall. CW = cell wall, H = hyphae, A = appressorium, AH = advancing hypha still enclosed in sheath, HC = hypha in cytoplasm, S = sheath.

BIOCHEMICAL DEFENCE

There are several articles and books on the biochemical defence mechanism in plants against infection. Some of the important one are those of Asada et al., (1982), Bailey and Deverall (1983) and Dixon et al., (1994) and Bowels (1990). Pre-existing Biochemical Defence Mechanism Inhibitors Released by the Plant in its Environment Walker and Link (1935) worked on onion smudge caused by Colletotrichum circinans. Varieties with red scales were resistant in contrast to varieties with white scales. This resistance is due to the fact that the onion varieties with pigmented outer scales contain catechol and protocatechuic acid which are phenolic compounds and which diffuse out from the dead red scales and prevent spore germination. According to Hafiz (1952), the blightresistant varieties of Cicer arietinum have more glandular hairs, which secrete more malic acid on the leaves than the susceptible varieties. A higher concentration of malic acid inhibits spore germination and retards the hyphal growth of the fungus.

How Plants Defend Themselves Against Infection 149

Fungitoxic substances have been detected in plant waxes, and cutin acids of citrus lime are toxic to Gloeosporium limetticola. Johnstone and Sproston (1965) have reported the resistance of Ginkgo biloba leaves with cuticular wax to disease. Timonin (1941) maintains that root exudates also protect plants against diseases. It was reported that the root exudates from the resistant varieties of flax excrete a glucoside, which on hydrolysis produces hydrocyanic acid inhibitory to the soil inhabitants but is less toxic to Trichoderma viride. Resistant varieties have T. viride, which helps flax to resist wilt caused by Fusarium oxysporum f. sp. lini. T. viride was earlier reported to produce two antibiotic substances, gliotoxin and viridin, but now it is known that the antagonistic effect of T. viride is not due to these two antibiotic substances which, in fact, it does not produce (Webster and Lomas, 1964). The antibiotic action of T.viride is due to the production of a volatile antibiotic substance known as trichodermin. Buxton (1957) has done classical work on the resistance of pea cultivars due to root exudates. Exudates from the roots of one pea variety reduce the germination of the spores of a race of Fusarium oxysporum f. sp. pisi, non-pathogenic to this variety. This inhibitor of unknown composition showed a far weaker effect on the pathogenic race. Inhibitory Substances Present in the Plant Cells before Infection In potato scab caused by Streptomyces scabies, higher concentrations of chlorogenic acid have been reported in potato tubers (Johnson and Sachaal, 1952) and in citrus lime leaves, and Martin, Baker and Byrde (1966) suggest that isopimpinellin (a furocoumarin) gives resistance to citrus lime leaves and protects them against Gloeosporium limetticola. Turner (1961) reported that oat leaves and roots contain a fluorescent glucoside (avenacin) which is inhibitory to the growth of several fungi. The growth of Ophiobolus graminis, the cause of the take all disease of wheat, is also inhibited by the sap exuded from the oat roots, but the growth of O. graminis var. avenae is not. Yet the purified inhibitor is equally toxic to both the strains. On the other hand, Turner (1961) has shown that a specific glucoside (avenacinase), produced by the strain var. avenae, pathogenic to oats while the wheat race completely inhibited by 5g/ml avenacin, lacks this enzyme. This enzyme destroys the biological activity of the inhibitor and hence the fungus is able to infect the host. Thus, the pathogenic ability and specificity of O. graminis var. avenea to oats is dependent on the inactivation of the inhibitor. According to Bergman (1966), the infection of tulips by F. oxysporum f. sp. tulipae mainly takes place during the last week before harvesting. The phase of susceptibility of tulip bulbs to F. oxysporum coincides with the drop in the tuliposide content. In the case of resistance of pears to fire blight caused by Erwinia amylovora, it was seen that resistance in certain tissues is related to the presence of phenolic glucoside arbutin. This compound is hydrolyzed by β-glucosidase of host origin to yield glucose and an antimicrobially active semiquinone. Arbutin is uniformly distributed in the tissues of the pear tree, yet these tissues vary in their susceptibility to E. amylovora. These investigators positively correlated those portions of the tree that were resistant (floral bracket) with the high levels of β-glucosidase activity. Likewise, those parts of the tree that were the most susceptible (nectaries of flowers) had decreased levels of β-glucosidase activity.

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According to Kuc’ (1963), phloridzin, the glucoside of phloretin, is present in the leaves of both resistant and susceptible apple varieties. There is no correlation between the contents of phloridzin and resistance to pathogens.

OH

HO

OH

HO

Glucose Phloridzin

O

b-glucosidase H2 O Hydrolysis

OH + Glucose

OH Phloretin

— —

O

— —

OH

O

The hydrolysis of phloridzin to phloretin by b-glucosidase

When the pathogen penetrates the leaves of a resistant variety, the plant cells around the point of penetration collapse and die. Apparently, the non-toxic glucoside phloridzin is hydrolyzed by a β-glucosidase to yield phloretin and glucose. The phloretin in turn is oxidized by phenoloxidases to yield highly fungitoxic compounds. In susceptible varieties, the fungus penetrates the leaves and grows extensively below the cuticle for 10 to 14 days without initiating the collapse of the host cells. Thus, phloridzin is not hydrolyzed and the pathogen is not contained. Phenolic Substances Phenolic compounds and their role in disease resistance have been reviewed by Nicholson and Hamanerschmidt (1992). Resistance to several fungal plant pathogens has been ascribed to higher concentrations of fungitoxic phenolic substances and their oxidation products and to increased polyphenol-oxidase (PPO) activity which generally, but not invariably, results from infection. In the case of Puccinia coronata, more that 60 years ago, Dufrenoy (1936) observed that when the progress of some pathogens is checked, phenolic compounds mostly tannins of the gallic group, develop in abundance within the vacuoles of adjacent cells, whereas in specialized parasites tending to become systemic, tannin is formed very slowly, in small amounts and only in vacuoles close to the parasite. The activity of polyphenoloxidase (PPO) seems to be important because it can oxidize phenolics to quinones which may be more fungitoxic. Polymerization into dark-coloured melanins may then occur. PPO produced by the pathogen may oxidize the host polyphenols to more highly fungitoxic substances which, may prevent further development of the pathogens. For example, a PPO-inhibitor reduces the toxicity of catechol to Cochliobolus miyabeanus (leaf blight of rice) and prevents melanin development, presumably by preventing enzymatic conversions of catechol into fungitoxic quinones. The melanin is apparently fungitoxic although in other cases insoluble pigments are relatively non-toxic to fungi. Germinated uredospores of Puccinia graminis release phenols and PPO and resistance of barley plants to Erysiphe graminis appears to be associated partly with the collapse of the mesophyll cells with an accompanying release of a phenolic substance which accumulates around the haustoria and inhibits further development of the fungus. Chlorogenic acid is partly responsible for the resistance of potato tubers to scab (Streptomyces scabies), perhaps by stimulating protective cambium activity (Johnson and Sachaal, 1952). Hare (1966) points out that aromatic substances, such as polyphenols, phenolic glucosides, flavonoids, anthocyanins, aromatic amino acids, and coumarin derivatives tend to accumulate in and around infected plant tissue and also in tissues adjacent to wounds where presumably, they might exert a fungistatic effect. Phenolic compounds are produced in plants primarily via the shikimic and acetic acid pathways. In the latter, phenolics are produced by a head-to-tail condensation of acetate units

How Plants Defend Themselves Against Infection 151

derived from the breakdown of sugars during respiration. In the shikimic acid pathway, phosphoenol pyruvate from glycolysis reacts with erythrose produced during the pentose pathway, the activity of which is increased in diseased plants, and forms dehydroquinic and then shikimic acid. From the shikimic acid and various intermediate compounds, some of which are still unknown, phenolic compounds are produced. Some of the most important phenolic compounds involved in the defence of plants are caffeic acid, ferulic acid, chlorogenic acid, phloretin and various phytoalexins. The high concentrations of chlorogenic acid in the roots of certain potato varieties have been considered the main mechanism of defence against the Verticillium wilt pathogen. Resistant varieties contain more chlorogenic acid than the susceptible younger roots and are thus not attacked, but they become susceptible later on. Chlorogenic acid is a competitive inhibitor of IAA oxidase and may be involved in diseases where resistance is increased with high IAA content of the plant. Chakravarty and Srivastava (1967) have attributed the resistance of carrot roots to Pythium aphanidermatum to an unidentified, apparently pre-formed phenolic substance. It is said that the phenolic content of rice is responsible for resistance in Pyricularia grisea. Polyphenols have been implicated in resistance to Venturia inaequalis (apple scab) and Venturia pirina (pear scab). Sproston (1957) holds that balsam (Impatiens balsamina) leaves are free from fungal diseases due to the abundance of phenolic substances or glycosides which are oxidized to fungitoxic quinones. Allen and Kuc’ (1968) presented evidence of the fact that sterols may be involved in defence mechanisms. The steroid glycoalkaloids, α-chaconine and α-solanin, were the major fungitoxic substances in potato peel, being present in at least eight times the amount necessary to inhibit 50% of the growth of Helminthosporium carbonum, a fungus which does not attack potato. Phytophthora infestans is not affected due to the lack of cell-membrane sterols, one of the two prerequistes for susceptibility to saponins. Tomatine, another steroid glycoalkaloids of solanaceous plants, was more toxic to non-pathogens than to pathogens of tomato and Septoria lycopersici (tomato leaf spot) was able to detoxify tomatine, whereas other Septoria spp., non-pathogenic to tomato, were unable to do so. These results provide some circumstantial evidence that tomatine may protect tomato leaves against certain pathogens and further investigation is needed. The mode of action of these fungitoxic steroid glycoalkaloids is not properly understood, but as suggested by Hendrix (1970) in his review on “Sterols in growth and reproduction in fungi”, this may be brought about by the disruption of permeability in fungi. Absence of Nutrients Required by the Pathogen Unless the host tissue supplies all the nutrients necessary for the growth of a pathogen, the latter will not grow and cause infection. Walker and Stahmann (1955) suggest that in such pathogens the availability of highly elaborate growth factors, such as vitamins, polypeptides, amino acids and proteins, and the presence of complex enzyme systems may be essential for successful parasitism, and that resistant plants may lack one or more of these factors. Some mutants of Venturia inaequalis which were deficient in cholone and ribloflavin were unable to cause scab unless these substances were supplied (Kline et al. 1957). A total of 54 biochemical mutants including those in which synthesis of various vitamins, organic bases and amino acids has been impaired have been studied in Venturia inaequalis and the effect of each mutation on the pathogenicity of the fungus has been determined. Of the vitamins requiring mutants only those in which metabolic reactions involving choline or riboflavin were affected, were found to have lost in pathogenicity. All mutants in which the metabolism of purines, pyrimidines and amino acids had been affected, incited only the fleck reaction and were non-pathogenic (Keitch, et al.1959). Lukezic and De Vay (1964) have correlated the resistance of plum varieties to Rhodosticta auercina (canker) with the amount of myoinositol in the bark of trees. This fungus is naturally deficient in

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myoinositol, and more of this was present in the bark of the susceptible cultivar President, than in the bark of the two resistant cultivars, Beauty and Duarte; further, the resistance of Duarte was reduced by adding myoinositol to the bark tissue. Resistance has also been attributed to the concentration of nutrients in plant tissues, as distinct from the absence of specific nutrients needed by the pathogen. Sugar content is perhaps the best known example of this. The high sugar content of tissues favours some pathogens, such as certain rusts and powdery mildews, whereas the reverse is true for other, including Alternaria solani (early blight of potato) and Ceratostomella ulmi (Dutch elm disease), as discussed by Horsfall and Dimond (1957). Plants presumably become resistant to high sugar pathogens when the content of their tissues falls to a certain level and this may be brought about, for example, by treatment with certain growth regulating substances. Boron facilitates the translocation of sugars so that these tend to accumulated in the leaves of borondeficient plants and the latter are apparently resistant to Cochliobolus sativus (barley leaf spot), a lowsugar pathogen, but susceptible to Erysiphe graminis, a high-sugar pathogen. Erwinia carotovora var. atroseptica which causes bacterial soft rot of potatoes is less severe on potatoes with low reducing sugar content than on potatoes with high in reducing sugars. Beever (1970) found that the xylem sap of trees (peach, plum) susceptible to Stereum purpureum contained more nitrogen and carbohydrate and was more favourable to the growth of the fungus than was the sap of resistant trees (apple, pear). There were also qualitative differences in that the main sugar and sugar alcohol in the sap from resistant trees were sucrose and sorbitol, neither of which supported good growth of S. purpureum, whereas the sap of susceptible trees also contained glucose and fructose which were more favourable to the growth of the fungus. pH There are few convincing cases of disease resistance directly attributable to pH, although the resistance of ripe tomatoes to Bacterium vesicatorium (Xanthomonas vesicatoria) may be due to their high acidity (pH 4-4.5) as reported by Gardner and Kendrick (1921). The pH of the plant tissues influences many physiological processes in both plants and parasites, notably enzyme activities, and resistance to certain pH-sensitive pathogens might be related to unsuitable pH of the plant tissue. Osmotic Pressure and Permeability Effects The cells of parasitic fungi have an unusually higher osmotic pressure than that of the surrounding host cells, this being necessary for absorption of water from the latter. In the case of Erysiphe cichoracearum (lettuce mildew), cells of resistant tissues or varieties have a higher osmotic pressure than those of the susceptible ones. Further, resistance can be modified by altering the mineral nutrition of the plant and shaded leaves or those cultured on distilled water become susceptible, whereas young lettuce seedlings with high osmotic pressure are resistant. Defence through Absence of Common Antigens

The gene-for-gene concept of the relationship between the host and pathogen evolved from the work of Flor (1955) with flax rust. The idea that the antigens shared by the host and pathogen may determine the nature of disease reactions has been explored by Doubly et al. (1960). Tests of globulin antigens from four races of rust and four from near isogenic lines of flax suggested that susceptibility depended on the presence, in the host, of a protein serologically similar to the one possessed by the pathogen. Some support for the common antigen hypothesis has been obtained from work on non-obligate parasites. Pathogenic races of Xanthomonas campestris pv. malvacearum which cause the angular leaf spot of

How Plants Defend Themselves Against Infection 153

cotton, had more antigens in common with those of cotton leaves than did the non-pathogenic races of bacteria and the black rot fungus Ceratocystis fimbriata which attacks sweet potato (De Vay et al., 1967). Phytoalexins Lasting immunity against certain viral diseases in humans and animals has been known to science for many years. However, there has been no evidence that plants possess anything like an antibody-forming mechanism which makes for recovery and protects against subsequent infections as in animals. The virus-diseased plants do not normally recover and in fact they remain infected till they die. Wingard (1928) for the first time presented evidence that there were symptoms of recovery in tobacco ring-spot-virus-infected plants. Wingard’s view that these infected plants acquired an immunity to the disease was vigorously supported by Price (1932, 1936 a, b). Later, workers opposed the use of the term immunity to this phenomenon of recovery, largely because the disease clearly had two phases – the acute and chronic. Moreover, the recovered plants contained the virus. Indeed, Wallace (1938, 1940) suggested that in the case of tobacco plants infected with the sugar beet curly top virus, recovery was due to the in vivo development of protective substances and considered that they could be transferred to other plants where they would confer a form of passive acquired immunity. This suggestion put forward by Wallace was followed soon after by a somewhat similar approach to a host reaction in the potato – Phytophthora disease resulting in the discovery of substances called phytoalexins (Muller and Boerger, 1940). During the last 60 years many papers have described the structure, synthesis and activity of such compounds, now called phytoalexins. The term was introduced by Muller and Boerger (1940) to describe the substances that inhibit fungus development formed when living plant tissues are invaded by a fungal parasite. It has since been redefined and broadened to include substances formed by host tissue “……. in response to injury, physiological stimuli, infectious agents or their products ….. (that) accumulate ……. to levels which inhibit the growth of micro-organisms” (Kuc, 1972). Day (1973) proposed to include substances that are formed in response to infection, that are toxic to, or which repel insect or nematode parasites. Phytoalexins can be described as lowmolecular-weight antimicrobial compounds that accumulate in plants as a result of infection or stress (Kuc, 1995). They may be associated with host-cell necrosis (hypersensitivity) at the infection site but not necessarily so when induced by chemicals. It was envisaged that the factor, which induces the formation is released by the parasite into the host cell. The role of the fungus and the nature of the fungal metabolites involved in host-parasite interactions, have as yet received very little attention from research workers in this field. Phytoalexins are now being increasingly investigated into and have been detected in several diseases, but more investigation of their overall significance as a resistance mechanism is needed. Among other papers are those by Muller and Boerger (1939), Muller (1956, 1961), Cruickshank (1963) and Kuc (1966,1976). There is a recent article on “Phytoalexins Stress metabolism, and disease resistance in plants,” by Kuc′ (1995). There are two hypotheses put forward to explain the mechanism of the induction of phytoalexins. 1. De novo synthesis of enzymes, or 2. Activation or activity control of enzymes already formed. In the field of phytoalexin research, only the end-products are known with certainty. The metabolic pathways and associated enzymes involved in the biosynthesis of these products are largely unexplored. The initial work of Muller and Boerger (1940) laid the foundation of the concept of phytoalexins. The conclusions of Muller and Boerger (1940) are given below: 1. A principle designated as phytoalexin which inhibits the development of the fungus in hypersensitive tissues is formed or activated only when the host cells come into contact with the parasite.

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2. The defence reaction occurs only in the living cells. 3. The inhibitory material is a chemical substance and may be regarded as a product of necrobiosis of the host cell. 4. The phytoalexin is non-specific in its toxicity towards fungi; however, fungal species may be differentially sensitive to it. 5. The basic response that occurs in resistant and susceptible hosts is similar. The basis of differentiation between resistant and susceptible hosts is the speed of formation of phytoalexin. 6. The defence reaction is confined to the tissue colonized by the fungus and its immediate neighbourhood. 7. The resistant state is not inherited. Phytoalexins are probably fungistatic rather than fungicidal but this no doubt depends partly on the quantity present. According to Cruickshank (1963), the rate of formation of phytoalexin is probably critical in combinations of host variety parasite strain (as in the reaction of the different varieties of potato to different races of Phytophthora infestans) but probably less so when combinations of pathogens and non-pathogens of specific hosts are involved. Host specificity of phytoalexins occurs between genera but probably not within a species or even between related species. The gene-for-gene relationship might be interpreted by the phytoalexin characteristic of the pathogen, both being genetically determined; a range of host-parasite interaction types might thus arise. Susceptibility could be related to the failure of the invading pathogen to stimulate the formation of phytoalexins or to its tolerance of the amounts produced, which in turn is related to its ability to degrade the phytoalexin. There is increasing evidence that some of the fungi which attack a plant are able to do so because they have the capacity to degrade its phytoalexin and the non-pathogens are not able to do this. Relatively few phytoalexins have been investigated for their contribution to varietal resistance. From the point of view of practical agriculture, the mechanism of varietal resistance and susceptibility are much more pertinent than a fundamental consideration of immunity. Ipomeamarone This is an abnormal sesquiterpinoid (tetrahydrofuran, C15H22O3, mol. wt. 230) that seems to be induced in sweet potato (Ipomea batatas) tissue infected with black rot fungus Ceratocystis fimbriata, by a possible disturbance of carbohydrate and fatty acid metabolism of the host (Uritani and Akazawa, 1959). These authors considered that the biosynthesis of ipomeamarone may involve mevalonic acid as a precursor and could perhaps also be induced by poisonous chemicals and pectinase (Tomiyama, 1963). The presumption was that the carbon sources being utilized for respiration and biosynthesis of phenolic compounds, may, by an inhibition being utilized for respiration and biosynthesis of phenolic compounds, may, by an inhibition of the TCA cycle and pathway of the biosynthesis of phenolics, be diverted to the biosynthesis of ipomeamarone. Ipomeamarone increases substantially in diseased tissues adjacent to infected tissues. Such tissues show terpenes which are also produced by slices of sweet potato in response to chemical stimuli. The interesting part is that ipomeamarone is toxic to both non-pathogenic and pathogenic fungi and to some bacteria, and there seems to be no correlation between its inhibitory effect and pathogenicity.

How Plants Defend Themselves Against Infection 155

Pisatin Pisatin has the chromocoumarin ring system and is a phenolic ether (C17H14O6, mol. wt. 314). Cruickshank and Perrin (1960) and Perrin and Bottomeley (1962) isolated pisatin from the pods of the garden pea inoculated with fungi, using the drop diffusate technique. Subsequent studies by Cruickshank and his colleagues are classic in phytoalexin research and provided the impetus for much of the research which followed thereafter. This is produced by the exposed endocarp of the detached pea in response to inoculation with many fungi or injury. When different fungi, pathogenic and non-pathogenic to pea, are used for inoculation, they induce the production of different concentrations of pisatin. The non-pathogenic fungi induce the formation of pisatin at concentrations high enough to inhibit their growth. If pea pathogens induce the formation of pisatin, it is at concentrations much below those that are toxic to the pathogen. Production of pisatin by pea pods inoculated with Monilinia fructicola, a non-pathogen, is reduced or delayed at high temperature on anaerobic storage, and the pea pods become susceptible to this fungus, while storage at normal temperature conditions results in the accumulation of high levels of pisatin and resistance. Pisatin can also be formed when dilute solutions of certain metallic salts (especially those of mercury, silver and copper) are placed on pea pod endocarp tissues and it is possible that phytoalexins are produced when metallic fungicides are applied to crops. Certain metabolic inhibitors such as sodium fluoride and sodium monoiodoacetate are reported to have similar effects. Other substances reported to induce pisatin formation by pea pods include ethylene, which at a concentration of 1 ppm induces a slight production. At certain concentrations, a number of antibiotics (actinomycin D, cyclohesimide, chloramphenicol, and others) and ribonuclease stimulate pisatin formation. However, they fail to do so at higher concentrations (Schwochau and Hadwiger, 1969). Many of these substances interfere with protein synthesis in plants. Species or races of fungal pathogens, pathogenic to a particular species seem to stimulate production of generally lower concentrations of phytoalexins than nonpathogens. In case of pea pods inoculated with the pathogen Ascochyta pisi, pea varieties produce concentrations of pisatin that are approximately proportional to the resistance of the variety to the pathogen.

Phaseollin Phaseollin is similar to pisatin in chemistry and function. It was isolated from detached, open bean pods, following inoculation into the non-pathogen Monilinia fructicola. More recently, a number of compounds structurally similar to phaseollin have been identified from diffusates or tissues inoculated with fungi, bacteria, and viruses. These are phaseollidin and phaseollinisoflavan and kievitone. According to Rahe et al. (1969), phaseollin is important in determining varietal resistance to Colletotrichum lindemuthianum, the cause of anthracnose of beans. It accumulates most rapidly in bean hypocotyls inoculated with an avirulent race. Inoculation with a virulent race produces a much lower level of accumulation. Three races of the same organism examined by Bailey and Deverall (1971) were equally sensitive to phaseollin, suggesting that if phaseollin had a role in race specific resistance, it would depend on the amount produced and not on differential toxicity.

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Glyceollin This is produced in soybean plants infected with the fungus Phytophthora megasperma f. sp. glycinea. Inoculation of fungal races on incompatible host cultivars resulted in higher concentrations of the phytoalexin glyceollin than in inoculations of fungal races on compatible cultivars. It has been suggested that the higher concentrations of glyceollin in incompatible host-pathogen combinations are the result of reduced biodegradation rather than increased biosynthesis

Trifolirhizin This is a new glucoside which has been isolated from the roots of red clover. Its aglycone has the formula, C16H12O5. Trifolirhizin is of interest here, as its structure indicates that it is chemically closely related to pisatin.

Orchinol and Hircinol Orchinol is a phenanthrene and one of the three compounds formed by the interaction of diffusible metabolities of Rhizoctonia repens with Orchis militaris tuber tissues (Gaumann et al., 1950). According to Gaumann, when there is a stimulus from outside, both Orchis militaris and Loroglossum hircinum synthesize concurrently two substances – orchinol and hircinol. O. militaris produces 50 to 100 times more orchinol than hircinol, whereas L. hircinum produces 100 times more hircinol than orchinol. Both substances are found in tissues of bulbs, roots and stalks under aerobic

How Plants Defend Themselves Against Infection 157

conditions, only as a defence mechanism from specific fungal infection, especially mycorrhizae, and not against common saprophytic or semiparasitic soil fungi (Gaumann, 1964). Gaumann considers that the stimulus causing the synthesis of hircinol is a microbially-induced antigenic effect resulting in antibody production or from lesions, a traumatic induction of antibody production. Antimicrobial spectrum of orchinol is broad and hardly specific.

Isocoumarin This has been isolated from carrot root tissues inoculated with a fungus non-pathogenic to carrot, Ceratocystis fimbriata (Condin and Kuc’, 1960). This compound is of special interest as it can be produced in tissues in response to a number of non-pathogenic microorganisms such as Ceratocystis ulmi, Helminthosporium carbonum, Fusarium oxysporum f. sp. lycopersici race 1 and Thielaviopsis basicola, whereas when Stemphylium radicinum, a pathogen of carrot, was inoculated into this tissue, no such response was noticed. Klarman and Sanford (1968) obtained a crystalline compound with absorption maximum at 288, 293, 315 nm from soybean (Glycine max). They suggested that these compounds may be chemically related to the pterocarpan phaseollin.

Medicarpin Alfalfa (Medicago sativa) inoculated with a series of pathogens and non-pathogens of this host has been studied by Higgins and Millar (1968). The antifugal compound was isolated from leaf diffusate solutions and has been identified as (-) dimethyl-homopterocarpin and given the name, medicarpin. Wyerone acid Purkayastha and Deverall (1965) and Deverall and Vessey (1969) have reported an ether soluble acid identified as wyerone acid, CH3—CH2-CH==CH. C==C.CC. C==CH. CH==C. CH==CHCOOH in broad bean. Infection of broad bean leaves by Botrytis fabae leads to an increase in wyerone acid and wyerone, its methyl ester.

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Cicerin Kunzru and Sinha (1970) reported the occurrence of a new phytoalexin associated with Ascochyta blight of gram. The inhibitory principle is composed of two fluorescent phenols or phenolic compounds. Phytoalexin diffuses into inoculation droplets during the course of interaction between Ascochyta rabie and the pod tissues of Cicer arietinum. The name cicerin was tentatively suggested by them. Rishitin Muller and Boerger (1940) and Muller (1953) were the first to show that the potato tubers carrying the gene R1 for late blight resistance responded when inoculated with avirulent race (race 0) of Phytophthora infestans by producing a phytoalexin that inhibited the development of a virulent race (race 1) applied a short time later. A number of other examples of host parasite systems in which inoculation with avirulent strain protects the plant against subsequent infection were reviewed (Matta, 1971). Two non-pathogens of potato (C. fimbriata and Helminthosporium sativum) also stimulated the accumulation of rishitin in both kinds of tuber tissues. In studies on potato tubers (Solanum tuberosum) infected with an incompatible race of Phytophthora infestans, isolated a new compound with phytoalexin-like properties. It is a bicyclic non-sesquiterpene alcohol (Katsui et al., 1968) and has been given the name, rishitin.

Gossypol Bell (1967) has reported the accumulation of several antifungal compounds as a result of the introduction of conidia of Verticillium albo-atrum and Rhizopus nigricans, heavy metal ions and various metabolic inhibitors into boll cavities of xylem vessels of the excised stems of two species of Gossypium and several cultivars of cotton (glandular and glandless). The compound has been identified as gossypol. Bell (1967) has identified it as an ether-soluble phenol. In 1969 he related the accumulation of gossypol and gossypol-like compounds in stem sections and intact plants to host resistance and inversely to the virulence of the pathogen. Other diseases in which there is evidence of the formation of phytoalexin-like substances include black spot of rose (Diplocarpon rosa), leaf spot of wheat caused by Septoria tritici, leaf disease of Primula caused by Ramularia primulae and Cercosporella promulae, leaf spot of Brassica species and viciatin from broad bean, the structure of which does not appear to have been characterized.

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Capsidiol Diffusates from pepper (Capsicum frutescens ) fruits inoculated with a number of nonpathogenic fungi accumulate a sesquiterpene phytoalexin capsidiol in concentrations that are sufficient to inhibit these fungi in vitro (Gordon et al., 1973).

Xanthotoxin This phytoalexin was isolated from the top millimetre of parsnip root discs inoculated with C. fimbriata, a non-pathogen of parsnip (Johnson et al., 1973). Inoculation with other non-pathogens of parsnip also resulted into the production of xanthotoxin. Camalexin is produced in Arabidopsis and Momilactone A phytoalexin has been reported in rice.

DEFENCE THROUGH INDUCED SYNTHESIS OF PROTEINS AND ENZYMES

Molecular biology of pathogenesis and induced systemic resistance has been reviewed by Vidhyasekaran (1998). In addition to the phenolic substances produced in plant tissues in response to infection, induced synthesis of proteins and enzymes seems to play a role in disease resistance. In the case of the black rot of sweet potato caused by the fungus Ceratocystis fimbriata, sweet potato tissues inoculated with the pathogen and with a non-pathogen that induces immunity, showed marked changes in 9 of the 13 enzymes examined by Weber and Stahmann (1966). The influence of host parasite interactions on proteins, enzymes and resistance has been discussed by Stahmann (1967). Nucleic acid and protein changes in wheat leaf nuclei during rust infection were studied by Bhattacharya et al. (1965). There were decreases in histones and marked increases in RNA and acidic proteins as early as two days after inoculation in the nuclei of the mesophyll cells of a susceptible wheat infected with Puccinia graminis tritici. Deese and Stahmann (1962a, 1962b) reported that pectic enzyme formation by Fusarium or Verticillium was greatly reduced when the pathogens were grown on tissues from resistant varieties as compared to that from susceptible varieties. In numerous incompatible host-parasite combinations, peroxidase activity is often several times higher than in compatible ones (Fric and Fuchs, 1970). In other cases, however, peroxidase activity is greater in compatible reactions (Fric, 1969; Grzelinska, 1969; Wood and Barbara, 1971). It is, therefore, clear that the activation of peroxidase in the host and parasite complex need not always be accompanied by the incompatible reaction.

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Plants defend themselves from pathogen infection through a variety of mechanisms that can be either local or systemic, constitutive or inducible. In most cases, the extent to which these various systems affect disease progression is poorly understood and is an area of extensive biochemical and genetic research (Kessman et al., 1994). One particular inducible systemic response, known as systemic acquired resistance (SAR) has become a subject of great importance and inquiry. SAR is a broad, physiological immunity that develops from infection with a necrogenic pathogen. In addition to this, certain natural and synthetic chemical compounds on trigger similar plant responses. The fresh carefully controlled laboratory study SAR was published 1961 when Ross (1961a, 1961b) used tobacco mosaic virus (TMV) on a local lesion host (Xanthi–nc tobacco) and found that subsequent infections were reduced in severity. Ross coined the term SAR for the resistance that developed in the distal, unheated portions of TMV-inoculated plants. Resistance that developed in the same leaf that had been TMV-inoculated he called local acquired resistance (LAR). Cruickshank and Mandryk (1960) showed high levels of resistance in field grown tobacco against blue mould (Peronospora tabacina) when plants were injected in the stem with the spore suspension of the same fungus. Cohen and Kuc (1981) subsequently showed that resistance takes approximately 3 weeks to develop. Heat killed conidia and other pathogens of tobacco failed to induce SAR. Other workers have shown that inoculation of tobacco leaves with Thielaviopsis basicola leads to necrosis and subsequent systemic resistance to other pathogens, TMV, and tobacco necrosis virus (TNV). The SAR response in tobacco gives a broad-spectrum disease resistance to fungal bacterial and viral pathogens but is ineffective against aphids and tobacco horn worm (Manduca sexta). Some of the examples where SAR have been recorded are for cucumber, muskmelon and watermelon. Localized infections of cucumber with either TNV, Pseudomonas lacrymans or Colletotrichum lagenarium lead to broad spectrum systemic resistance. In monocotyledonous plants, there are well developed SAR systems established both in rice and wheat. In rice, Smith and Metraux (1991) reported that the inoculation of the first, fully developed leaf with Pseudomonas syringae pv. syringae, which causes a rapid hypersensitive necrosis results in systemic resistance against Pyricularia grisea (P. oryzae) the causal agent of blast of rice. There are several other examples of SAR and LAR in various host-pathogen combinations. Mechanism of SAR (SELF ACQUIRED RESISTANCE)

In 1970, the groups of Van Loon and Van (1970) and Gianinazzi et al., (1970) showed that viral infection of tobacco induced the accumulation of a distinct set of proteins called pathogenesis-related proteins (PR proteins). PR-Proteins Pathogenesis-related proteins often called PR proteins are generally a group of plant proteins that are toxic to invading fungal pathogens or other pathogens. Several groups of PR proteins have been classified according to their function, serological relationship, aminoacid sequence, molecular weight, and certain other properties. PR proteins are either extremely acidophilic or extremely basic and therefore are highly soluble and reactive. Some better known PR proteins are PR1 proteins, β,1, 3,glucanases, chitinases, lysozymes, PR4 proteins, thaumatin-like proteins, osmolur-like proteins, cystein-rich proteins, glycine-rich proteins, proteinase inhibitors, proteinases, chitosanases, and peroxidases (Agrios, 1997). Although healthy plants may contain trace amounts of several PR proteins, it is attack by plant pathogens, treatment with defence elicitors, wounding or stress that induces transcription of a battery of genes

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that code for PR proteins. It has been shown that β,1,3 glucanase and chitinase, diffuse towards and affect (break down) the chitin-supported structure cell walls of several but not all pathogenic fungus, whereas lysozymes degrade the glucosamine and muramic acid component of the cell wall of bacterial plant pathogens. Plants which have been genetically engineered and in which chitinase gene has been introduced show good resistance against Rhizoctonia solani. Some characteristics of systemic acquired resistance (SAR) are that: 1. It is induced by agents/pathogens causing necrosis, for example, local lesions. 2. There is a delay of several days between induction and full expression. 3. Production is conferred on tissues not exposed to induced inoculation 4. Is expressed as reduction in lesion number, size, spore production, and so on. 5. Production is long lasting 6. Production is non-specific. 7. Development of SAR is associated with expression of several gene families for example, PR proteins. 8. The signal for SAR is translocated and graft transmissible. 9. The protein not passed on to the next progeny, the transmission to clonal tissues is unresolved so far. Most current interest in SAR concerns the possible identity of the translocable signal. It has been known for sometime that the signal is graft transmissible and presumably moves in the vascular system. In addition to developing as a result of a primary infection, resistance can be induced by treatments of plants with such compounds such as viral coat proteins, bacterial or fungal proteins, lipoproteins, polysaccharides, and yeast RNA and by synthetic molecules mostly polyanions such as polyacrylic acid, salicylic acid (aspirin) and 2-chloroethyl phosphonic acid, 2,6-dichloro-isonicotenic acid (CGA-41396) and its methylated ester (CGA-41397) both referred to as INA. These substances act as in access of local resistance in plants when they are applied through petioles and roots. In general, the greater the concentration of the inducer, the faster and more effective the induced resistance in the host. Localized induced resistance in inoculated (treated) leaves develops 2-3 days after primary infection (treatment) while systemic induced resistance, if present, usually develops 7 or more days after the primary infections and may persist for 3 to 5 weeks. It is interesting to note that induced resistance may be prevented if the plants are treated with actinomycin D. Actinomycin D prevents transcriptions of the cell DNA to messenger RNA and therefore prevents productions of new proteins (enzymes). Further evidence that salicylic acid plays an important part in the signal pathway has come from the work on Arabidopsis mutants altered in SAR expression and transgenic plants engineered to break down salicylic acid. The signal molecules implicated in the induction of plant defence are given as follows: These are salicylic acid, Acetyl salicylic acid, Dichloro-iso-nicotinic acid Ethylene, xylanase, the polypeptide systemin, jasmonic acid and probably others.

Val Ala H2N

Gln Ser Lys Pro

Pro

Ser Lys Arg

Pro Asp

Systemin

Pro

Lys Met

Gln Thr Asp COOH

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COOH OH

COOH

COOH

O O

Salicyclic acid

Acetyl salicylic acid

Cl

N

Cl

DCINA (Dichloro-iso-nicotinic acid)

Ethylene, xylanase, the polypeptide systemin, jasmonic acid and probably others. A number of reports have claimed resistance inducing activity for certain chemicals or extracts, but have failed to demonstrate that the compound in question fulfills the criteria previously established for SAR activators. Fosetyl-AL, metalaxyl (Ward, 1984) and more recently triazoles (Hauthal, 1993) are among these compounds. Fosetyl-AL shows very weak antifungal activity in traditional in vitro tests. Plants pretreated with these compounds accumulate phytoalexins faster and the effectiveness of metabolic inhibitors like glyphosate decreases. These experiments led to speculations that fosetyl-AL was a resistance-inducing compound. There are several other compounds which elicit the defence mechanism of plants such as probenazole, a systemic compound used successfully to protect rice against Pyricularia grisea (P. oryzae) and weakly against Xanthomonas oryzae. An astonishing case of induced resistance was reported by Arimoto et al. (1991) in which they claimed that rice plants can be protected for generations when the seeds are soaked in a 500 ppm solution of DL-alanine dodecylester HCl. However no further information of this has come from other quarters. Cohen and co-workers (1993) reported that jasmonic acid and its methylester induced resistance in tomato towards Phytophthora infestans. DEFENCE THROUGH FORMATION OF SUBSTRATES RESISTING THE ENZYMES OF THE PATHOGEN

The resistance of green immature apple fruit to several fungi which can infect the mature fruit has been attributed to the presence in the green fruit of a pectin-protein-polyvalent cation complex which is resistant to hydrolysis by fungal hydrolytic enzymes and suppresses the production of pectin methyl esterase and polygalacturonase by the fungi. An induced mechanism of resistance through increased resistance of the substrate to enzymes by the accumulation of polyvalent cations has been reported for Rhizoctonia-infected bean tissues. In the resistant variety, calcium ions accumulate in and immediately around developing Rhizoctinia lesions, inhibit further tissue maceration by polygalacturonase, and therefore, play a role in confining the pathogen to lesions of a limited size. Fungal PME demethylate pectic substances near and in advance of the

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pathogen and result in the formation of pectate salts with polyvalent cations, particularly calcium, which in turn are resistant to hydrolysis by fungal polygalacturonase. Disease resistance introduced through artificial application of auxins to infected plants has been reported for Fusarium wilt of tomato and late blight of potato. The resistance to disease in auxin-treated plants is accompanied by higher levels of insoluble pectates and lower levels of water soluble pectins that are found in untreated, susceptible plants. The resistance of cider apples to wound infection by the brown rot fungus Sclerotinia fructigena seems to be due to the ability of phenolic oxidation products in these apples to inactivate extracellular pectinolytic enzymes produced by the fungus. The phenolics produced in the juice of the resistant variety do not inhibit the growth of the fungus, but do inhibit the activity of its polygalacturonase. DEFENCE THROUGH DETOXIFICATION OF PATHOGEN TOXIN

Detoxification may operate in resistance of vascular wilt diseases caused by Fusarium oxysporum. Its significance depends on the importance properly ascribed to toxins in these diseases. Lakshminarayanan (1955) reported that varieties of cotton resistant to F. oxysporum f. vasinfectum contain relatively high concentrations of cystine and that cystine reduces the toxicity of the complex formed when ferric iron is added to filtrates from the cultures of the parasite. When shoots are allowed to absorb cystine from solutions before the complex is transferred to the former, they are less damaged than are plants not pretreated with cystine. In the case of the toxin, piricularin, Tamari (1959) found that chlorogenic acid could detoxify the piricularin when applied to rice seedlings in equimolar concentrations. Tamari et al. (1962) also showed that piricularin was detoxified with ferulic acid, which was oxidized by peroxidase, and its oxidized products were also shown to detoxify the piricularin. Those varieties of rice which have more have more chlorogenic or ferulic acid are more resistant to the blast disease. The partially purified toxin produced by Alternaria kikuchiana is inhibited by various kinds of oxidizing substances, including dehydroascorbic acid (Mori, 1962). Mori (1962) showed that the resistant varieties of pear contained more dehydroascrobic acid and more ascorbic acid oxidase than the susceptible varieties. DEFENCE THROUGH ALTERED RESPIRATION

The relation of increased respiration to disease resistance and susceptibility is unclear. In some incompatible host-pathogen combinations (resistance), the increase is initially greater than in compatible combinations (susceptibility), although the increase may be of greater total magnitude and longer in duration in susceptible reactions (Uritani, 1962). From observations, however, indicating that substances (narcotics) inhibiting respiration also decrease resistance to disease in the host, it is suggested that increased respiration is often a necessary condition for the development of a defence reaction in the host. A marked increase in oxygen uptake has been reported for the hypersensitive defence reactions. This is also accompanied by the accumulation and oxidation of phenolic compounds and by the activation of phenoloxidases.

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DEFENCE THROUGH HYPERSENSITIVE REACTION

Hypersensitivity reaction or hypersensitivity functions as a defence reaction in plants against pathogens. It occurs only in incompatible host-parasite combinations. This is a significant and common form of reaction in plants. Usually, a small part of the plant tissue quickly dies after infection, resulting in a minor or local lesion or one as extensive as to cause the death of the plant. Hypersensitive reaction is believed to be caused by the disturbance of the balance between the oxidative and reductive processes in the cells resulting in excessive oxidation of phenolic substances and leading to the breakdown of cellular structures (Fig. 6.8) The hypersensitive response is the culmination of the plant defence response indicates by the recognition by the plant of specific pathogen-produced signal molecules (elicitors). Recognition of the elicitors by the invaded host plant elicited a cascade of biochemical reactions in the plant attacked and surrounding cells leads to altered cell function and new or greatly activated defence-related compounds. z G

H

H

H PS a

b

c

d

e

f

NC

Fig. 6.8 Different stages in the development of necrotic defence reaction (hypersensitivity) in a cell of a very resistant potato variety infected by Phytophthora infestans. N = nucleus, Z = zoospore, PS = protoplasmic strands. H = hypha. G = granular material. NC = necrotic cell. (Redrawn from K. Tomiyama (1956). Ann. Phytopath. Soc., Japan. 21: 54-62.)

THE CONCEPT OF PHYTONCIDES

In 1930, at the All-Union Conference of Zoologists in Kiev, USSR, Tokin reported the results of his preliminary observation on the antibiotic properties of plants. Counting this work, he published a monograph in 1942 on antibacterial substances formed by plants, which he called phytocides. This term was later extended by other Russian worker to include the substances of plant origin active against other organisms, both animals and plants. Phytoncides, by definition, include phytoalexins, and pre-formed and post-infectionally formed substances which give resistance to plant pathogens, in addition to those which deter or destroy insect pests. Rubin and Arzichowskaja (1963) discuss this aspect of phytoncides in detail. It is felt by plant pathologists that the term is too all-embracing to be of much use and the individual categories of chemical substances known so far are adequately covered by more specific terms.

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REFERENCES Agrios, G.N. (1997), Plant Pathology, Fourth Edition Academic Press, New York, pp. 635. Akai, S. (1959), “History of defense in plants”, in: Plant Pathol.—An Advanced Treatise, J.G. Horsfall and A.E. Dimond (Eds.), Academic Press, London, pp. 435–467. Akai, S. and M. Fukutomi (1980), “Preformed internal physical defenses”, in Plant Disease An Advanced Treatise Vol. IV, J.G. Horsfall and E.B. Cowling (Eds.), Academic Press, New York, pp. 139–160. Allen, E.H. and J. Kuc (1968), “Solanine and chaconine as compounds in extracts of Irish potato tuber”, Phytopathol., 58: 776–781. Arimoto, Y., Y. Homma, R. Yoshina, S. Saito (1991), “Generational accession of DL-alanine dodecylester HClinduced resistance to blast disease in rice plants”, Ann. Phytopathol. Sci. J., 57: 522–525. Asada, Y., W.R. Bushnell, S. Ouchi, and C.P. Vance (Eds.) (1982), Plant Infection: The Physiological and Biochemical Basis, Springer-Verlag, Berlin and New York. Asada, Y., W.R. Bushnell, S. Ouchi, and C.P. Vance (1982), “Studies on the mode of action of tomatine as fungitoxic agent”, Pl. Pathol., 43: 683–686. Bailey, J.A. and B.J. Deverall (1971), “Formation and activity of phaseollin in the interaction between bean hypocotyls (Phaseolus vulgaris) and physiological races of Colletotrichum lindemuthianum”, Physiol. Pl. Pathol., 1: 435–449. Bailey, J.A. and B.J. Deverall (1983), The Dynamics of Host Defense, Academic Press, New York. Bell, A.A. (1967), “Formation of gossypol in infected or chemically irritated tissue of Gossypium sp.”, Phytopathol., 57: 759–764. Bell, A.A. (1981), “Biochemical mechanisms of disease resistance”, Ann. Rev. Plant Physiol., 32: 21–81. Bergman, B.H.H. (1966), “Presence of a substance in white skins of young tulip bulbs which inhibit growth of Fusarium oxysporum”, Neth. J. Pl. Pathol., 72: 222–230. Bergman, B.H.H. and J.C.M. Beijersbergen (1968), “A fungitoxic substance extracted from tulips and its possible role as a protectant against disease”, Neth J. Plant Pathol., 74: 157–162. Beever, D.J. (1970), “The relationship between nutrients in extracted xylem sap and susceptibility of fruit trees to silver leaf disease caused by Stereum purpureum,” Ann. Appl. Biol., 65: 85–92. Bhattacharya, P.K., J.M. Naylor and M. Shaw (1965), “Nucleic acid and protein changes in wheat leaf, nuclei during rust infection”, Science, 150: 1605–1607. Blackhurst, F.M. and R.K.S. Wood (1963), “Resistance of tomato plants to Verticillium albo-atrum”, Trans. Brit. Mycol. Soc., 46: 385–392. Bowels,D.J. (1990), “Defense related protein in higher plants”, Ann. Rev. Biochemistry, 59: 873–907. Buxton, E.W. (1957), “Some effects of pea root exudates on physiologic races of Fusarium oxysporum f. sp. pisi”, Trans. Brit. Mycol. Soc., 40: 145–154. Cohen, Y., U.Gisi and T. Niderman (1993), “Local and systemic protection against Phytophthora infestans induced in potato and tomato plants by jasmonic acid and jasmonic acid methyl ester”, Phytopath., 71: 783–787. Condon, P. and J. Kuc (1960), “Isolation of fungitoxic compound from carrot tissue inoculated with Ceratocytis fimbriata”, Phytopath., 50: 267–270. Cruickshank, I.A.M. (1963), “Phytoalexins”, Ann. Rev. Phytopath., 1: 351–374. Cruickshank, I.A.M. and D.R. Perrin (1960), “Isolation of a phytoalexin from Pisum sativum L.”, Nature, 187: 799–800. Cruickshank, I.A.M, and M. Mandryk (1960), “The effect of stem infestation of tobacco with Peronospora tabacina adam. on foliage reaction to blue mould”, J. Aust. Inst. Agric. Sci., 26: 369–372. Day, P.R. (1973), “Genetic variability of crops”, Ann. Rev. Phytopath., 11: 293–312.

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Jhooty, J.S. and W.E. McKeen (1965), “Studies on powdery mildew of strawberry caused by Sphaerotheca macularis”, Phytopath., 55: 281–285. Johnson, C., D. Brannon and J. Kuc (1973), “Xanthotoxin: a phytoalexin of Pastinaca sativa root”, Phytochem., 12: 2961–2962. Johnson, G. and L.A. Sachaal (1952), “Relation of chlorogenic acid to scab resistance in potatoes”, Science, 115: 627. Johnstone, N.W. and T. Sproston (1965), “The inhibition of fungus infection pegs in Ginkgo biloba”, Phytopath., 55: 225. Katsui, N., A. Murai, M. Takasugi, T. Masamune and K. Tomiyama (1968), “The structure of Rishitin, a new antifungal compound from diseased potato tubers”, Chem. Com., pp. 43–44. Klarman, W. and J. Sanford (1968), “Isolation and purification of an anti-fungal principle from infected soybeans”, Life Science, 7: 1095–1103. Keith, G.W., D.M. Bone and J.R. Shay (1959), “Genetic and nutritional controls of host parasite interactions in apple scab”, in: Plant Pathology—Problems and Progress-1908–1958, C.S. Holton et al. (Eds.), Univ. of Wisconsin Press, Madison, pp. 157–167. Kessman, H., T. Staeeb, C. Hofmann, T. Maetzke, J. Herzog, (1994), “Induction of systemic acquired disease resistance in plants by chemicals”, Ann Rev. Phytopathol., 32: 439–59. Kline, D., M. Boone and G.W. Keitt (1957), “Venturia inaequialis-XIV: Nutritional control of pathogenicity of certain induced biochemical mutants”, Am. J. of Bot., 44: 791–803. Kuc, J. (1963), “Role of phenolic compounds in disease resistance”, in: Perspectives of Biochemical Plant Pathology, S. Rich (Ed.), Coon, Agr. Exp. Sta. (New Haven), Bull., 663: 20–25. Kuc, J. (1966), “Resistance of plants to infectious agents”, Ann. Rev. Microbiol., 20: 337–370. Kuc, J. (1972), “Phytoalexins”, Ann. Rev. Phytopath., 10: 204–232. Kuc, J. (1976), “Phytoalexins”, in: Physiological Plant Pathology, R. Heitefuss, and P. H. Williams (Eds.), Springer-Verlag, Berlin, Heidelberg and New York, pp. 632–652. Kuc, J. (1995), “Phytoalexins, stress metabolism and disease resistance in plants”, Ann. Rev. Phytopathol., 33: 275–297. Kunzru, R. and S. Sinha (1970), “Cicerin, a new phytoalexin associated with blight of gram”, in: Pl. Dis. Prob., Proc. First. Int. Symp. On Pl. Pathol., Indian Phytopathological Society, IARI, new Delhi, S.P. Raychaudhary, et al. (Eds.), pp. 724–731. Lakshaminarayanan, K. (1955), “Studies on Fungal Enzymes with Special Reference to the Fusarium Wilt of Cotton”, Ph. D. Thesis, Univ. of Madras, Madras. Lukezic, F. L. and J. E. DeVay (1964), “Effect of myo-inosital in host tissues on the parasitism of Prunus domestica by Rhodisticta quircina”, Phytopath., 54: 697–700. Martin, J. T. (1964), “Role of cuticle in the defense against plant diseases”, Ann. Rev. Phytopath., 2: 81–100. Martin, J. T., E.A. Baker and R.J.W. Byrde (1966), “The fungitoxicities of cuticular and cellular components of citrus lime leaves. The fungitoxicities of plant furocounmarins”, Ann. Appl. Biol., 57: 491–500, 501–508. Matta, A. (1971), “Microbial penetration and immunization of uncongenial host plants”, Ann. Rev. Phytopath., 9: 387–410. Melander, L.W. and J.H. Craigie (1927), “Nature of resistance of Barberis spp. to Puccina graminis”, Phytopath., 17: 95–114. Mori, R. (1962), “Studies on resistance of Japanese pears to the black spot disease”, Lib. Arts. J., Tottori Univ., 13: 53–119 Muller, K.O. (1953), “The nature of resistance of the potato to blight Phytophthora infestans”, J. Neth. Inst. Agr. Bull., 6: 346–360. Muller, K.O. (1956), “Einige einfache Versuche zum Nachweis von Phytoalexinen”, Phytopath. Z., 27: 237–254. Muller, K. O. (1961), “The phytoalexin concept and its methodological significance”, in: Recent Advances in Botany, D.L. Bailey (Ed.) Univ. of Toronto Press, Toronto, 1: 396–400.

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Walder, J. C. and K.P. Link (1935), “Toxicity of phenolic compounds to certain onion bulb parasites”, Bot. Gazette, 96: 468–484. Walker, J.C. and M.A. Stahmann (1955), “Chemical nature of disease resistance in plants”, Ann. Rev. Pl. Physiol., 6: 351–366. Wallace, J.M. (1938), “Acquired tolerance of curly top in Nicotiana tabacum”, Phytopath., 28: 674. Wallace, J.M. (1940), “Evidence of passive immunization of plants from curly top”, Phytopath., 30: 25–27. Weber, D. and M. Stahmann (1966), “Induced immunity to Ceratocystis infection in sweet potato root tissue”, Phytopath., 56: 1066–1070. Webster, J. and N. Lomas (1964), “Does Trichoderma viride produce gliotoxin and viridin”, Tran. Brit. Mycol. Soc., 47: 535–540. Wingard, S.A. (1928), “Hosts and symptoms of ringspot: a virus disease of plants”, J. Agr. Res., 37: 127–153. Wood, R.R. and D.J. Barbara, (1971), “Virus multiplication and peroxidase activity in leaves of cucumber (Cucumis sativa L.) cultivars systemically infected with W strain of cucumber mosaic virus”, Physiol. Pl. Pathol., 1: 73–81.

QUESTIONS The cuticle acts as a physical or chemical barrier to infection. Comment. What are the different morphological defence mechanisms in plants? Give suitable examples. What is a biochemical defence mechanism? Explain why in onion smudge caused by Colletotrichum circinans, varieties with red scales are resistant in contrast to varieties with white scales. 5. Give an example of a soil borne disease in which root exudates are implicated in the protective mechanism. 6. What is the role of phenolic substances in the resistance mechanism in plants? 7. Give an example of a host pathogen combination where: i. Absence of nutrients brings about resistance to disease. ii. Resistance is attributable to pH. iii. Resistance is attributable to osmotic pressure. iv. Is the defence mechanism in plants akin to that in animals? v. Who suggested the theory of gene-for-gene responsible for host specificity? vi. What are phytoalexins? Are phytoalexins responsible for host specificity? vii. What is hypersensitivity? How does it bring about resistance to pathogens? viii. How does altered respiration bring about resistance? ix. Give a summary of the potential ways in which metabolic pathways altered by infection play a role in disease and pathogen development. x. What is the concept of phytoncides? Who evolved this concept? xi. What is induced resistance? What is its possible mechanism and what are some of the induced chemical signals?

1. 2. 3. 4.

7 Genetics of Plant Pathogen Interaction

GENETICS OF HOST-PARASITE INTERACTION

Each host-parasite interaction is a struggle for survival between two organisms. In nature, there exists a state of balance and surviving plants and their parasites are capable of coexistence. The attack and defence mechanisms discussed earlier are determined genetically in the pathogen, some of which have different pathogenic abilities and may be able to attack hitherto resistant plants. In both organisms, the more effective races and varieties tend to survive, the less effective ones tend to disappear. Effective disease develops when a virulent race meets a susceptible variety but not when the pathogen is avirulent or the plant is resistant. The genetics of the host parasite interaction have been discussed by Day (1974) and Ellingboe (1976). The molecular genetics of pathogenicity of phytopathogenic bacteria was reviewed by Daniels et al. (1980). Advances in molecular genetics of plant microbe interaction have been elucidated in an edited volume by Hennecke and Verma (1991). The molecular characterization of the gene-for-gene system in plant-fungus interactions and the application of avirulence genes in control of plant pathogens has been reviewed by deWit (1992); the role of gene-for-gene interaction in the determination of host specificity has been reviewed by Heath (1991); molecular mechanisms of fungal pathogenicity in plants has been reviewed by Schaffer (1994); plant resistance genes in signal perception and transduction has been reviewed by Lamb (1994); molecular genetics of plant disease resistance has been recently reviewed by Staskawicz et al. (1995); and molecular approaches to manipulation of disease resistant genes has been reviewed by Michelmore (1995). Hahn (1996) have reviewed microbial elicitor’s and their receptors in plants. RESISTANCE AND SUSCEPTIBILITY

A plant is either immune to a pathogen, that is, it is not attacked at all by the pathogen even under the most favourable conditions, or it may show varying degrees of resistance ranging from immunity to complete susceptibility. Resistance may be conditioned by a number of internal and external factors

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which reduce the chance and degree of infection. There are several kinds of resistance: it may be physical or chemical, it may depend upon the lack of a nutrient or a substance that the parasite needs for development, or it may be due to a toxic or repellent substance that is either preformed or formed only in response to infection. Irrespective of whether the morphological or biochemical defence mechanism is involved in the resistance of a particular host-parasite combination, it is ultimately the genetic makeup of the two that determines whether a particular host will be susceptible or resistant under given environmental conditions. The mechanisms by which genes control the physiological processes that lead to disease resistance or susceptibility are not yet clear, but they are presumably no different from the mechanisms controlling any other physiological process in a living organism. Under the influence of the genetic material (DNA) a specific protein, which may be a structural protein or an enzyme, may be produced. The produced enzyme, of course, under certain conditions will participate or initiate biochemical reaction related to one or the other cellular process, and may result in the production of certain morphological characteristics or accumulation of a certain chemical substance. Most of the approximately 100,000 known species of fungi are strictly saprophytic and degrade dead organic material for their nutrients. Only 8000 can cause disease to one or more species and only 100 are pathogenic to humans and animals. It is the same for all other groups of plant pathogenic organisms such as bacteria, viruses, mycoplasms. What then distinguishes this small minority of pathogenic fungi or other pathogens from the majority of saprophytic fungi or other microorganisms? The fungus Fusarium oxysporum f. sp lycopersici that causes tomato wilt attacks tomato and has absolutely no effect on wheat or mango. Similarly the fungus Phytophthora infestans causing late blight of potato and tomato does not infect wheat or apple and the fungus Puccinia graminis tritici infects wheat only and not apple or potato. Interactions between a host and pathogen are mediated by environmental conditions but are ultimately determined by genotype. Flor (1956) studied the genetics of flax cultivars and isolates of the linseed rust pathogen, Melampsora lini in field observations. He demonstrated a close genetical relationship between the host and pathogen and formulated the gene-for-gene concept, which is still regarded as an important hypothesis in plant pathology. The concept states that for each gene conditioning avirulence or virulence in the pathogen there is a corresponding gene conditioning resistance or susceptibility in the plant. Usually, although not always, genes for resistance in the host are dominant (R) and those for susceptibilty (lack of resistance) are recessive (r). In the pathogen, genes for avirulence are dominant (A) and those for virulence (a) are recessive. Where the gene-for-gene hypothesis operates therefore, four gene combinations are possible in host-pathogen interaction. These combinations are: R-a, r-A, r-a, and R-A. Three combinations R-a, r-A and r-a give rise to compatible reaction (susceptible) and infections are successful. One combination, R-A results in an incompatible reaction and no infection occurs (resistant). TABLE 7.1

Gene for gene combinations and disease reaction types in a host-pathogen system in which the gene for gene concept operates

Virulence or avirulence genes in the pathogen A (avirulent) dominant a (virulent) recessive + = Compatible (susceptible) reaction – = Incompatible (resistant) reaction

Resistance or susceptibility genes in the plant R(resistant) r(susceptible) dominant recessive AR(–) Ar(+) aR(+) ar(+)

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Genes for virulence appear in pathogens in response to new genes for resistance in the host. New resistance in the host leads to a situation which selects new virulence in the pathogen. Hosts and pathogens are in a continuing state of coevolution. Exceptions to this hypothesis have been seen, particularly where virulence or resistance is controlled or modified by more than just a few genes. It is also possible that not all host-pathogen interactions are represented in this way. According to Issac (1992) the genefor-gene concept remains largely theoretical and provides information, which is important in epidemiological terms but it does not consider the biochemical or molecular basis of resistance. According to Issac (1992) although a number of theories have been put forward to explain the specificity of host-pathogen interactions in biochemical terms, very little is known about the genes involved and their regulation. Few gene products have been analysed but modern molecular biological techniques now provide means by which these factors may be investigated. In an incompatible reaction, molecular recognition leads to the activation of a range of defence mechanisms. In a compatible reaction however, recognition does not occur, or is supressed, and successful host penetration and colonization of the host may be achieved by this invading pathogen. In an incompatible reaction structural and biochemical host defence mechanisms are initiated by the activation of the defence response genes. Crop plants are endowed with numerous defence genes which encode various proteins responsible for synthesis and accumulation of defence arsenals in plants. The defence genes are involved in the production of antimicrobial compounds such as phenolics, phytoalexins, pathogen-related (PR) proteins, and active oxygen species or involved in the reinforcement of the cell wall by accumulating hydroxyproline-rich glycoproteins, callose, lignin, and wall-bound phenolics (Vidhyasekaran,1997). Some of them show antifungal action, some show elicitor-releasing activity, some increase the mechanical strength of host cell walls, and some others are involved in the release of secondary messengers and systemic signal molecules.While defence genes are fuctional genes, resistant genes are regulatory genes which regulate the functions of the defence genes. Defence genes are quiescent in healthy plants, but activated when the pathogens come into contact with the plants, releasing signals. These signal molecules are transferred to the host plant nucleus through a signal transduction pathway activating defence genes. Several disease resistant genes have been cloned and characterized (Baker et al. 1997). Products of most of the resistance genes share striking structural similarities and they appear to be involved in the signal transduction system to activate defence genes. Analyses of several cloned resistance genes revealed the presence of leucine-rich repeats (LRR), leucine zippers and nucleotide-binding sites (NBS). Analogues of resistance genes with LRR, NBS and protein kinase regions have been recently detected in barley, wheat, lettuce, soybean, pepper, chickpea, common bean and Brassica napus (Vidhyasekaran, 1998). LRRs are contained in a variety of proteins and have been suggested to be involved in protein-protein interactions in a downstream step of the signal transduction pathway. Leucine zippers and LRRs serve as sites for protein-protein binding, leucine zippers may directly interact with DNA. The NBS may direct transport of the signals to the plant nucleus. Protein kinases are involved in protein phosphorylation and the transcriptional activation of plant defence genes is modulated by phosphorylation. It is now established that signals are required to activate defence genes which are commonly present in both susceptible and resistant plants. There is no plant in the world without defence genes. These signal molecules have been detected in fungi, bacteria, and viruses, irrespective of whether they are pathogens or non-pathogens and are released by host enzymes when the pathogens come into contact with plants. Endogenous elicitors of host origin, pectate fragments, are released by pathogen enzymes. These signals have to be transferred to the plant nucleus to generate mRNA for the synthesis of various defence-related proteins.

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Although the area of host-pathogen specificity has been the subject of many previous investigations the basic molecular mechanisms have yet to be discovered. Past strategies have often employed a comparative biochemical approach in which a pathogen is typically inoculated on near isogenic lines of the host plant and a difference in a biochemical process between the susceptible and resistant host plant is studied. This has led many researchers to hypothesize the role of compounds such as phytoalexins and lectins as determinants of host-pathogen specificity. Thus most researchers are looking for the gene products that determine specificity and have ignored the physical isolation of the genes that encode for these products. The closest approach to the identification of gene products which determine disease reactions and account for pathogen specificty come from the work of selective pathotoxins. The role of cell wall-degrading enzymes has been implicated with host specificity in certain hostpathogen combinations. Initial results with the bean anthracnose system indicated that a-galactosidase was produced in greater quantities when the pathogen was grown on cell walls from susceptible plants than when grown on those from resistant plants. However, further work with this and other systems failed to provide clear evidence for such selectivity. Evidence for Specific Inducers of Resistance

Despite extensive research there is little evidence for materials produced under the control of specific avirulence genes which, on the basis of Flor’s concept, should have selective effects on resistant plants. Work in Holland which indicated that filtrates of avirulent cultures of a certain strain of Venturia inaequalis were selectively toxic to resistant plants, was not confirmed in later work (Day, 1974). THE COMMON ANTIGEN HYPOTHESIS

The idea that plants and pathogens which interact to give susceptible (compatible) reactions have more antigens in common than those that give resistant (incompatible) reactions was explored first with the flax rust system. Further attempts to explore the hypothesis that antigens shared by the plant and pathogen may account for specificity have been carried out with several types of pathogens. Pathogenic races of Xanthomonas campestris pv. malvacearum, which causes angluar leaf spot of cotton, had more antigens in common with those of cotton leaves than did nonpathogenic races of bacteria. However, in another system, Fusarium wilt of cotton, such a relationship was not observed. In this case loss of virulence in the pathogen did not result in a marked change in antigenic relationships between the plant and pathogen. Taken together, serological data suggest that common antigens may play a role in pathogen specificity. THE PHYTOALEXIN-INDUCED HYPOTHESIS

Phytoalexins are low molecular weight antimicrobial compounds produced de novo in plants as a results of infection or abiotic stress. The degree of resistance of hosts has been correlated with the rate of production/accumulation of phytoalexin in the host or detoxification/suppression of phytoalexin by the pathogen. Although in a few cases (bean-Colletotrichum lindemuthianum and potato-Phytophthora

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infestans) there was no correlation between concentration of phytoalexin and resistance of the host (Agrios,1997) but in most of the cases, a positive correlation did exist between the two. VERTICAL AND HORIZONTAL RESISTANCE

True host resistance is genetically controlled. It has been shown that resistance may be controlled by a single gene or very few genes (2-3) genes (oligogenic resistance) or many genes (polygenic). Van der Plank (1963) introduced the concept of vertical resistance to describe the condition in which plants showed a very high level of resistance to a particular physiological race of the pathogen but showed very little resistance to other races of the pathogen. Such hosts very often show a hypersensitive response, usually very early in the infection, and the pathogen is not able to establish or multiply within host tissues. This is also known as race-specific resistance. Thus vertical resistance is complete but not permanent in nature as any change in the race flora will make the host plants susceptible if the host does not possess genes for resistance against the new races which may be more virulent than the other race or races of the pathogen present in that particular area. Some plants have a general level of resistance to pathogens, which is race non-specific and controlled by a larger number of genes (polygenic resistance). Equal resistance is shown to all pathogen races. Although this resistance called horizontal resistance, may not totally protect plants from infection, it does reduce susceptiblity of the host plants and slows down the establishment of the pathogen. It is also known as field resistance. Thus horizontal resistance is incomplete but is of a permanent nature as some resistance in the host will always be present to all the races of the pathogen. Breeders try to bring in this resistance into the crop plants. To understand and control plant diseases, it is essential to know not only what a pathogenic organism looks like and what it is called, but also its behaviour and that too not only in a single plant under one set of conditions, but on populations of different kinds of plants under all conditions in which plants grow. The mechanisms which bring about variations in plants principally hybridization and mutation, will not be considered here. Those which bring about variations in fungi are perhaps more diverse and include mutation, hybridization, heterokaryosis, parasexual recombination and possible adaptation. MUTATION

Mutations are abrupt changes in the genetic material of a cell, which is then transmitted in a hereditary fashion to the progeny. Mutations occur spontaneously in nature in all living organisms which reproduce only sexually or only asexually and also in those which reproduce both sexually and asexually. Mutations, apparently, are the results of unavoidable but rather infrequent accidents that take place during cell division and result in irregularities in the replication or rearrangement of minute parts of the genetic material of the cells. Mutations can also be induced at a high rate artificially by exposing the organisms to either physical agents, such as ultraviolet light, X-rays, g-rays and extreme temperatures, or to highly reactive chemicals such as the nitrogen and sulphur mustards, epoxides, peroxides, phenols, alkaloids and alkylating agents (e.g., ethyl methane-sulphonate, methyl methane-sulphonate, diethyl sulphate, etc). One highly potent mutagenic chemical, often styled as a magic bullet, is N-methyl-N nitro-N nitrosoguanidine.

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The pathogenic abilities of parasites as well as their morphological and other characteristics can be changed in this way and there is evidence that these are important in the development of new strains. In a haploid fungus, such mutations should have immediate expression but this may be modified by epistatic or other effects. If the cell in which gene mutation occurs contains more than one nucleus, the expression will be modified by the other nuclei present and in such heterokaryotic fungi, pathogenicity and other characteristics will have a composite effect. New races attributed to mutations have been reported in Cladosporium fulvum (tomato leaf mould) where step mutation (each mutation overcoming the protective effect of a single gene) is thought to have occurred and similar mutations have been achieved experimentally by ultraviolet irradiation. Pathogenic variation due to mutation has also been reported in Phytophthora infestans, Puccinia graminis, Melampsora lini and various smuts, to mention but a few. Many of these mutations are likely to be recessive and their expression will be dependent on sexual reproduction with an accompanying recombination of pathogenicity factors. Anastomosis between hyphae and germ-tubes may be followed by an exchange of nuclei and this may also permit the expression of recessive pathogenic mutations. This could be of considerable significance in Fungi Imperfecti and in those Chytridiomycetes, Oomycetes, Ascomycetes and Basidiomycetes in which the perfect stage is rarely formed as in some Erysiphaceae in which cleistothecia fail to develop under certain climatic conditions. Although the inadequate information available suggests that mutations affecting pathogenicity are fairly rare, they are doubtless an effective mechanism for variations when the enormous numbers of spores produced by many pathogens are taken into account. For example, Watson (1957) reported increased pathogenicity due to mutations in P. graminis when large numbers of urediniospores were involved. Mutation can also bring about reduced pathogenicity or other changes disadvantageous to the pathogen. The practical implications of mutation in the aggressiveness of pathogens are difficult to assess. The sudden appearance of new races of increased pathogenicity in a pathogen which has hitherto been of only moderate pathogenicity has been ascribed to mutation and this may well be true. The appearance of a new, highly destructive race of Helminthosporium maydis (southern leaf blight of maize) in the USA in 1970 may be cited as an example of this. Mutations also provide the basic pathogenicity changes, which are subsequently brought out by hybridization or similar recombination mechanisms and without mutation the latter would be of a more limited scope. Beadle and Tatum (1946) demonstrated the role of mutations in producing hereditary variants in fungi. Several workers have discovered mutations in plant pathogenic viruses, bacteria, and fungi. HETEROKARYOSIS

Hetrokaryosis and variability in the plant pathogenic fungi has been ably summarized by Parmeter et al. (1963). Hansen and Smith (1932) defined heterokaryosis as a term which “… precisely describes the condition of a cell containing two or more genetically different nuclei.…” In a later classification of the use of the term heterokaryosis, Hansen (1942) pointed out that the term may be used whenever such a condition exists, not only in a single cell, but also when it exists in any or all cells of an individual thallus. This clearly implies that heterokaryosis involves the occurrence of dissimilar nuclei in a single cell, even if it is only one cell in a thallus.

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Nelson et al. (1955) used the term “heterokaryosis” to designate the association of more than two genetically different kinds of nuclei in a vegetative cell or spore. Considerable information on the potential advantage of heterokaryosis is available in the existing literature. It is agreed by most workers that heterokaryosis provides a haploid organism with many of the advantages of heterozygosity enjoyed by diploid organisms. Nuclear changes could provide additional somatic flexibility with changing environments (Davis, 1966). However, the main advantage of heterokaryosis appears to be the fact that it is a requisite step for genetic recombination brought about by interchanges of whole chromosomes or through mitotic crossing over. Herterokaryosis may thus play an important role in homothallic and imperfect fungi. This aspect was reviewed by Caten and Jinks (1966). Heterokaryosis can arise in several ways: 1. Gene mutation: These are not easily detected at a gross level since their frequency in nuclei is very small. 2. By fusion of vegetative cells: Anastomosis is undoubtedly important in the initiation of heterokaryosis. Anastomosis, as it is related to the establishment and development of heterokaryosis, involves fusion of hyphae, movement of one or more nuclei into one or the other of the fused cells, and the establishment of a compatible heterokaryotic state. Failure to establish heterokaryons may involve any one of these steps. 3. Heterokaryosis may take place at the time of spore formation in many fungi, such as inclusion of + and – nuclei in the same ascospores of Neurospora tetrasperma or Podospora anserina. Apart from dikaryotics in smuts, rusts, and Hymenomycetes, heterokaryosis has been reported in pathogens belonging to the Fungi Imperfecti, Ascomycetes and to a lesser extent, in lower fungi, but not all these reports are entirely convincing (Parmeter et al., 1963). Heterokaryosis appears to be necessary for pathogenicity in heterothallic rusts, but not in infection by basidiospores as the latter are normally uninucleate and hence necessarily homokaryotic. There is considerable evidence that heterokaryosis not only conditions pathogenicity but also enables the effects of mutation on pathogenicity (often recessive) to be expressed. Buxton (1956) produced nearly avirulent race of Fusarium oxysporum f. sp. pisi by ultraviolet irradiation and showed that heterokaryons were recovered by culturing from the diseased plants. Buxton (1960) concluded that some of the increases in the virulence of pathogens in nature may be due to heterokaryosis between weakly virulent strains. The plant pathogenic role of heterokaryosis in producing new strains with altered pathogenicity has been discussed by several workers. Nutritional deficiencies can affect pathogenicity as shown by Hrushowvitz (1957), who demonstrated that repeated culturing of H. sativum on media supplemented with certain amino acids led to a progressive loss in pathogenicity in some cases. The mechanism involved is uncertain but such repeated culturing may have led to the selection of deficient nuclei, thalli containing such deficient nuclei being less pathogenic. The increased pathogenicity due to heterokaryosis in the mycelium would not be inherited by the spores unless the latter contained a full complement of the parental nuclei and would not be obtained with uninucleate conidia. Heterokaryosis is thus unlikely to bring about any permanent inherited change in pathogenicity which would persist after sporulation, although parasexual recombination in which heterokaryosis is the first step, could do so. Heterokaryosis had been discussed by Davis (1966). Heterokaryosis is certainly a way in which avirulent strains may acquire virulence. One of the best documented cases is that of Thanatephorus cucumeris. Heterokaryosis between avirulent strains, both spontaneous and induced, or between avirulent and virulent strains were all highly pathogenic, in the latter case more so than either component.

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Heterokaryosis may give rise to races or strains with novel characteristics. One of the best documented examples is P. striiformis (Little and Manners, 1969). PARASEXUAL RECOMBINATION

This process in which genetic recombination occurs in the vegetative thallus in the absence of a sexual stage has been investigated chiefly in species of Aspergillus and Penicillium in the laboratory of Pontecorvo and his co-workers (Pontecorvo et. al., 1963; Pontecorvo and Sermonti, 1954; Pontecorvo, 1956; Bradley, 1962). The sequence of events in a complete parasexual cycle is as follows: 1. Formation of heterokaryotic mycelium. 2. Fusion between two nuclei: (a) Fusion between like nuclei. (b) Fusion between unlike nuclei. 3. Multiplication of diploid nuclei alongside haploid nuclei. 4. Occasional mitotic crossing over during the multiplication of diploid nuclei. 5. Sorting out of diploid nuclei. 6. Occasional haploidization of the diploid nuclei. 7. Sorting out of the new haploid strains. The results are similar to those achieved by meiosis, a much more precise and regular process. The frequency of these events appears to be low and there is little information about the significance of this somatic recombination in nature, specially about fungi which attack plants. Parasexuality, however, produced new races of F. oxysporum f. sp. pisi and has also been reported in Ascochyta imperfecti responsible for black stem of alfalfa, Verticillium albo atrum, F. oxysporum f. sp. cubense responsible for banana wilt, Cochliobolus sativus and others. There is evidence that somatic recombination may occur in smuts and rusts as in Ustilago maydis (Rowell, 1955), Puccinia graminis and Puccinia recondita (Vakili and Caldwell, 1957). ADAPTATION

Adaptation in fungi has been reviewed by Buxton (1960) and Person (1968). The possibility that pathogens might adapt themselves to their host plants has been debated for many years. There seems to be no incontrovertible evidence that this occurs although the evidence for adaptation to chemicals is stronger. Apparent adaptation to host plants might be due to mutation or to the selection of pre-existing strains which are better able to attack the host. Changes in the enzymic capabilities of a pathogen may be mediated by the presence of appropriate substrates (adaptive enzymes) and this might modify its pathogenicity. Many years ago Ward (1903) suggested the concept of a bridging host by means of which a pathogen is able to pass from a susceptible to a hitherto resistant host: for example, in Puccinia dispersa the pathogen becomes adaptive to resistant species of Bromus. After propagation in some species of grass, somewhat similar changes were reported by Salmon (1904) in powdery mildews, and more recent work indicates that passage through some host plants can sometimes affect pathogenicity. Repeated passage through a resistant plant sometimes enhances the aggressiveness of a pathogen and

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vice versa, as has been demonstrated in several fungi and bacteria: for example, Phytophthora infestans and Xanthomonas stewarti. Changes in the pathogenicity of F. oxysporun f. sp. pisi is brought about by exudates. Although there is increasing evidence in favour of parasitic adaptation, the mechanism involved remains obscure. Host selection of more aggressive biotypes from a mixture of biotypes, together with adaptation, mutation (perhaps by some mutagens in the host) and cytoplasmic variation may be involved in different cases. SALTATION

This phenomenon, also described as dissociation or sectoring, refers to the appearance of morphologically different sectors in fungal colonies. It occurs frequently in some fungi including some isolates of Fusarium and Helminthosporium, but infrequently in others. Some such sectors remain fairly stable when sub-cultured while others are unstable. Saltation may be influenced by the compaction and thickness of the culture medium and can affect such characteristics as colony colour and topography; rate of growth and sporulation; and size and shape of the spore. Pathogenicity might also be affected and this has been studied in Fusarium species by several investigators. The results have been somewhat conflicting; saltants tended to be less aggressive than the parent cultures, but occasional ones were more aggressive and these may be of significance where the saltation rate is high. No new pathogenic races with altered host ranges have been reported in saltants. The mechanisms involved in saltation are varied and not well understood. CYTOPLASMIC VARIATION

There is a great deal of evidence for the existence in fungi of genetical determinants located in the cytoplasm outside the nucleus. Inheritance of such cytoplasmic determinants differs from that of nuclear genes. Cytoplasmic inheritance has been attributed to plasma genes. Cytoplasmic RNA (possible some ribosomes) is the genetic material detached from the chromosomes (episomes). There is little information as to the possible significance of cytoplasmic factors in bringing about variations in the pathogenicity of plant pathogens, although Johnson (1954) has described the cytoplasmic control of pathogenicity of Puccinia graminis f. sp. tritici; this is a subject which needs investigation. Cytoplasmic inheritance had been discussed by Jinks (1966) and Fincham and Day (1965). SEXUAL RECOMBINATION

The origin of new physiological races through meiotic recombination is well attested in many pathogenic fungi, rusts, smuts, powdery mildews, and the potato blight fungus. Hybridization is likely to be encouraged by incompatible mechanisms which prevent or reduce inbreeding, and many pathogens which are heterothallic. Hybridization can be intraspecific, interspecific or even intergeneric and the resulting hybrids may have different pathogenic abilities than the parental races. Often the hybrids are intermediate in pathogenicity between the two parental races but some may be more pathogenic than others and similar considerations apply to other inherited characteristics.

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Extensive recombination of genes occurs in autoecious rusts such as Melampsora lini (flax rust) in which all the spore stages occur in the same plant. Interspecific hybridization has been discussed by Nelson (1963). TRANSFORMATION

In addition to sexual recombination, there are a number of possibilities for the production of new variants in micro-organisms by asexual means. One of these, a sexual type of combinations of genetic characters, is bacterial transformation. The observation of Avery et al. (1944) called attention to this process. They were successful in converting a non-pathogenic non-capsule-forming strain of Diplococcus pneumoniae into a pathogenic strain which forms a carbohydrate capsule. This was done by culturing the non-pathogenic strain in the presence of the biologically-active fraction of the capsule forming a pathogenic strain. It was later demonstrated by these workers that this transformation was brought about by the introduction of purified deoxyribonucleic acid (DNA of the donor). Thus transformation is a specific form of recombination, involving the naked genes or DNA. Several workers have demonstrated the production of new strains of plant pathogenic bacteria by means of transformation. Corey and Starr (1957) were able to change the type of colonies and resistance of Xanthomonas phaseoli (a pathogen of beans) to streptomycin by transformation. Similarly, Klein and Klein (1953) have shown that the tumour-inducing ability of Agrobacterium tumefaciens can be transferred from virulent donor strains to avirulent strains. In this case, the material transforming the avirulent cells was also deoxyribonucleic acid (DNA). TRANSDUCTION

One of the most recently discovered processes of recombination different from sexual recombination is transduction. This process has been found in a variety of bacteria where DNA is transferred from cell to cell through the agency of temperate viruses. Bacteria need not necessarily be destroyed by their infective viruses (bacteriophages). In certain cases after infection of the bacterium with a bacteriophage, the phage (temperate) may produce in the host an immunity that prevents multiplication of the phage and lysis of the bacterial cell. Such an association is called the lysogenic state. Under the influence of different chemicals and physical treatment (e.g. ultraviolet irradiation) lysogenic bacteria liberate their latent phages by lysis. These are infective viruses with the ability to enter the new bacterial cells. During symbiosis, the phage becomes the genetic material of the host, with phage DNA coming into close contact with the host DNA. This makes it possible for the DNA molecule, carrying the gentic factor of the donor bacterium to be transformed to a recipient bacterial cell through the vehicle of a phage particle. Pathogenicity can also be transduced by bacteriophages. Therefore, transduction is an important mechanism for genetic recombination in bacteria. This interesting mechanism of genetic exchange was discovered by Zinder and Lederberg (1952) in Salmonella typhimurium. Its importance was also demonstrated in a plant pathogenic bacterium (Pseudomonas solanacearum) by Okabe and Goto (1955). One can suppose that transduction has a role in producing new biotypes of plant pathogenic bacteria in nature, however, this remains to be proved.

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Viruses also show considerable variability in several characteristics, such as infectivity, longevity, symptoms produced on the host plant, host range, and specificity to vectors. These variations are possibly due to some sort of mutation process and are inherited in that they are perpetuated during the transfer of the virus from one plant to another. The mechanism involved in these changes in not quite well understood. In virus-resistant plants, the relationship is, in effect, between the nucleic acids of the resistant genes of the host plant and RNA of the virus particle. PHYSIOLOGICAL SPECIALIZATION

Many factors in host-parasite interactions present the fundamental problems of plant disease. The major factor towards the elucidation of host-parasite interactions is the genetic aspect. The rediscovery of Mendel’s laws of heredity in 1900 and the subsequent rapid development of the basis science of genetics gave a strong impetus to the study of plant pathology. The genetic basis of disease resistance was analyzed in many cases. Although the genetics of the host received most attention initially, advances in the knowledge of the sexuality of fungi led to the the development of materials and methods for genetic studies of pathogenic fungi. There is now positive evidence that the general pattern of the laws of inheritance are the same in fungi as in other groups of plants. The fungi undergo heritable variations brought about by segregation and recombination of genes, mutations, heterokaryosis, cytoplasmic inheritance, etc. Not only this, because of their great variety in sexual and asexual reproduction and their unique suitability for experimental manipulation, micro-organisms now afford the best available material for many types of fundamental studies in genetics, biochemistry, pathology, and other fields. Despite all these advances, however, knowledge of the actual physico-chemical mechanisms that determine parasitism, pathogenicity or disease resistance is still very meagre. In a nutshell we can say that disease resistance is a genetic character as regularly inherited as any other character. However, the genetic behaviour of resistance usually differs in different cases. Resistance to one disease does not necessarily imply resistance to any other disease. Thus, Thatcher wheat, while resistant to stem rust is highly susceptible to leaf rust (P. recondita, P. triticina). Various kinds of genetic behaviour have been observed with respect to disease resistance, for example, simple monohybrid inheritance of resistance, in cabbages to yellows (Fusarium conglutinans), lettuce to downy mildews (Bremia lactusae) and peas to wilt (Fusarium oxysporum sp. pisi). In these cases, resistance is a dominant characteristic. Resistance as a recessive characteristic in simple monohybrid inheritance is seen in powdery mildew of barley (E. graminis), smut of sorghum (Sphacelotheca sorghi), and speckled leaf blotch of wheat (Septoria tritici). Two gene segregations appear to be involved in resistance to anthracnose of beans (Colletotrichum lindemuthianum) and some cereal rusts. Many genes which are more difficult to analyze are known to be involved in a large number of diseases. And finally, there may be a number of alleles (instead of two) for one gene, giving a complex series of resistance relationships when several parents are studied.

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Physiological Specialization in Fungi

Complexity of Organisms Like all other organisms, fungi are also complex and variable. This complexity is governed by the genetic material contained in the chromosomes. The crop plants are similarly complex, for example, there are 14,000 varieties of wheat; similarly there are innumerable varieties of apples, beans, grams, roses, corn, and so on. Gene Action When fungal species parasitize a number of host varieties they often develop physiological specialization, which is the development of certain character in the fungus which are not morphological but physiological, and can be detected only when the effect is noted on the host. A special species of fungus such as the black stem rust, Puccinia graminis, may include a number of “physiological races” which are similar in morphological structure but may be different in their capacity to produce disease. So the difference between them is physiological and not morphological. Such races are commonly encountered in many groups of parasites such as rusts, smuts, powdery and downy mildews, and so on. Gene action expresses itself in various morphological features and it also always governs the various physiological processes of the organism. The interaction between the host and parasite is actually the interaction between the enzyme systems of the two (now proved by Dodge and others in Neurospora) and the result is the effect produced. The details of this phenomenon can be easily explained by taking the example of the black stem rust, Puccinia graminis. The discovery of pathogenic specialization is fundamental to studies on pathogenic variability. Eriksson (1894) showed that P. graminis on cereals and grasses is composed of several pathogenically different strains which he described as specialized forms (firmae speciales). These are now known as varieties. There are at least six varieties of P. graminis which can be recognized by the size of the spores and also by their pathogenicity on different hosts: TABLE 7.2 Variety P. graminis tritici P. graminis secalis P. graminis avenae

Urediniospores 32¥20 mm 27¥17 mm 28¥20 mm

P. graminis phleipratensis P. graminis agrostidis P. graminis poae

24¥17 mm 22¥16 mm 19¥16 mm

Host attacked Wheat, barley and many wild grasses Rye, barley and many wild grasses Oats and wild grasses (Phleum pratense) Timothy and other wild grasses Argrostis spp. Poa pratensis and other grasses

The general characteristics of all these varieties of P. graminis are so alike that it will not be possible to differentiate between them unless microscopic examination is conducted and measurements are taken. Each variety, for example, P. graminis tritici, is further subdivided into pathogenic units called physiological races. These races cannot be distinguished by morphological examinations but by noting the different effects they produce on various varieties of wheat. The only method by which the nature (identity) of physiological races can be known is to grow them on a number of host varieties and note the symptoms which will be different if the isolates differ in physiological capacity. In order to have a

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standard method for the identification of a physiological race, some host varieties are fixed, which are called “standard differentials”, and the record of each race is taken. These differentials, now used to distinguish Puccinia graminis tritici and Puccinia graminis avenae, were selected about sixty years ago. Whenever the necessity arises new ones are added. For identifying the races, three varietal reaction classes are used. These are resistant (R), susceptible (S), and mesothetic (M). Further degrees are recognized within these three classes, for example, immune, practically immune, extremely resistant, moderately resistant, moderately susceptible, completely susceptible, and heterogenous mixed reaction (x). In addition to these infection types, a plus (+) or a minus (–) sign or two plus (++) or two minus (– –) signs are included to indicate variations of a slightly greater or lesser extent. Infection Types According to the degree of spotting produced, each type of infection is indicated by one of the six given symbols: Type 0 Very faint, light flecks; host therefore considered immune. Type 1 Uredosori very minute; host strongly resistant. Type 2 Small to medium; host resistant. Type 3 Medium size; host moderately susceptible. Type 4 Large and confluent; host very susceptible. Type x A mixed reaction; uredosori of variable size, mostly those of types 1 and 4. To identify a race of Puccinia graminis, the isolate is inoculated on 12 standard differentials and the symptoms produced are noted and classified into infection types. These are then compared with the existing records and the race is identified. Key numbers are given each on the basis of its identity. Production of New Races

All cereal rusts are heteroecious. An aeidial stage is never produced on a cereal host. As long as the fungus reproduces itself by means of uredospores, it breeds true except for the occasional appearance of a mutation. On the alternative hosts (barberry, buckthorn and so forth) the sexual stage of the fungus is developed (Cragie,1927) and this gives an opportunity for hybridization. This could result in the formation of heterozygous characteristics with a difference in physiological specialization. When such a heterozygous race forms the basidiospores it segregates again and different recombinations may result again. In this way the appearances of new races are recorded more in those regions where Berberis spp. plays an active role, as in the USA and Europe. On the other hand, in countries where Berberis spp. play no part or little part in the life cycle, the number of races is much less, as in Australia, India and Kenya. In P. graminis tritici alone there are about 225 races in the USA, and 32 races and biotypes in India. In other species of Puccinia, the number of physiological races is lesser. In Puccinia recondita there are 90 in the USA and 28 in India. In Puccinia striiformis there are 30 in the USA and 14 in India. Biotypes A biotype is a population of individuals that are genetically identical. The descendants of a single non-sexually produced stem rust spore constiute a biotype. The biotype will remain strictly pure unless a mutation occurs. Most species of pathogenic fungi are now known to have many genotypes. The most closely related races are similarly grouped into varieties and finally the varieties into species. Actually, a reverse procedure is followed. The larger groups (varieties) are recognized first and then smaller groups (races and biotypes) are easily distinguished by more refined methods. Examples of the

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occurrence of biotypes can be given in the case of race 59 in which several biotypes are known to exist. Biotypes show enough similarities to be placed within one race, but when different biotypes are studied very closely, they show minor but consistent differences in their effects on some differentials, proving that they are genetically pure, though with different identities. Such races are, therefore, know to have biotypes designated as 59, 59A, 59B, 59C. Another instance may be given. Race 15 of wheat stem rust was discovered in 1918. It was a fairly homogenous rust for a long time and was identified as such. Later, some dissimilarities in isolates from different regions were noted. These differences were consistent enough and 15A and 15B were recongnized as biotypes. Race 15B which was known to occur occasionally near barberry bushes became widespread in 1950 and attacked the well-known hitherto resistant varieties. Besides rusts, specialization of parasitism is found in other fungi also. In smuts, as in rusts, races and biotypes are known to occur in different species. As a matter of fact, smuts offer better material for the study of genetics. In smuts (Ustilago zeae) the chlamydospores undergo reduction division and the sporidia produced are haploid structures which can be grown into haploid mycelium in culture media and matings done as per plan. The colonies from haploid sporidia are genetically pure and differ from one another in characters such as size, colour and surface pattern. It is now known that in Ustilago zeae there are thousands of haploid biotypes within the species. When we talk of races in rusts, we consider dikaryotes, but in smuts, we consider haploid biotypes. Biotypes may differ from one another in one or more of all known characters, including pathogenicity. Similar though less elaborate work has been done on other smuts, for example, Ustilago segetum var. tritici, Ustilago nuda, Sphacelotheca sorghi, Ustilago avenae and Ustilago hordei. Investigations in rusts and smuts are complementary. In rusts only the binucleate uredial stage can be propagated clonally, while in smuts it is only the haplophase which can be propagated in this way. In both smuts and rusts three distinct phases correspond with the nuclear condition, the haplophase, the dicaryophase and the diplophase. In rusts, both dikaryophase and haplophase are parasitic in life, but in smuts, the haplophase is generally saprophytic. The dikaryophase, both in smuts as well as rusts, can be hybrid in character. This character is multiplied (in an equational manner) vegetatively through the urediniospore stage. In many cases, rusts (in India) can go on repeating indefinitely from year to year. In smuts, such multiplication does not occur and sexuality is an indispensable process. Variability in smuts is, therefore, more common, and hence there is a very large number of races, but these races have to be expressed in terms of haploid strains and not in the dikaryotic phase, as in rusts. No sooner is the chlamydospore formed in smuts, than the dikaryotic phase comes to an end and is not propagated clonally (by urediniospore, like conidia) as in rusts. Erysiphe graminis is also strongly specialized into physiological races. ADAPTATION OF FUNGI TO DIFFERENT HOSTS The Gene-for-Gene Theory of Host Parasite Relations

Flor (1956) in his studies on rust disease of flax (Melampsora lini),came to the conclusion that for each gene that governs resistance in the host there is a specific and related gene in the pathogen that governs its virulence (pathogenicity). In other words, there is a genetic inter-relationship between the host and pathogen. This is the complementary gene-for-gene theory. The mechanism for the evolution of this

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relationship in a host-parasite system is an automatic consequence of the distribution of new resistance genes by mutation and selection in the host, and virulence genes in the parasite population. The genetic definition of specificity embodies the so-called “quadratic model” in which genes in the host are clearly defined in terms of resistance or susceptibility and the complementary genes in the pathogen in terms of virulence or avirulence. The relationship between the host and parasite induces a genetic polymorphism in both partners (Person, 1959, 1968). This genetic variability, exhibiting a self regulation automatic mechanism, is the reason for the vicious circle that consists of the alternative prevalence of the resistant host and the virulent pathogen. The gene-for-gene hypothesis was proposed by Flor (1942, 1955, 1956) as the simplest explanation of the results of studies on the inheritance of pathogenicity in the flax rust fungus ( Melampsora lini). In the varieties of flax (Linum usitatissimum) that have a gene to resist the avirulent parent race, F 2 cultures of the fungus segregate into monofactorial ratios. In the varieties that possess 2, 3 or 4 genes for resistance to the avirulent parent race, the segregation in the F2 cultures is in bi, tri or tetrafactorial ratios. Flor (1955, 1956) summarized the evidence for flax rust in the gene-for-gene hypothesis. This states that during their evolution the host and parasite develop complementary gene systems so that “for each gene conditioning rust reaction in the host there is a specific gene conditioning pathogenicity in the parasite” (Flor, 1956). The current status of the gene-for gene concept has been reviewed by Flor (1971). This hypothesis is applicable to most host-parasite systems in which resistance is conditioned by major (vertical resistance) genes and virulence increases in a step-wise manner. Hatchett and Gallun (1970) showed that the ability of specific races of Hessian fly to survive on certain varieties of wheat was controlled by genetic systems in the host and insect that were complementary. There is considerable literature on the inheritance of pathogenicity and resistance, including smuts, Venturia inaequalis and Phytophthora infestans. In the case of Phytophthora infestans, Black (1952) in Scotland identified four major dominant genes, R1, R2, R3 and R4. At least nine major genes are now known to control resistance. This polygenic resistance is apparently conditioned by different factors in the case of tuber rot compared with foliage blight. The reactions of host plants in the different pathogenic races are controlled by many genes, and within them, an even larger number of alleles exist to enable resistance. Resistance in plants is often dominant over susceptibility to disease. It is generally found that resistance of plant cultivars to disease is not a permanent character. The reason for this phenomenon is to be found in the self-regulating nature of genetic variability in host-parasite relations. The evolution of new genes for virulence in the pathogens is the result of the selective action of resistant cultivars on populations of the pathogens which are genetically plastic. Similarly, the genes for virulence in parasites promote the selection of genes for resistance in the host population. This automatic regulation of population of the host and the parasite explains the fact that in many cases the utilization of resistant cultivars is very limited. For example as a rule, rust resistance is lost in three to seven years. This is valid, first of all, for pure line cultivars of a self-pollinated plant species that is, for wheat which suffers from attack by a new rust race much more heavily than the cross-pollinated plants (e.g . cultivars of corn). Cultivars of cross-pollinated plants are composed of a mixture of different lines (biotypes). This means that some biotypes will be resistant host population to selection effect (attack) of the virulent gene of the parasite. However, there are very few, if any, resistant biotypes in the susceptible population of pure line cultivars, since they are largely eliminated during the breeding process which produces a high degree of

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homozygosity. Thus pure lines will be highly vulnerable to attack by the parasite containing genes for virulence. On the other hand in cross-pollinated plants, the buffering capacity of the genetic pool is relatively high compared to the self-pollinated ones. In recent years plant breeders have tried to incorporate greater variability into self-pollinated plants such as wheat, by developing multiline cultivars. The various lines in the multiline cultivars have different gene-for-gene systems against the virulent genes of rust fungi. By continually manipulating intra-varietal heterogeneity within available resistance genes, it should be possible to provide a suitable control of widespread pathogen inoculum potential. The advantage of using multiline cultivars, according to Sadasivan (1975), is that they enable quick synthesizing of well buffered “horizontal resistance” cultivars (HR). They are capable of utilizing, unlike pureline cultivars, several resistance genes at the same locus. The useful life of a resistant gene can be extended indefinitely and the breeding programme can be cut to size by this method. There is an even chance for the cultivars to stabilize themselves in a given environment. This method helps to distribute developed varieties on a wide geographical scale without any risk of homogenizing the pathogen population. The multiline approach is thought to be expensive, agronomically conservative and a breeding ground for new races, possibly even a super race. Another drawback is that because their variability is synthetic it is polarized. The resistant cultivars of cross-pollinated plants remain resistant for considerable time. One such example, according to Borlaug (1965), is the relative stability of resistance in corn cultivars to the rusts, Puccinia sorghi and Puccinia polyspora. These two rusts of corn are much less important in limiting corn production than the rusts of wheat. The cross-pollinated corn usually possesses sufficient variability, that is, sufficient resistant lines are present in the population of the cultivar to resist the selective action of virulent genes of new rust races. This means that the development of rust epidemics in crosspollinated corn is almost impossible.

REFERENCES Agrios, G.N. (1997), Plant Pathology, Fourth Edition, Academic Press, London, New York, pp. 635. Ark, P.A. (1957), “Phenotypic variations induced by chemicals in Corynebacterium michiganense and Xanthomonas juglandis”, J. Bact., 61: 293–298. Avery, O.T., C.M. MacLeod and M. McCarty (1944), “Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a DNA fraction isolated from Pneumococus type III”, J. Exp. Med., 79: 137–158. Backer, B., P. Zambryski, B. Staskawiez and S.P. Dinesh Kumar (1977), “Signalling in plant-microbe ineraction”, Science, 276: 726–733. Beadle, G.W. and E.L. Tatum (1946), “Neurospora-III: Method of producing and detecting mutations concerned with nutritional requirements”, Am. J. Bot., 32: 678–686. Black, W. (1952), “A genetical basis for the classification of strains of Phytophthora infestans”, Proc. R. Soc. Edinb., 65(8): 36–51. Borlaug, N. (1965), “Wheat, rust and people”, Phytopath., 55: 1088–1098. Bradley, S.G. (1962), “Parasexual phenomena in micro-organisms”, Ann. Rev. Microbiol., 16: 35–52. Buxton E.W. (1956), “Heterokaryosis and parasexual recombination in pathogen strains of Fusarium oxysporum”, J. Gen. Microbiol,. 15: 133–139.

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Buxton E.W. (1960), “Heterokaryosis, saltation and adaptation”, in: Pl. Pathol.-An. Adv. Trea., J.G. Horsfall and A.E. Dimond (Eds.), Academic Press, New York 2: 359–407. Caten, C.E. and J.L. Jinks (1966), “Heterokaryosis its significance in wild homothallic ascomycetes and fungi imperfecti”, Trans. Brit. Mycol. Soc., 49: 81–93. Corey, R.R. and M.P. Starr (1957), “Genetic transformation of colony type in Xanthomonas phaseoli”, J. Bact., 74: 141–145. Cragie, J.H. (1927), “Discovery of the function of the pycnia of the rust fungi”, Nature, 120: 765–767. Daniels, M.J., J.M. Dow and A.E. Osbourn (1988), “Molecular genetics of pathogenicity in phytopathogenic bacteria”, Ann. Rev. Phytopathol. 26: 285–313. deWit, P.J.G.M. (1992), “Molecular characterization of gene-for-gene system in plant-fungus interactions and the application of a virulence genes in control of plant pathogens”, Ann. Rev. Phytopathol., 30: 391–418. Davis, R.H. (1966), “Heterokaryosis”, in: The Fungi-An Adv. Tre., G.C. Ainsworth and A.S. Sussman (Eds.), Academic Press, New York, 2: 567–588. Day, P.R. (1957), “Mutation to virulence in Cladosporium fulvum”, Nature, 179: 1141–1142. Day, P.R. (1960),“Variation in phytopathogenic fungi”, Ann. Rev. Microbiol., 14: 1–16. Day, P.R. (1974), Genetics of Host-Parasite interaction, W.H. Freeman and Company, San Francisco. Dodge, B.O. (1927), “Nuclear phenomena associated with heterothallism and homothallism in the ascomycetes, Neurospora”, J. Agr. Res., 35: 289–305. Dodge, B.O. (1928), “Production of fertile hybrids in the ascomycete”, Neurospora”, J. Agr. Re., 36: 1–14. Ellingboe, A.H. (1976), “Genetics of host parasite interactions”, in: Encyclo. of Pl. Physiol. (New series), R. Heitefuss and P.H. Williams (Eds.), Springer-Verlag, Berlin, Heidelberg and New York, 4: 761–778. Eriksson, J. (1894), “Uber die Spezialisierrung des Parasitismus bei den Getreiderost-pilzen”, Ber. Dtsch. Botan. Ges., 12: 292–331. Fincham, J.R.S. and P.R. Day (1965), Fungal Genetics, Blackwell, Oxford, p. 326. Flor, H.H. (1942), “Inheritance of pathogenicity in a cross between physiologic races 22 and 24 of Melampsora lini”, Phytopath., 32: 5. Flor,H.H. (1955), “Host-parasite interaction in flax rust- its genetics and other implications”, Phytopath., 45: 680–685. Flor, H.H. (1956), “The complementary genic system in flax and flax rust”, Adv. Gen., 8: 29–54. Flor, H.H. (1971), “Current status of the gene-for-gene concept”, Ann. Rev. Phytopath., 9: 275–296. Gordon, F.B. (1950), “Genetics of viruses”, Ann. Rev. Microbiol., 4: 151–168. Hahn, M.G. (1996), “Microbial elicitors and their receptors in plants”, Ann. Rev. Phytopathol, 34: 387–412. Hansen, H.N. (1942), “Heterokaryosis and variability”, Phytopath., 32: 639–40. Hansen, H.N. and R.E. Smith (1932), “The mechanism of variation in imperfect fungi-Botrytis cinerea”, Phytopath., 22: 953–964. Hatchett, J.H. and R.L. Gallum (1970), “Genetics of ability of the Hessain fly, Mayetiola destructor to survive on wheat having genes for resistance”, Ann. Ent. Soc. Am., 63: 1400–1407. Heath, M.C. (1991), “The role of gene for gene interactions in determination of host specificity”, Phytopathology, 81: 127–130. Hennecke, H. and D.P.S. Verma (Eds.) (1991), Advances in Molecular Genetics of Plant Microbe Interactions, Vol. 1, Kluwer, Dordrecht, The Netherlands, Hrushowitz, S.B. (1957), “Effect of amino acids on the virulence of Helminthosporium sativum to wheat seedling”, Phytopath., 47: 216–264. Issac, S. (1992), Fungal Plant Interactions, Chapman & Hall, London, pp. 418. Jinks, J.L. (1966), “Mechanism of inheritance”, in: The Fungi, G.C. Ainsworth and A.S. Sussman (Eds.), Academic Press, London, II: 619–660.

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Johnson, T. (1954), “Selfing studies with physiological races of wheat stem rust Puccinia graminis var. tritici”, Can. J. Bot., 35: 506–522. Johnson, T. (1960), “Genetics of pathogenicity”, in: Plant Pathology, J.G. Horsfall and A.E. Diamond (Eds.), Academic Press, London and New York, 2: 407–579. Klein, D.T. and R.M. Klein (1953), “Transmittance of tumour inducing ability to a virulent in Agrobacterium tumefaciens”, J. Bact., 66: 220–228. Lamb, C.J. (1994), “Plant disease resistance genes in signal perception and transduction”, Cell, 76: 419–422. Little,R. and J.G. Manners (1969), “Somatic recombinations in yellow rust of wheat (Puccinia striiformis)—The production and possible origin of two new physiologic races”, Trans. Brit. Mycol. Soc., 53: 251–258. Michelmore, R. (1995), “Molecular approaches to manipulation of disease resistant genes”, Ann. Rev. Phytopathol., 35: 393–427. Nelson R.R. (1963), “Interspecific hybridization in the fungi”, Ann. Rev. Microbiol., 17: 31–48. Nelson R.R., R.D. Wilcoxon and J.J. Christensen (1955), “ Heterokaryosis as a basis for variation in P. graminis var. tritici”, Phytopath., 45: 639–643. Okabe, N. and M. Goto (1955), “Studies on Pseudomonas solanacearum- X: Genetic change of the bacterial strains induced by the temperate phage T-c 200”, J. Fac. Agr. Schizuoka. Univ. pp., 57–62. Parmeter, J.R., Jr., W.C. Synder and R.E. Reichle (1963), “Heterokaryosis and variability in plant pathogenic fungi”, Ann. Rev. Phytopath., 1: 51–76. Person, C. (1968), “Genetical Adjustment of fungi to their environment”, G.C. Ainsworth and A.S. Sussman (Eds.), Academic Press, London, III: 395–415. Pontecorvo, G.C. (1956), “The parasexual cycle in fungi”, Ann. Rev. Microbiol., 10: 393–400. Pontecorvo, G.C. and G. Sermonti (1954), “Recombination without sexual reproduction in Pencillium chrysogenum”, Nature, 172: 126–127. Pontecorvo, G.C., J.A. Roper and E. Forbes (1963), “Genetic recombination without sexual reproduction in Aspergillus niger”, J. Gen. Microbiol., 8: 198–210. Rowell, J.B. (1955), “Functional role of compatibility factors and in vitro test for sexual compatibility with haploid lines of Ustilago zeae”, Phytopath., 45: 370–374. Sadasivan, T.S. (1975), “Breeding for disease resistance in plants”, Proc. Indian Acad. Sci., 81(B): 229–248. Salmon, E.S. (1904), “Recent research on the specialization of parasitism in the Erysiphaceae”, New Phytologist, 3: 55–60. Schaffer, W. (1994), “ Molecular mechanisms of fungal pathogenicity in plants”, Ann. Rev. Phytopathol, 32: 461–477. Staskawicz, B.J. et al. (1995), “Molecular genetics of plant disease resistance”, Science, 268: 661–667. Vakili, N.G. and R.M. Caldwell (1957), “Recombination of spore colour and pathogenicity between uredial clones of Puccinia recondita f. sp. tritici”, Phytopath., 47: 356. Van der Plank, J.E. (1963), Plant Disease Epidemics and Control, Academic Press, New York. Vidhyasekaran, P. (1997), Fungal pathogenesis in plant and crops, Molecular biology and host defence mechanisms, Marcel and Dekker, New York, pp. 553. Vidhyasekaran, P. (1998), “Molecular biology of pathogenesis and induced systemic resistance”, Indian Phytopath. 51(2): 111–120. Ward, H.M. (1903), “Further observations on the brown rusts of Bromus caused by Puccinia dispersa (Erikss.) and its adaptive parasitism”, Ann. Mycol., 1: 132–151. Watson, I.A. (1957), “Mutation for increased pathogenicity in P. graminis var. tritici,” Phytopath., 47: 507–509. Zinder, N.D. and J. Lederberg (1952), “Genetic exchange in Salmonella”, J. Bact., 64: 679–699.

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QUESTIONS 1. What are the various mechanisms which bring about variations in fungi? Give examples of diseases in which mutations have been instrumental in increased pathogenicity. 2. What are heterokaryosis and somatic hybridization? 3. How does the parasexual cycle differ from the normal sexual cycle? 4. Fungi Imperfecti do not have a normal sexual cycle and yet they are quite abundant and successful in nature. Why is this so? 5. What are transformation and transduction? 6. What is physiological specialization? 7. Identify the biotypes in Puccinia graminis tritici. 8. What is the gene-for-gene concept? 9. Write in brief what you know of host-pathogen specificity. What are the various hypotheses in this regard?

8 Effect of Environmental Factors and Nutrition on Disease Development

The weather and soil conditions influence the seasonal development and geographical distribution of plant diseases. About 75 years ago, Jones (1924) directed increased attention towards the relation of the environment to the inception and development of disease. The effects of environmental factors on plant diseases have been reviewed by Colhoun (1973). The connection between nutrition and susceptibility to parasitic and non-parasitic diseases in plants has been discussed by Karl Boning (1976). The influence of environment on the development and the control of disease has been reviewed by McCartney (1997). The “environment” connotes all external conditions affecting the life and development of an organism, which include temperature, light, and moisture and may also include living factors, such as competing micro-organisms or vectors. The environment can affect the development of plant disease in different ways. It can affect the perpetuation or over-wintering of the pathogen from one growing season to the next, the build-up of both primary and secondary inoculum, the dissemination of inoculum, germination, and host penetration. The environment can affect growth development of the host prior to being infected in such a way as to affect its susceptibility. Finally, it can influence the actual development of disease after the host has become infected. The development of all parasitic diseases also obviously varies with the pathogen, host, interactions between the pathogen and host, and environmental factors in the air and soil. When we take into account the environment, we have to distinguish between macroclimate, and microclimate, between weather in general and the particular weather under which specific diseases develop. For example, temperature and humidity in the open atmosphere may be quite different than in the foliar canopy of dense stands of growing plants. EFFECT OF TEMPERATURE ON THE DEVELOPMENT OF A DISEASE

Temperature is undoubtedly one of the most important factors, which influences the occurrence and development of many diseases. The late blight of potato is usually considered a disease which occurs in the northern latitudes, but it can become destructively epidemic when potatoes are grown during winter in the sub-tropics. Some pathogens develop best in low temperature regions. Taphrina pruni and

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T. deformans are confined to the cool, wet parts of the globe. Similarly, Spongospora subterranea, the powdery scab, is limited to cold areas. Puccinia striiformis also thrives in the cold and in India it over summers at heights above 7000 feet above sea level while black rust survives at 5000 feet above sea level. Cercospora beticola, the cause of sugar beet leafspot, needs a temperature 15oC for sporulation and for germination and infection. Urocystis cepulae, the cause of onion smut, is spreading throughout the USA on infected onion sets. It is absent in the extensive southern onion growing regions, but is an established disease in the cooler, northern onion growing areas of the USA and Europe. Walker and Wellman (1926) have explained this in terms of temperature effects on the pathogen. On the other hand, there are diseases which occur in the warm belts of the globe, for example, Fusarium oxysporum f. sp lini, F. oxysporum f. sp lycopersici, F. oxysporum f. sp. conglutinans causing wilts of flax, tomato and cabbage, Erwinia amylovora, the fire blight bacterium, Pseudomonas (Xanthomonas) solanacearum causing bacterial wilt of potato, tomato, tobacco, brinjal, pepper, and so on, Sclerotium rolfsii, various rusts, Macrophomina phaseoli, Phymatotrichum omnivorum causing Texas root rot of cotton. Pseudomonas solanacearum which causes bacterial wilt and brown rot of potato is most severe in warm regions. Potato crops raised in winters on the plains is not affected by this disease but it may occur when the crop is planted in January/February. A variety of this bacterial pathogen causes wilt of eggplants (brinjal) at high temperatures. The best development of the disease occurs at 37°C. The stages of infection are also affected by changes in temperature. Temperature can also effect the rate of penetration of the pathogen into the host. In epidemiology this is often quantified as the incubation period. Both infection and latent periods are influenced by temperature. Latent periods tend to decrease with increasing temperature, provided other factors are favourable. The latent period for Erysiphe graminis decreases from about 14 days at 5 oC to 3 days at 18 to 25 oC. In the case of black stem rust of wheat the incubation period is 22 days at 4.5 oC, 15 days at 10 oC, 9 days at 19 oC, and only 5days at 24 o C. In the sunhemp wilt (F. oxysporum f. sp. crotolariae) the optimum growth is accomplished at 25-28o C. This coincides with the maximum development of the disease when the soil temperature is between 25 oC and 28 oC. Above and below this temperature, the disease diminishes. The seedling blight of maize and wheat are caused by Gibberella zeae, but the optimum temperature for the disease is different in the two crops. Blight of wheat is caused at a comparatively higher temperature that is 20 to 28 oC. In the case of maize, severe disease is caused when soil temperature is between 16 oC and 20 oC. It should be mentioned here that wheat normally grows at lower temperatures than maize, and the temperature which produces the maximum amount of disease of these two crops is actually (too high for wheat and too low for maize) unfavourable for the growth of these two crops. Histological and biochemical studies of roots have shown that at these unfavorable temperatures the cell walls of the roots remain for a considerable time in the primary pectic condition and are easily penetrated by the fungal hyphae. This is a case in which the influence of temparature on the disease acts mainly through the host. The incidence of cereal rusts depends mainly on humidity and temperature. The incubation period and the germination of urediniospores of Puccinia graminis tritici is favoured by a temperature of 20°C. In the Indian plains this usually occurs in the period between February and March when the weather just begins to warm up. If the weather becomes cloudy and moist during this period, the rust incidence is immediately high, causing huge damage. According to Stakman and Harrar (1957), the time required for the production of urediniospores of Puccinia graminis tritici on a susceptible wheat cultivar, can range

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from 85 days at 0°, to 5 days at 24o C. The incubation period of Pseudoperonospora humuli can vary from 3 days at 21-25o C to 23 days at 5 oC or 11days at 29 o C. Thielaviopsis basicola which causes black root rot of tobacco is highly damaging when the soil temperature is between 17 o C and 23 o C but causes little damage when the temperature is between 21oC and 30 o C. This has been attributed to the formation of a phellogen layer at the higher temperature, which arrests the spread of the fungus. Many viral diseases are more virulent when the temperature is between 20o C and 25 o C. The most favourable temperature for disease is between 20 o C and 25 o C; these include the tobacco mosaic virus, potato mosaic virus, cabbage virus, potato yellow dwarf and others. Late blight of potatoes generally does not occur in the plains of India, where potatoes are grown as a winter crop. At this time, the conditions necessary for the growth of fungi do not exist in plains. But in the hills where the crop is grown in the rainy season, late blight is very severe. Musk melons are relatively resistant to seedling blight and wilt caused by Fusarium oxysporum f. sp. niveum when soil temperature is at an optimum of 30 o C or higher for the growth of melons. However, the disease is abundant and destructive at temperatures below 27 o C which is optimum for the growth of the pathogen. The resistance at high temperatures appears to be due to the greater ability of the host to exclude the pathogen from the vascular bundles by the formation of the periderm. Sunhemp wilt, due to Fusarium oxysporum var. crotolariae occurs in soils in India when the soil temperature is low—between 19 o C and 29 o C. HUMIDITY AND MOISTURE

Atmospheric humidity and precipitation in the form of rain, fog, dew, and so on determine disease incidence to a great extent. Likewise, the amount of soil moisture determines the severity of the disease in soil. The speed of germination of inoculum, its entry into the host, and the period of incubation are very much affected by the amount of moisture available. In case of soil diseases the amount of moisture will also affect the soil aeration and the concentration of various salts in the soil. Downy mildew of grapes is very much favoured by moist weather and high temperatures. Similarly, late blight of potato is favourably affected by high moisture content. A large number of diseases are favoured by increased humidity, such as the late blight of potatoes, Koleroga of arecanuts, downy mildew of cereals, blast of rice, early blight of potatoes, scab of apples and circular leaf spot of coffee. Precipitation and dew play their own part in increasing atmospheric humidity and cannot be considered in isolation. Heavy dewfall favours the germination of the spores of the pathogen and infections in diseases, such as downy mildew and rusts. Soil moisture appears to have a decisive effect on the amount of the big vein disease of lettuce caused by a virus that persits in the soil. In experiments performed 85% of the plants became infected in soil containing 95% of its moisture holding capacity, and there was a progressive decrease in infection with decreasing moisture, until only 10% of the plants became infected at 35% moisture. Diseases such as the root rot of pear caused by Aphanomyces euteiches, the damping off of seedling caused by Pythium ultimun, Pythium debaryanum and others, the club root disease of crucifers (Plasmodiophora brassicae) are greatly increased by an increase in the moisture content of the soil. But on the other hand, diseases like common scab of potatoes (Streptomyces scabies) and white rot of onion (Sclerotium cepivorum) usually spread under dry conditions. According to Beach (1949), the severity

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of damping off caused by Rhizoctonia solani increases with increasing moisture up to 65% of saturation and then decreases abruptly. Moderate soil moisture is most favourable for head smut of Sorghum caused by Sphacelotheca reiliana and pea foot rot due to Fusarium solani f. pisi. Soil moisture appears to have a decisive effect on the amount of big vein disease of lettuce, caused by a virus which persists in the soil. The disease is favoured with light moisture content up to 85%. Moist weather (coupled with suitable temperature) favours the spread of rusts and downy mildew in general. The powdery mildews (Erysiphales), such as powdery mildew of pea caused by Erysiphe polygoni and mildew of Dalbergia sissoo caused by Phyllactinia corylea, are not so dependent on moisture and they are known to flourish even in conditions of drought. Karnal bunt of wheat (Neovossia indica), which is air borne, occurs as an epiphytotic only when there is heavy rain and air and soil temperature is around 15–20 oC, two to three weeks before flowering time. The rain water soaks the spores thoroughly and the low temperature promotes germination. The disease is rare in the absence of both these conditions. Colocasia blight due to Phytophthora colocasiae occurs only in the rainy season, especially when there is a continuous spell of moist and cloudy weather. Plants recover as soon as dry condition return. Soil moisture also materially affects the cause of disease in Phytophthora foot rot of Piper betle and gummosis of Citrus. The moisture content of the air is usually referred to as relative humidity. This is defined as the ratio of the actual amount of moisture in the air to the amount of moisture the air could hold if the latter were saturated at such a temperature. However, since the amount of water the air can hold varies with temperature (warm air can hold more moisture than cold air), relative humidity is not the best measure of moisture in the air when we are concerned with its effect on the pathogen. A better measure is vapour pressure deficit (VPD). The VPD is measured in terms of pressure of atmosphere and is the difference between the actual vapour pressure of water and the vapour pressure of water in a saturated atmosphere. Lack of atmospheric humidity is also the reason for the relative absence of certain geographical areas of foliage diseases, particularly those of beans, such as anthracnose (Colletotrichum lindemuthianum), bacterial blight (Xanthomonas campestris pv. phaseoli). Beans are grown for seed in the arid portions of the USA to avoid seed infections by those organisms which require free moisture on plant parts. Late blight of potato seldom occurs in drier areas, except sometimes in those under irrigation. The powdery mildew fungi, a group of pathogens, are affected by moisture in an unusual manner. Germination of condia of the lettuce powdery mildew fungus (Erysiphe cichoracearum) greatly decreases at 100% relative humidity (VPD = 0 mm Hg), germinates best at 93% RH (VPD = 2 mm Hg), and germinates to some extent at 0.1% relative humidity (VPD = 25 mm Hg). Thus it is not hard to see why powdery mildews develop in the driest of climates. It is believed that the reason these conidia germinate under such dry condition is that they contain about 70% water and they carry their own water supply with them. Soil moisture does not appear to have the same controlling effect on the development of disease as soil temperature. In fact, in judging the effect of soil moisture, it is difficult to consider this factor in isolation from other soil factors, such as physical condition, water holding capacity, drainage, retentivity and so on. All these factors tend to make up a microclimate which influences the course of disease collectively and not individually. Disease favoured by wet soil or high relative humidity are damping off of papaya and vegetable seedlings, foot rot of Piper betle caused Phytophthora nicotianae var. parasitica or other Phytophthora species, collar rot of citrus caused by Phytophthora citrophthora, late blight of potato, cereal rusts,

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downy mildew of bajra (pearl millet), sorghum or maize bacterial blight and streak of rice and many others. Diseases favoured by dry soil or water deficient soils are common scab of potato (Streptomyces scabies) seedling blight of cereals caused by Fusarium species. Fusarium wilt of tomato, peas and cotton are more severe in wet soils in comparison to dry soils. There is a very close relationship between plant pathogenic nematodes and the amount of soil moisture in the soil. SOIL pH

The effect of alkalinity and acidity of the soil is so pronounced in many cases as to level the incidence and distribution of certain diseases and to be the most important factor in their control. Alkaline soils are conducive to the spread of the scab of potatoes (Streptomyces scabies). It usually develops between pH 5.2 and 8.0. Its incidence fails sharply when the soil pH falls below 5.2 . Successful attempts have been made to control this disease by acidifying the soil with sulphur or sulphuric acid. The club root disease of crucifers (P.brassicae) is favoured by acidic soil. It develops most at about pH 5.7 and drops sharply if pH is raised so that it totally fails to develop at 7.8. Liming, therefore, helps to control the disease. The influence of pH in this case is on the swarming myxamoebae. Other pathogens which are favoured by the alkaline conditions of soil are Texas root rot pathogen, Phymatortichum omnivorum, which attacks more than 1000 spp. of hosts, G. (Ophiobolus) graminis, the take all of wheat and Verticillium albo-atrum vascular parasite of many kinds of plants. Other diseases which are associated with acidic soils are F. oxysporum f. sp. lycopersici and F. oxysporum f.sp. vasinfectum which causes tomato and cotton wilts, Synchytrium endobioticum which causes potato wart and Spongospora subterranea, the cause of powdery scab of potatoes. High soil acidity is inimical to the development of tobacco root rot caused by Thielavia basicola, while such a condition is highly favourable to wilt of tomatoes. The behaviour of F. oxysporum f.sp. vasinfectum which causes the cotton wilt in the USA and India was studied by Mundkur (1936), who found that the American strain was favoured under acidic conditions, while the Indian strain developed best in alkaline reaction. Root rot of cotton caused by Rhizoctonia destruens is favoured by light soils with acid reactions, while in India Fusarium wilt of cotton develops best in heavy alkaline soils. Covered smut of oats develops at a pH value of 7.8 and falls sharply with either a decrease or increase in the soil pH; it is moderate at 6.5 pH. Although the pH of the soil affects the growth and reproduction of some pathogens directly and this determines disease development, it sometimes is only an indicator of certain other conditions or it acts indirectly by affecting certain biological and chemical processes in the soil. Just as plant pathogens are affected differently by soil pH, so many saprophytic soil organisms are also affected. Thus, the pH may sometimes influence disease development through its effect on soil microflora. A still more common indirect effect probably is its role in determining the availability of nutrient elements. It is well known, for example, that phosphates, calcium, iron and manganese may be present but in insoluble form and therefore may be unavailable. SOIL TEXTURE

In the black cotton soils which are clayey and heavy, cotton wilt (F. oxysporum f. sp vasinfectum) occurs abundantly and root rot due to Rhizoctonioa bataticla does not occur.

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On the other hand, in regions of lighter soils (Gujarat) the case is reversed, that is only root rot occurs and there is no wilt disease. Both these types of soils are alkaline and the determining influence in this case appears to be texture (clayey or sandy); Rhizoctonia cannot survive poor aeration. It is very sensitive to oxygen tension. EFFECT OF LIGHT

Although light can influence the development of some diseases, it is rarely a limiting factor in their seasonal or regional occurrence. It probably is not usually a determining factor in the development of epidemics nor in the geographical distribution of diseases. Light intensity and length of day, however, may affect the survival of inoculum, prepenetration processes, possibly the entrance of some pathogen/s length of the incubation period, abundance of sporulation and sometimes the type of pathogenic effect. Light intensity and day length also affect the development of many host plants and can be the determining factors in their viability as media for the growth of pathogens and their relative resistance or tolerance to pathogenic effects. Although wheat stem rust develops well at light intensities ranging from 500 to 10,000 foot candles, the incubation period is likely to be shorter and sporulation to be more abundant with increasing light intensity. In experiments on Melampsora lini, a flax rust, uredinia appeared 9 days after inoculation when plants were kept under normal light and after 14 days under reduced light, It has been found that high light is most favourable for leaf rust of rye, Puccinia rubigovera var. secalis. Light is necessary for photosynthesis and thus the general well-being of the plant is affected by both the quantity and quality of light. Several viral diseases are known to be influenced by light intensity. Reduced light intensities generally increase the susceptibility of plants to viral infection. For example, tomato plants will get infected by sugar beet curly top virus when they are grown under conditions of low light intensity, that is in shaded areas, whereas in full sunlight they will die. Apparently the concentration of carbohydrate in the leaf affects virus symptom expressions. The intensity of light also affects several fungal diseases. Powdery mildews often develop better under shaded conditions. Although it is difficult to separate the response of the host from that of the fungus to light, it has been shown by Masri and Ellingboe (1966) that low light intensities (25 to 50 foot candles) favour maturation of appressoria of Erysiphe graminis. While high intensities (7200 foot candles) and darkness inhibit appressorial maturation. The quality of light may also affect plant pathogenic fungi. Leach (1962, 1967) has shown that the ultraviolet portion of the light spectrum initiates and stimulates the sporulation of several plant pathogens for example, Botrytis cinerea and Hemminthosporium avenae. Sclerotia of Sclerotnia sclerotiorum, when placed in moist saw dust and in the dark, only produce apothecial initials and not apothecia. They develop apothecia as soon as they are exposed to light Ultraviolet light and direct sunlight inhibit the germination of the teliospores of Puccinia graminis. If the dishes are covered with green light filters, germination normally occurs. Collectrichum lagenarium (anthracnose of cucurbits) produces perfect stages in the culture if exposed to ultraviolet light. Prolonged exposure to ultraviolet light is harmful to fungi for it has fungicidal properties.

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EFFECT OF OXYGEN AND CARBON DIOXIDE CONCENTRATION

The ratio of oxygen to carbon dioxide in the atmosphere is a almost fixed and generally there is no marked affect of these gases on the foliar pathogens. However in the soil-borne diseases the soil aeration O2 and CO2 are the two gases which are of importance. The oxygen present in the soil also influences the root growth of host plants and carbon dioxide in the soil also influences the general microbiota and also the growth of roots in the soils of the host plants. The role of available oxygen in the soils also appears most important in determining the population limit reached by the nematode species. Increased oxygen concentration stimulates activity of larvae of nematodes while the reverse is seen when carbon dioxide concentration is increased. The amount of oxygen is not as important as the concentration of carbon dioxide. Rhizoctonia solani infecting various crops is inhibited if the concentration of CO2 in soil is more. Fusarium culmorum and Gebberella zeae cause root rot and seedling blight of wheat. These fungi can tolerate higher CO2 concentration, plants become weak and increased activity of fungal pathogens cause more diseases. Many species of Phytophthora require some oxygen for their growth and zoospore production. The availability of oxygen determines the vertical distribution of fungi in soils which has marked relevance to the occurrence of disease. EFFECT OF NUTRIENTS

The relation between nutrition and susceptibility to parasitic and non-parasitic diseases in plants has been discussed by Karl Boning (1976). We can distinguish three main types of reactions in the behaviour of the host plants towards the parasite. These are susceptibility, resistance and reduced receptivity. These different kinds of reactions to parasite attack are correlated in the following way with the different forms of nutrition. Susceptibility is usually most evident where there is potash deficiency, resistance where there is nitrogen deficiency and reduced receptivity where there is phosphoric acid deficiency. When one nutrient is given in excess there is also a relative deficiency of the two other main nutrients. Usually, however, only one deficiency factor predominates and it behaves in line with the excess factor as regards the kind of reaction involved. Thus, one-sided nitrogen excess usually reacts in a susceptible way as in the case of potash deficiency, although, of course, where nitrogen feeding is very one-sided, behaviour may also resemble that associated with phosphoric acid deficiency. The relative phosphorus deficiency component acts in a similar way, as resistance and reduced susceptibility frequently have similar effects. Excess phosphorus tends to behave more or less like nitrogen deficiency although the potash deficiency component may also show itself. Generally, we find that nitrogen and potash determine the reaction of the plant to the disease organism more than phosphoric acid does. This may have something to do with the fact that phosphoric acid deficiency represents a combination of two excess factors that act in a contrary manner. This is also true where there is excess phosphorus and where the two deficiency factors, again potash and nitrogen also have effects. The effect of different levels of application of lime is considerably less than that of different levels of nitrogen and potash. Nitrogen abundance results in the production of more vegetative and succulent growth of the plants. It is known that fertilizer application with large amounts of nitrogen increases the susceptibility of the

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pear to Erwinia amylovora, cause of fire blight, rusts of wheat caused by Puccinia species and powdery mildew. While reduced availability of nitrogen increases the susceptibilty of tomatoes to Fusarium wilt, early blight of potato, and tomato caused by Alternaria solani, wilt of solanaceous plans caused by Pseudomanas solanacearum, and damping off of seedlings due to Pythium species. Not only the amount of nitrogen but the form of nitrogen has its bearing on certain diseases. For example Plasmodiophora brassicae, Sclerotium rolfsii and Pyrenocheata lycopersici are more serious when ammonium nitrogen fertilizer is applied; on the other hand Gaeumannomyces graminis, Phymatotrichum omnivorum and Streptomyces scabies are more serious when nitrate nitrogen is supplied in the soil. The effect of each nitrogen form appears to be related to soil pH influences (Agrios, 1997). Phosphorus has been shown to reduce the severity of take all disease of barley caused by G. graminis and potato scab caused by S. scabies. Potassium reduces the severity of several diseases. Stem rust of wheat, stalk rot of corn are reduced if more of potassium is available to the plants. However blast disease of rice caused by Pyricularia grisea and root knot of nematodes are increased due to higher amounts of potassium. Calcium reduces the severity of several diseases such as diseases caused by Rhizoctonia, Sclerotium and Fusarium species. But black shank of tobacco and common scab of potato are enhanced by calcium. Micronutrient applications have tremendous impact on the occurrence of several diseases such as copper application enhances take all and ergot diseases caused by G. graminis and Claviceps purpurea. Addition of silicon to the nutrient sloutions has been shown to reduce the blast of rice and brown leaf spot of rice. ROLE OF BIOTIC ENVIRONMENT

For the foliar pathogens the phyllosphere is the region which influences the disease in the aerial parts and for the root diseases it is the rhizosphere which greatly influences the activity and growth of several pathogens. Phylloplane is natural habitat on the leaf surface which supports a heterogenous population comprising both pathogens and non-pathogens. Last (1955) termed the niche of microbes as phyllosphere (now known as phylloplane). The phylloplane microbes are of special interest from various points of view. For example, some of them have antagonistic action against fungal pathogens, degrade plant surface wax and cuticles, produce plant hormones, decompose plant material, activate plants to produce phytoalexins, and influence growth behaviour and root exudates of plants. Thus phylloplane exerts a great influence on the diseases caused by aerial plant pathogens. Andrews (1990) has reviewed the work on biological control in the phyllosphere. The microbiota in the soil influence the growth of plant roots as well microbial population in the rhizosphere or rhizoplane. A rich flora and fauna of soil microbiota lessens the danger of epiphytotic outbreaks of diseases caused by soil borne pathogens. There are antagonistic and associative effects on the plant pathogens in the soil. This aspect has been discussed at great length in the chapter on root diseases (Chapter 21).

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ROLE OF ENVIRONMENTAL FACTORS IN EPIPHYTOTICS

A plant disease is called epidemic or epiphytotic when the amount of disease present increases rapidly from a low level to a high level. In an endemic disease the disease level remains fairly constant. The science dealing with this area is called epidemiology. Environmental factors which predispose the plants to infection and leading to the occurrence of epiphytotics are temperature, light, relative humidity and moisture, wind and the presence of inoculum or introduction of a new pathogen species or race. Thus a combination of environmental factors both at macro and microlevel has great relevance in the causation of plant disease epiphytotics. These have been discussed at the appropriate places in Chapter 10.

REFERENCES Agrios, G.N. (1997), Plant Pathology, Fourth Edition, Academic Press, New York, pp. 635. Andrews, J.H. (1990), “Biological control in the phylloplane realistic goal or false hope”, Can. J. Plant Pathol., 12: 300. Beach W.S. (1949), “The effects of excess solutes, temperature and moisture upon damping off”, Pa. Agr. Exp. Sta. Bull., 509. Boning, Karl (1976), “Relation between nutrition and susceptibility to parasitic and non-parasitic diseases in plants”, Pl. Res. and Development, Institute for Scientific Cooperation, Tubingen, 4: 24–33. Colhoun, J. (1973), “Effects of environmental factors on plant diseases”, Ann Rev. Phytopath., 11: 343–364. Conant, G.H. (1927), “Histological studies of resistance in tobacco to Thielaviopsis basicola”, Am. J. Bot., 34: 457–480. Jones, L.R. (1924), “The relation of environment to disease in plant”, Am. J. Bot., 11: 601–609. Last, F.J. (1995), “Seasonal incidence of Sporobolomyces on cereal leaves”, Trans. Brit. Mycol. Soc., 38: 221–239. Leach, C.M. (1962), “Sporulation of divese species of fungi under near-ultraviolet radiation”, Can. J. Bot., 40: 151–161. Leach, C.M. (1967), “Interaction of near ultraviolet light and temperature on sporulation of the fungi Alternaria, Cercosporella, Fusarium, Helminthosporium and Stemphylium”, Can. J. Bot., 45: 1999–2016. Masri, S.S. and H.H. Ellingboe (1966), “Germination of conidia and formation of appressoria and secondary hyphae in Erysiphe graminis f. sp. tritici”, Phytopath., 56: 304–308. McCartney, A. (1997), “The influence of environment on the development and control of disease”, in: Enviromentally safe Approaches to Crop Disease Control, N.A. Rechcigl and J.E. Rechcigl, (Eds.), CRC Press, Boca Raton, New York, pp.3–31. Mundkur, B.B. (1936), “Resistance of American cottons to Fusarium wilt in India”, Proc. Indian Acad. Sci., 3(B): 498–501. Stakman, E.C. and J.C. Harrar (1957), Principles of Plant Pathology, Ronald Press, New York. pp. 581. Walker, J.C. and F.L. Wellman (1926), “Relation of temperature to spore germination and growth of Urocystis cepulae”, J. Agr. Res., 32: 133–146.

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QUESTIONS 1. Give examples of plant diseases which are influenced by soil temperature. 2. Why does late blight of potato generally not occur in the plains of northern India? 3. How does pH of soil influence the occurrence of a disease? Give examples of diseases which occur:

(i) in alkaline soils (ii) in acidic soils. 4. 5. 6. 7. 8. 9. 10. 11.

Name the environmental factors necessary for the development of Karnal bunt of wheat. Explain why wilt of cotton occurs in clayey soils while root rot generally occurs in sandy or loamy soils. Give examples of diseases which are influenced by light conditions. How does nutrition affect the incidence of disease? Name some plant pathogens which require high relative humidity throughout their development. Name some plant pathogens which can cause infection at low relative humidity. Give an example of a disease in which high levels of phosphorus bring about resistance in the plants. How to phylloplane and rhizosphere organisms influence diseases in plant?

9 Dispersal of Plant Pathogens

Distribution implies the spread of a plant pathogen into new geographical areas and its establishment there. The spread of a plant pathogen within the general area in which it is established is termed dissemination or dispersal. Contamination—the movement of inoculum (even if only a few inches) to susceptible plant—is a kind of dissemination. Knowledge of the methods by which pathogens are dispersed is usually essential before effective control measures can be devised. There are essentially two types of dissemination of plant pathogens: 1. Direct transmission 2. Indirect transmission In direct transmission, the dispersal takes place along with the seeds and vegetative parts, while indirect transmission may be autonomous, that is, by wind, water, animals or human beings. Autonomous dispersal by plant pathogens has been discussed by Muskett (1960), dispersal of inoculum by insects and other animals including man, has been discussed by Broadbent (1960) and liberation and dispersal of fungal spores have been ably discussed by Ingold (1971) in his book “Fungal Spores”. 1. Direct Transmission a. Germinative Transmission b. Vegetative Transmission c. Adherent Transmission 2. Indirect Transmission a. Autonomous transmission b. Wind dispersal of pathogerns—Anemochory c. Water dispersal of pathogens—Hydrochory d. Animal dispersal of pathogens—Zoochory e. Human dispersal of pathogens—Anthropochory DIRECT TRANSMISSION Germinative Transmission

This takes place along with the seeds or propagules. Bean mosaic is transmitted through pollen grains and carried in the seeds. In the case of loose smut of wheat and barley caused by Ustilago segetum

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(U. tritici) var. tritici and U. nuda respectively, the seeds get infected without producing any symptoms in the parent carrier. The seeds carry the intraseminal mycelium. When such apparently healthy-looking seeds are sown, the symptoms appear when the ears emerge. The pathogen develops systemically inside the plants. Alternaria leaf blight of wheat is both internally as well as externally seed-borne. (Prabhu and Prasada, 1966). The Helminthosporium blight of wheat and barley is seed-borne. The seed-borne infection is present in the form of mycelium within the seed including the endosperm and embryo. The fungus has been found to remain viable for up to seven years on barley seed (Christensen, 1922). Bacterial blight of paddy caused by Xanthomonas campestris pv. oryzae is believed to be seed-borne. Vegetative Transmission

Transmission of a large number of plant pathogens takes place through the vegetative parts of plants used as seed, such as tubers, cuttings, runners, grafts, and so on. Ring rot of potato caused by Clavibacter michiganense subsp. sepidonicum (Corynebacterium sepidonicum) is carried through infected potato tubers to newer areas as in the case of the pathogen Pseudomonas solanacearum responsible for brown rot of potatoes. A large number of viruses are transmitted through vegetatively propagated parts. Dormant mycelium of Phytophthora infestans is carried through the potato tubers. Seed setts of sugar cane carry the pathogens and when such apparently healthy-looking vegetative parts are sown, the pathogen develops systemically. Whip smut of sugar cane caused by Ustilago scitaminea and red rot of sugar cane caused by Collectotrichum falcatum are ‘seed’ borne (i.e. through the diseased cane setts). Adherent Transmission

In this case, the propagules of the pathogens are carried over the surface of the seed or vegetatively propagated parts. Many pathogens are present on or in seeds, transplants, hard woods or nursery stock and are disseminated by them as the latter are transported to other fields or areas or are sold and transported to other areas far and wide. Bunt of wheat caused by Tilletia foetida and T. caries is carried through the seeds externally, as in the case of covered smut of barley, where the smut spores attach themselves to the surface of the seeds at the time of threshing. There are many other fungi whose spores remain adherent, such as Synchytrium endobioticum responsible for potato wart and species of Helminthosporium, Rhizoctonia solani (black scurf) sclerotia on the tuber surface of potato and Alternaria which causes various diseases. INDIRECT TRANSMISSION Autonomous Transmission

Autonomous dispersal has been discussed in great detail by Muskett (1960). Plant pathogens can be spread over short distances by this method. Autonomous transmission takes place by active growth of hyphae or hyphal strands. It is characteristic of wood-rotting fungi, such as Armillaria, Fomes, Ganoderma, Polyporus spp belonging to the Hymenomycetes which migrate independently through the soil from plant to plant or even from field to field by the active growth of their hyphal strands.

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Armillaria mellea rhizomorphs spread from one root to the other in forest trees. Hyphal mats or strands grow out to short distances in the soil from the sclerotia of Rhizoctonia solani and Sclerotium rolfsii. The rate of such spread in Phymatotrichum omnivorum, responsible for Texas root rot of cotton crop, is estimated at 5 to 30 feet per season and 2 to 8 feet per month in alfalfa crop. It may be emphasized that in Armillaria mellea, the cause of the tree root disease, Gaeumannomyces (Ophiobolus) graminis, the cause of the take all disease of wheat, Phymatorichum omnivorum, the cause of root rot of cotton, the pathogens do not grow and ramify through the soil as do the non-specialized types, but require a food base such as the roots or crop debris of the host plant which they attack. Wind Dispersal of Pathogens

This type of spread is termed by Gaumann (1950) as ‘anemochory.’ Some plant pathogenic bacteria are carried to short distances by the wind. The seeds of some angiospermic plant parasites are effectively carried by the wind. All fungal pathogens which produce spores or conidia on the surface are disseminated by the wind, such as downy mildews, powdery mildews, rusts, smuts, leaf-spot causing pathogens, sooty moulds, and so on. The pathogen must produce numerous spores which are successfully liberated, dispersed and deposited in a viable condition on susceptible plants under conditions conducive to infection; in other words production, liberation, dispersal, and deposition of spores is involved. Many fungal pathogens are remarkably well adapted to wind dissemination for they produce and liberate into the air countless numbers of small and very light spores that are carried to long distances. Propagative spores are produced in many different ways in sporangia on simple or complex conidiophores in or on ascocarps and basidiocarps, by budding or fusion, within pycnidia, perithecia or sori of various types, and in other ways as described in books on mycology and plant pathology (Stakman and Harrar, 1957; Hughes 1953; Hawker, 1960). Fungi show a great diversity in their methods of producing propagules and many different ways of more efficient production and liberation of spores seem to have been explored. Spore production per unit area of infected leaf tissue may be increased by close aggregation of sporophores, branched or whorled sporophores, production of spores in chains or clusters, by a more or less continuous production of spores within the sorus, or by successive crops of spores. The number of spores produced can be astronomical. Powdery mildews may produce several thousand conidia per square centimeter of infected leaf surface, a fairly modest output compared with 100,000 or more in some downy mildews. A single smut sorus may contain millions of spores, a heavily infected barberry bush is said to produce up to about 70,000 million aeciospores of Puccinia graminis at one time (and there may be several crops of spores in the spring), and a relatively small apothecium of Sclerotinia can produce about 30 million ascospores. The spores produced by large polypores, such as Fomes are even more numerous. Heavily rusted wheat fields produce so many spores that spore clouds may be seen rising during harvesting and implements too may be layered with a fine reddish deposit of spores. The duration and periodicity of sporulation is as important as the number of spores. Some pathogens produce a more or less simultaneous crop of propagules followed by other crops, as in the case of sporangiophores of Peronospora. The sporophores of others may bear several successive propagules, for example, those of Phytophthora, and Cercospora. The capacity to produce a steady stream of infective propagules over a prolonged period of time is likely to be advantageous to the pathogen. Some fungi show a diurnal periodicity in sporulation, as nocturnal production of sporangia by some species of

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Sclerospora. This is probably chiefly due to temperature moisture effects, but might in some cases be related to periodicity in susceptibility of the host plant. Similar periodicity in spore discharge (Hirst, 1953) and in the germination and formation of appressoria (Yarwood, 1936) has been reported Apart from spores, bits of mycelium and nematode cysts are also sometimes disseminated by wind. In India the molya disease of wheat and barley (Heterodera major) is believed to have been introduced into Haryana from Rajasthan . According to Schrodter (1960), the problem of dissemination of plant diseases where propagules cannot move themselves is more amenable to theoretical treatment than any other problem in phytopathology. Fungal spores in air behave as inert particles, perhaps similar to those in smoke, with terminal velocities ranging from about .05 to about 2.5 cm per second, the larger spores falling more rapidly than small ones. Although fungal spores are generally more abundant in the air near the earth than at high altitudes, several investigators have encountered clouds of spores, numerous bacteria, and other minute objects several thousand feet above the earth. Urediniospores of Pucinia graminis tritici have been caught as high as 14,000 feet above infected grain fields, living spores of various fungi were caught from aeroplanes above the Carribean sea 600 miles from their nearest possible source, and living spores of several common moulds were caught in a spore trap released from the balloon Explorer II, at 72,500 feet and set to close at 36,000 feet. Long distance dissemination of rusts is a well established phenomenon. In the Indian subcontinent, rust spores are known to makes big jumps from the source areas to the plains (Mehta, 1940; Nagarajan and Singh, 1974,1975). Studies by Nagarajan and Singh (1973, 1974, 1975) have shown that for south and central India the spores are wind-borne, rain-deposited and are transported from an altitude of around 700 mb (3030 m). The urediniospores get transported by upper winds from the Nilgiri and Palney hills and are then washed down over Central India by rain. Such a spread occurs to a distance of 600 km or more without infecting the fields in between. Turbulence redistributes spores and affects the progressive dilution of spores with increasing distance from their source. There is ample evidence that with normal wind and turbulence conditions, spores especially small ones, can travel considerable distances. Those of Phytophthora infestans have been reported by various observers to travel distances of 200 m to more than 60 km depending upon the wind and degree of turbulence. An interesting example of wind dispersal is the case of fire blight bacterium (causing diseases in apples and pears). The pathogen produces fine strands of dried bacterial exudate containing bacteria, and these strands may be broken off and disseminated by wind. Water Dispersal of Pathogens (Hydrochory)

Water as an agent of dissemination appears to be relatively less important as compared to wind. Although it sometimes facilitates dissemination in various ways, it does not furnish the motive power for long-distance dissemination, except as streams and rivers. Flood and irrigation water may carry inoculum, especially that in or near the soil. It is clear that water separates and distributes spores in a microenvironment. Spores that are extruded in gelatinous tendrils that may harden when dry, can be separated from each other and washed down trees and other kinds of plants during rain or heavy dews.

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The splashing and splattering of water during heavy rains may result in distributing inoculum to plant parts near the soil and may distribute bacteria and spores to different parts of the same plant or neighbouring plants. Bacteria, nematodes and spores and mycelial fragments of fungi present in the soil are disseminated by rain or irrigation water that moves on the surface or through soil. In some places, rain seems to be more important than insects in disseminating the fire blight bacteria, Erwinia amylovora, by its washing or splattering effect. The secondary spread from initial infection of Xanthomonas campestris causing black rot of cabbage and other crucifers, of X. translucens which attacks cereals and grasses, and of bacterial blight of tomato, Corynebacterium michiganense, results largely from splashing during rains. Xanthomonas campestris pv. malvacearum, the bacterium that causes angular leaf spot of cotton, can be spread by wind blown rain. Surface water resulting from rains or irrigation is known to disseminate some pathogens and undoubtedly a large number of spores. The bacterium causing angular leaf spot or black arm of cotton is spread largely by irrigation water or surface water in Arizona, USA, and Uganda. Plasmodiophora brassicae, the organism causing club root of crucifers is spread by surface water, and Phytophthora infestans, causing late blight of potatoes has been observed to spread from plant in dry areas by means of irrigation waters. Colletotrichum coffeanum which produces spores within the acervuli in the maturing bark of coffee twigs and on diseased berries is disseminated by water. Water, then is an important agent of local dissemination, but it is not as important as the wind for long distance dissemination. Large drops of water in the form of rain or overhead irrigation, falling at their terminal velocity, are probably most active in splash dispersal, much more so than small drops. In splash dispersal, the spores are carried and often deposited on the plant surface in a droplet of water which could provide the moisture required especially in humid weather. Splash dispersal, although usually considered a mechanism for liberating and dispersing slimy spores, may also play a part in the dispersal of roughened spores which adhere strongly to each other or the plant surface. An example of this is provided by Hemileia vastatrix (coffee rust) in which, according to Nutman et al. (1960), the roughened urediniospores cling together and are broken up by water. They then float singly to the surface of the water drop, from which they are splash-dispersed by further rain drops. The cells of many plant pathogenic bacteria and the slimy spores of such important genera of fungal plant pathogens as Septoria, Fusarium, and Colletotrichum are splash-dispersed and it is surprising that relatively little information on splash dispersal is available. The eelworm disease of potato and Piper longum have been known to be carried through surface water from field to field. The spread of infection in the red rot of sugar cane caused by Colletotrichum falcatum and the disemination of the sclerotial bodies of Sclerotium rolfsii through soil are chiefly brought about by irrigaion water. Water is also an important agency of dissemination for the seeds of flowering parasites such as Orobanche, dodder (Cuscuta) and Striga through the soil. Short distance transmission of semi-aquatic pathogens from one leaf to another of an adjacent plant occurs through splashing rains accompanied by wind such as for Phytophthora arecae, P. parasitica, P. infestans and other downy mildew fungi. These fungi are well adapted to wind as well as water dissemination due to the formation of deciduous conidia capable of germinating either by zoospores or germ tubes and thus develop into dangerous pathogens capable of widespread damage within a short period.

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Dissemination by Insects, Mites and Nematodes Insects Since the discovery by Waite (1891) that bees and wasps can transmit bacteria causing fire blight of apple and pear, Erwinia amylovora, much information has been accumulated on the role of insects and other small animals in the dissemination of plant-pathogenic viruses, bacteria and fungi. Many small animals help in the dissemination of some pathogens occasionally and incidentally but insects fall into a special category. Insects not only transmit and disseminate many plant pathogens but are sometimes agents of inoculation also. Moreover, some pathogens multiply within insects and some are present in them during periods when host plants are dead or dormant. Various interesting relationships between micro-organisms including plant pathogens and insects had been discussed in detail by Leach (1940) and Carter (1962). Shorter accounts have been given by Broadbent (1960), Gäumann (1950) and Austwick (1957) on fungi, by Bawden (1964) on viruses, and by Madelin (1966,1968) on entomogenous fungi. The largest number of insect vectors have sucking mouth parts and belong to the Homoptera. Within this order the largest number of species known to transmit one virus or the other is contained within two families, Aphididae (aphids) and Cicadellidae (leaf hoppers). A few belong to the Aleyrodidae (white flies), the Cercopidae (spittle insects), the Coccidae (mealy bugs), and the Membracidae (tree hoppers) families. Thrips transmit the spotted-wilt virus. Some viruses are transmitted by both chewing and sucking insects, while a few are transmitted only by the former. Squash mosaic, cowpea mosaic and turnip yellow mosaic viruses are transmitted by beetles (Coleoptera) while the last is transmitted also by grass hoppers (Orthoptera), and earwigs (Dermaptera). It is of interest to note that practically all vectors of the so-called yellows group of viruses are leaf hoppers while most vectors of the mosaic group are aphids. In other words, viruses which are more readily transmitted mechanically are aphid-borne and those which do not yield to such transfers are leaf hopper-borne. More than fifty species of aphids are known to transmit viruses of plants. Some species can transmit only a few kinds and others transmit many; Myzus persicae, the green peach aphid, alone is the vector of fifty different viruses. Some of the diseases transmitted by aphids are; Katte or Marble disease of cardamom, mosaic and grassy shoot of sugar cane, mosaic streak of wheat, maize mosaic, barley mosaic, bean mosaic, cowpea mosaic, bunchy top of bananas, banana mosaic, papaya mosaic, potato necrosis (viral), potato leaf roll, tomato mosaic, and chilli mosaic. Some of the diseases transmitted by the white fly are tobacco leaf curl, mung (Phaseolus aureus) yellow mosaic, double bean (Phaseolus lunatus) yellow mosaic, and yellow mosaic of Dolichos lablab. Tungro of rice is transmitted by leaf hopper vectors, while bottlegourd mosaic is transmitted by the red pumpkin beetle. Three important questions arise with respect to the transmission of viruses by aphids: 1. How long must the insect feed on infected plants before it can transmit the virus to healthy plants? 2. Is an incubation period in the insect necessary before the virus can be transmitted? 3. How long do the insects remain infective after having acquired the virus? Some viruses are quickly acquired and lost, others are acquired more slowly and are quite persistent. Some require an incubation period and others do not. In general, the viruses that require no latent or

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incubation period in the insects are relatively nonpersistent and those that require one or more are persistent. Insects are important in the dissemination, inoculation, and persistence of certain bacterial pathogens of plants. Erwinia tracheiphila, the cucurbit wilt organism is completely dependent on cucumber beetles for its spread. Erwinia (Xanthomonas) stewarti, the corn wilt pathogen, overwinters inside the corn flea beetle (Chaetocnema pulicaria). The cucumber beetles, Diabrotica vittata and D. duodecimpunctata, chew the plant parts affected by E. tracheiphila and carry the bacteria inside the body where they overwinter, passing on the host in the next season. It is believed that the relationship between the insects is symbiotic. Beetle transmission of viruses has been reviewed by Fulton et al. (1987). Insects are important agents of dissemination, and inoculation of Erwinia carotovora, causing black leg of potato. The pathogen survives in the seed tubers, which can account for primary infection in the spring. It was originally thought that the organism did not survive in the soil and there was no secondary spread in the field. Circumstantial evidence later indicated that there must be a secondary spread, but the agents remained unknown until Leach in 1926 proved that the seed corn maggot, Hylemya cilicrura, is a very important agent of effective dissemination and inoculation. The seed corn maggot and closely-related species are the pests of many kinds of plants, such as corn, cabbage, beets, beans, pear, radish, onion, tomato, and so on. These insects and certain others contribute to the development of soft rot caused by Erwinia carotovora in some of these crops in much the same way as has been described for black leg. Erwinia amylovora, the cause of fire bright of apples, pears and certain other members of the Rosaceae, was the first plant disease known to be caused by bacteria and transmitted by flies and ants. Xanthomonas campestris pv. citri which causes the well known citrus canker is carried by the leaf miner from diseased to healthy plants. Erwinia (Xanthomonas) stewartii and X. campestris pv. vasculorum which cause wilt of corn and gummosis of sugarcane, respectively are transmitted by insects and can persist inside the vectors. Erwinia stewartii is also transmitted on seed and X. campestris pv. vasculorum on sugarcane setts used for planting. The corn wilt organism, however, is carried from plant to plant in the fields by flea beetles, Chaetocnema pulicaria and C. denticulata by Diabrotica duodecimpunctata, mentioned in the discussion on cucurbit wilt and probably by other insects as well. Moreover, the bacteria can live during the winter inside the corn flea beetle, C. pulicaria, and can live for some time in the adult Diabrotica beetle. The big-vein disease of lettuce has been known since the 1930s. The disease-causing agent was not identified at that time but was known to be soil-borne. In the 1950s. the chyridiomycetous fungus, Olpidium brassicae, was found to be intimately associated with this disease. Campbell and Grogan (1964) showed that the causal agent could be transmitted by grafting in the absence of Olpidium. Thus, this disease is now known to be caused by a virus. The work by Campbell and Grogan (1964) showed that this virus is transmitted by the zoospores of Olpidium and survives within the resting spores of this fungus. Olpidium also transmits other viruses that are soil-borne. They are also known to be transmitted by various chytridiomycetes. According to Austwick (1957), about 66 species of fungi causing plant diseases are transmitted by more than a 100 species of insects belonging to at least six orders. Transmission occurs in various ways with or without injury to the plant. Spores of Ustilago violaceae (anther smut of Caryophyllaceae) and Botrytis anthophila (anther mould of clover) are thus carried from the infected anthers to uninfected flowers by pollinating insects.

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The conidia of Claviceps are spread from infected ovaries to healthy ones by insects which feed on the sugary honey dew on which the conidia are produced. Other fungi infecting the shoots and roots of plants are likely to be fortuitously disseminated by insects which move or are carried from plant to plant. Richardson and Saunders (1968), for example, attribute the partial control of potato blight by aphidicide sprays to the destruction of aphids which carry mycelial fragments and sporangia of Phytophthora infestans on their bodies and leg bristles, and so spread the pathogen over short distances. More frequently, however, transportation is accompanied by injury to the plant. The insect carries the fungus and places it in the wounds, thus facilitating infection. The injury may be caused by mechanical breaking, by other animals or by the insect itself. The spores of Ceratocystis fagacearum (oak wilt) are carried by various insects, chiefly nitulid and scolytid beetles, to existing wounds caused by breaking, wood peckers, squirrels, and other causes. A mycelial mat of the fungus develops beneath the bark, the crack is open and gives off a characteristic odour which apparently attracts insects. These feed on the fungus and also carry small spores which fertilize the receptive hyphae and so lead to the development of perithecia of the fungus. In some cases the insect or the fungus is adapted to insect transmission. Female Siricid (Sirex) wood wasps carry hyphae and conidia of Stereum sanguinolentum (heart rot of conifers) within the intersegmental sacs at the anterior end of the long ovipositor. The eggs are contaminated with the fungus during laying and are deposited within the wood of the tree and the fungus is reported to precede the larva as the latter bores through the wood. The fungus is contained in the hypoplural organ modifications of the larval integument. Ceratostomella ulmi (Dutch elm disease) grows within the egg galleries of bark beetles (Scolytus) and contaminates emerging adults, which also inject the slimy spores and thus spread the fungus to adjacent healthy trees. More sprecific are the diseases (stigmatomycoses) in which the fungus is injected into plant tissue by the insect vector. Several parasitic yeasts, notably Nematospora, are transmitted in this way, the acicular ascospores being sucked up the feeding stylet of stainer bugs (Dysdercus, Nezara) whence they lodge in the stylet pouches and are injected into young cotton bolls causing internal boll disease due to Nematospora gossypi (Frazer, 1944), bean pods (N. phaseoli, yeast spots) or coffee berries (N. coryli, N. gossypi). It is thought that Nematospora is almost entirely dependent on plant bugs for dissemination and entry into the plant. In the case of chestnut blight caused by Endothia parasitica, the insects responsible for the disease are European bark beetle (Scolytus multistriatus) and the native elm bark beetle (Hylurgopinus rufipes). Many insects attack the fruits of plants and fruit-rotting fungi often enter through the wounds. The spores of Aspergillus niger, which causes smut of fig fruits, are reported to be introduced by certain beetles and fruit flies, and Sclerotinia sp. (brown rot of various fruits) can enter through feeding and oviposition wounds made by insects. Some fungi causing stem rot (e.g. Glomerella tucumanensis, red rot of sugar cane) also invade through insect wounds and it is suspected that some leaf attacking fungi behave in a similar way. Pycnostysanus azaleae (bud blast of Rhododendrons) is associated with the leaf hopper Graphocephala coccinea and the disease is very slight in the absence of the insect (Howell and Wood, 1962). Little is known of dissemination of the root pathogen by insects but it seems likely that this occurs in the soil and that insects feeding on roots also introduce fungi into them. The original insect damage may thus be greatly aggravated by subsequent fungal invasions. Insects which feed on cotton plants have been found to contain F. oxysporum f. sp. vasinfectum (cotton wilt), the fungus having been recovered in a viable condition from faecal pellets of various grass hoppers. It seems possible that such insects disseminate this normally slowly-spreading fungus which might explain the occasional appearance of the disease in hitherto disease-free fields or parts of fields, but other explanations are possible, and the relationship between the soil-borne pathogens and insects needs further investigation.

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Insects which act as diploidizing agents for some heterothallic fungi, transfer fertilizing spores (spermatia, oidia) between compatible strains and so bring about the formation of the perfect state. The best-known example of this diploidization is that of receptive hyphae of rusts by insect-borne spermatia, but similar fertilization occurs in some Ascomycetes and other Basidiomycetes. This phenomenon has obvious similarities with insect pollination in higher plants. Mites These, even though wingless, transmit some viruses (reversion disease of currants) which are perhaps carried by winged insects or wind. Transmission of fungal pathogens by mites has not been extensively studied. Nematodes Some viruses as well as some bacterial and fungal plant pathogens are transmitted by nematodes. Corynebacterium fascians, leafy gall of various herbaceous plants, is carried by ectoparasitic nematodes (Aphelenchoides) and typical cauliflower symptoms develop only when both nematode and bacterium are inoculated into strawberry plants (Cross and Pitcher, 1952). Yellow ear rot of wheat caused by Clavibacter (Corynebacterium) tritici is dispersed by the ear cockle nematode (Anguina tritici) There is also evidence that some root-infecting pathogens, particularly fungi, may enter roots through wounds made by nematodes and this may perhaps be of more frequent occurrence then is at present realized. Cotton wilt caused by Fusarium oxysporum f. sp. vasinfectum is an example of this, the pathogen being associated with root knot nematodes and probably others (Smith 1953). The nematodes may also affect the physiology of the root, making it a more suitable medium for the fungus (Powell and Nusbaum, 1960; Powell, 1963). The reverse has also been reported; the fungus (Pyrenochaeta lycopersici) causing tomato brown root rot reduced the invasion of tomato roots by the potato root eelworm Heterodera rostochiensis (James, 1968). Bacterial wilt of carnations, caused by Pseudomonas caryophylli, is reported to be more severe in the presence of root knot nematodes which, probably facilitate the entry of the bacteria (Stewart and Schindler, 1956). Pitcher (1965), Fielding (1959), Christie and Perry (1959), Leach (1940) have discussed this. In 1958, Hewilt et al. demonstrated that the grapevine fan leaf virus is disseminated by the nematode Xiphinema index. Both polyhedral and tubular viruses have since been shown to be nematode-transmitted, the former by species of Xiphinema and Longidorus and the latter by Trichodorus spp. Transmission of viruses by plant nematodes has recently been reviewed by Brown et al. (1995). Human Dispersal (Anthropochory)

Man is, to a large extent, responsible for the dissemination of plant pathogens which he does in two ways—through his person and through the objects he transports from one place to another. Local dissemination of pathogens on man’s person is illustrated by the angular leaf spot disease of cucumber caused by the bacterium Pseudomonas lachrymans. When cucumbers are grown for pickles, the fruit are picked. When a person comes in contact with or picks an infected plant, his hands become contaminated, particularly when the plants are wet with dew. The pathogen is thus spread down the row of plants. Another pathogen spread by man is Clavibacter michiganense Subsp. michiganense (Corynebacterium michiganense), the cause of bacterial canker on tomatoes. This pathogen is seedborne, and when it develops on tomato seedlings to be used as transplants, the workers handling the

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transplants may contact the bacterium on their hands. Since the handling of transplants often causes small injuries to the plant, the worker placing the transplants into the ground can easily inoculate healthy plants. In California, USA, this disease has become a rarity since most tomato fields are no longer planted with transplants but are direct seeded. The viruses that are spread by animals can also be spread by man. Tobacco mosiac virus is known to survive in dry tobacco leaves. Hence, workers who handle tobacco plants in the field are advised not to smoke while they are working and to thoroughly wash their hands before entering the field. The long distance dissemination of plant pathogen by man is usually accomplished by the transport of infected plant parts, such as seeds, nursery stock or timber products. The cause of some of the worst plant disease epidemics is the introduction of a pathogen into a new area. In many cases the plants growing in these areas are very susceptible since they do not develop resistance through evolution in the presence of the pathogen. TABLE 9.1

Examples of plant diseases which have been introduced to India from other countries Disease

Leaf rust of coffee caused by Hemileia vastatrix Late blight of potato caused by Phytophthora infestans Flag smut of wheat caused by Urocystis tritici Downy mildew of grapes caused By Plasmopara viticola Powdery mildew of cururbits caused by Erysiphe cichoracearum Powdery mildew of maize caused By Sclerospora phillipinensis Black rot of crucifers caused by Xanthomonas campestris Powdery mildew of rubber caused by Oidium heveae Black shank of tobacco caused by Phytophthora nicotianae Crowngall of apples and pears caused by Agrobacterium tumefaciens Bunchy top of banana (viral) Hairy root of apple (viral) Onion smut caused by Urocystis cepulae Bacterial blight of paddy caused by Xanthomonas campestris pv. oryzae Golden nematode of potato caused by Heterodera rostochiensis

Original home

Year of introduction

Sri Lanka

1879

England

1883

Australia

1906

Europe

1910

Sri Lanka

1910

Java

1912

Java

1929

Malaya

1938

Holland

1938

——

1943

Sri Lanka England Europe

1940 1940 1958

Philippines

1959

Europe

1961

Dispersal of Plant Pathogens

TABLE 9.2

209

Examples of plant diseases introduced to some other countries

Disease Late blight of potato caused by Phytophthora infestans Powdery mildew of grapes caused by Uncinula necator Blister rust of pine caused by Cronartium ribicola Downy mildew of grapevine caused by Plasmopara viticola American Gooseberry mildew caused by Sphaerotheca morsuvae Chestnut blight caused by Cryphonectria parasitica Citrus canker (Bacterial) Paddy blast caused by Pyricularia grisea Striga asiatica Rust of coffee caused by Hemileia vastatrix Dutch elm disease caused by Ophiostoma ulmi Fire blight caused by Erwina amylovora Golden nematode of potato caused by Heterodera rostochiensis

Introduced Home Europe

Original Home S. America

Year of Introduction 1830

England

N. America

1845

Europe

1910

France

USA

1878

England

N. America

1899

USA

Asia

1904

USA India

Asia Sri Lanka

1907 1918

N. America Brazil

Africa or Asia Africa and Asia

1970

USA

Holland

1928-30

New Zealand USA, Mexico Peru, India

N. America

1919

Europe USA

1881

USA

Agarwal et al. (1990) have listed major plant diseases introduced and established in India from other countries and have also listed destructive plant pathogens of quarantine importance not yet established in India. Man has repeatedly carried pathogens to areas which otherwise would have remained free of them. Almost all countries have suffered to such an extent from pathogens introduced by man that the Food and Agricultural Organization of the USA and several international committees of plant pathologists are promoting studies and regulations designed to reduce the danger. Man is an agent of transportation of pathogens from continent to continent, from country to country and even from plant to plant on his own farm. Eternal vigilance is necessary to protect economic plants against the pathogens distributed by man.

REFERENCES Agarwal, P.C., A. Majumdar and D.B. Parakh (1990), “Plant Pathogens of Quarantine Importance to India”, Pamphlet, published by the Division of Plant Quarantinee, National Brueau of Plant Genetic Resources, New Delhi, pp. 1–6.

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Austwick, P.K.C. (1957), Quoted in: Biological Aspects of the Transmission of Disease, Horton-Smith (Ed.), Oliver and Boyd, Edinburgh, pp. 73–79. Bawden, F.C. (1964), Plant Viruses and Virus Diseases, Fourth Edition, Ronald Press, New York, p. 361. Broadbent, L. (1960), “Dispersal of inoculum by insects and other animals including man”, in: Pl. Pathol.-An Adv. Tre., J.G. Horsfall and A.E. Dimond (Eds.), Academic Press, London and New York, III 58–83. Brown, D.J.F., W.M. Robertson and D.L. Trudgill (1995), “Transmission of viruses by plant nematodes”, Ann. Rev. Phytopath., 33:223–250. Campbell, R.N. and R.G. Grogan (1964), Acquisition and transmission of lettuce big-vein virus by Olpidium brassicae, Phytopathology, 54: 681. Carter, W. (1962), “Insects in Relation to Plant Diseases, Wiley, New York, pages pp. 705. Christensen, J.J. (1922), “Studies on the parasitism of Helminthosporium sativum”, Tech. Bull. Minn. Agr. Exp. St., vol. no. 11. Christie, J.R. and V.G. Perry (1959), “Mechanism of nematode injury to plants”, in: Pl. Pathol.- Problems and Progress, 1908-1958, C.S. Holton (Ed.), Univ. of Wis. Press, Madison, pp. 419–426. Cross. J.E. and R.S. Pitcher (1952), “Studies in relationship of Eelworms and bacteria to certain plant diseases”, Ann. Appl. Biol., 39:475. Fielding, M.J.F. (1975), “Nematodes in plant disease”, Ann. Rev. Microbiol., 13:239–254. Frazer, H.L. (1944), “Observations on the method of transmission of internal boll disease of cotton caused by cotton stainer bug”, Ann. Appl. Biol., 31:271–290. Fulton, J.P., R.C. Gergerich and H.A. Scott (1987), “ Beetle transmission of plant viruses”, Ann. Rev. Phytopath., 25: 111-124. Gaumann, E. (1950), Principles of Plant infection, (English transt.), W.B. Briesley (Ed.), Crosby Lockwood, London. pp. 543. Gregory, P.H. (1952), “Fungal spores”, Trans. Brit. Mycol. Soc., 35:1–18. Hawker, L.E. (1960), “Reproduction of bacteria, actinomycetes and fungi”, in: Pl. Pathol.-An Adv. Tre., J.G. Horsfall and A.E. Diamond (Eds.), Academic Press, London and New York, 2:117–165. Hirst, J.M. (1953), “Change in atmospheric spore content diurnal periodicity and the effects of weather”, Trans. Brit. Mycol. Soc., 36:375–393. Hewilt, W.B., N.H. Loomis, J.P. Overcash and G.K. Pavus (1958), “Pierce disease virus in Mississipi and other southern states,” Pl. Dis. Reptr., 42:207–210. Howell, P.J. and R.K.S. Wood (1962), “Some factors affecting rhododendron bud blast and its control”, Ann. Appl. Biol., 50:723. Hughes, S.J. (1953), “Conidiophores, conidia and classification”, Can. J. Bot., 31:577–659. Ingold, C. (1971), Fungal Spores—Their Liberation and Dispersal, Clarendon Press, Oxford. pp. 302. James, G.L. (1968), “The interrelationship of the causal fungus of brown root rot of tomatoes and potato root eelworm, Heterodera rostochiensis Woll”, Ann. Appl. Biol., 61:503. Leach, J.G. (1940), Insect Transmission of Plant Diseases, McGraw-Hill, New York. Madelin, M.F. (1966), “Fungal parasites of insects”, Ann. Rev. Entomol., 11:423–448. Madelin, M.F. (1968), “Fungal parasites on invertebrates”, in: The Fungi, G.C. Ainsworth and A.S. Sussman (Eds.), Academic Press, London and New York, 3:253–269. Mehta, K.C. (1943), “Further studies on wheat rust in India”, Sci. Monog. Coun. Agr. Res. India, 14:224. Muskett, A.E. (1960), “Autonomous dispersal”, in: Pl. Pathol, J.G. Horsfall and A.E. Dimond (Eds.), Academic Press, London and New York, 3: 57–96. Nagarajan, S. and H. Singh (1973), “Satellite television cloud photography as a possible tool of plant disease spread”, Curr. Sci., 42: 273–274. Nagarajan, S. and H. Singh (1974), “Satellite television cloud photography—a new method to study wheat rust dissemination”, Indian J. Gen., 34(A):486–489. Nagarajan, S. and H. Singh (1975), “Indian stem rust rules-an epidemiological concept on the spread of wheat stem rust”, Pl. Dis. Reptr., 59:133–136.

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Nutman, F.J., F.M. Roberts and K.R. Bock (1960), “Method of uredospore dispersal of coffee leaf rust fungus Hemileia vastatrix”, Trans. Brit. Mycol. Soc., 43:509–515. Pitcher, R.S. (1965), “Interrelationships of nematodes and other pathogens of plants”, Helminthological Abstract, 34:1–17. Powell, N.T. (1963), “The role of plant-parasitic nematodes in fungal diseases”, Phytopath., 53:28–35. Powell, N.T. and C.J. Nusbaum (1960), “The black shank root-knot complex in blue-cured tobacco”, Phytopath., 50:899. Prabhu, A.S. and R. Prasada (1966), “Pathological and epidemiological studies on leaf blight of wheat caused by Alternaria triticina”, Indian Phytopath., 19:95–112. Richardson, D.E. and P.J.W. Saunders (1968), “Possible roles of aphids in the dispersal and incidence of late blight of potatoes”, J. Natl. Inst. Agr. Bot., 11:343. Schrodter. H. (1960), “Dispersal by air and water-the flight and landing”, In: Pl. Pathol.-An Adv. Tre., J.G. Horsfall and A.E. Dimond (Eds.), III:169-227. Smith, A.L. (1953), “Fusarium and Nematodes on cotton”, In: Yb. Agr. US Deptt. Agr., pp. 292–298. Stakman, E.C. and J.G. Harrar (1957), Principle of Plant Pathology, Ronald Press, New York. Stewart, R.N. and A.F. Schindler (1956), “The effect of some ectoparasitic and endoparasitic nematodes on the expression of bacterial wilt in carnation”, Phytopath., 46:219–222. Waite, M.B. (1891), “Results from recent investigation in pear blight”. Bot. Gaz., 16:259. Yarwood, C.E. (1936), “The diurnal cycle of the powdery mildew Erysiphe polygoni”, J. Agr. Res., 52: 645–657.

QUESTIONS 1. What is the essential difference between distribution and dissemination? 2. Give some examples of viral diseases which are carried in the seeds. 3. Give examples of plant diseases in which dissemination of the plant pathogens is dependent upon special types of insects. 4. Which insects act as diploidizing agents for some heterothallic fungi? 5. Name the diseases which have been introduced in India from other countries.

10 Plant Disease Epidemiology and Plant Disease Forecasting

INTRODUCTION

The forecasting of plant diseases has become considerably important in the present day situation in India and other agriculturally advanced countries of the world due to large-scale adoption of new agricultural strategies for increasing food production. The new approach which involves use of fertilizers, intensive cropping, change in cultural practices as well as in cropping pattern, and cultivation of high-yielding varieties has no doubt paid rich dividends by increasing the production but has also disturbed the natural agrosystem. This has resulted in the increased incidence of diseases, which sometimes results in heavy losses or even near-failure of crops. Modern agriculture being cost oriented requires greater vigilance than before to ensure stable and good quality yields and for reducing expenditure on all types of inputs including chemicals for disease management. This is possible only if reliable disease forecasting systems are developed at least, for some of the destructive diseases of major crops (Bedi, 1986). Forecasting the future was one of most rewarding professions in ancient times. A forecaster was considered a prophet, a saint and often referred to as jyotishi and the prophet had his troubles. Prediction, forecasting, prognosis, forewarning or foretelling has been used in the literature of plant pathology. The word prognosis is used mainly to describe the expected progress of disease in humans. Taken literally it means knowing beforehand. Forecasting means to foresee or to calculate beforehand. Thus, the calculation of probabilities is implicit in the meaning of any word related to forecasting. Gaumann (1950) defines epidemic as “the massive occurrence of a disease in a limited time period” as influenced by the large number of factors, that interact with this increase. Van der Plank (1963) defines epidemiology as the science of disease in population. But according to Kranz (1973) epidemiology is the science of the population of host disease resulting under the influence of environment and human interference. Later, Kranz (1974) defined it as the science that deals with the increase or decrease of plant disease in a host population in time and space. The ultimate aim of the science of plant pathology is to have a complete understanding of plant diseases—host, pathogen, and environment interactions—so that it becomes possible to control diseases economically. The most efficient and most economical way to control diseases is usually to obtain a

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thorough knowledge of the disease epidemiology. We can not effectively control certain diseases without some prior indication as to how they will behave in the coming period that is, without some knowledge as to where and when they will occur, which will enable preventive action to be taken. Unless we know where and when the sprays or dusts are to be applied we are likely to waste them. For certain diseases, no effective or practicable chemical control measures are available. Forecasting gives us an opportunity to revise our plans in order to avoid or reduce losses. The practical implications of forecasting have been incorporated in the definition proposed by Miller and O’Brien (1952) who state: “Forecasting involves all the activity in ascertaining and notifying the growers of a community that conditions are sufficiently favourable for certain diseases, that application of control measures will result in economic gain, or on the other hand, and just as important that the amount of disease expected is unlikely to be enough to justify the expenditure of time, energy and money for control”. Miller and O’Brien thus include a warning service for plant disease prevention in their definition of forecasting. There are excellent reviews and books covering this aspect and related areas, such as those of Bourke (1953) and Cox and Large (1960), concerned with the forecasting, from weather data, of potato blight and other plant diseases and pests. Bourke (1970) has dissertated on the “Use of weather information in the prediction of plant disease epiphytotics,” Miller (1967) on “Plant disease epidemics — their analysis and forecasting”, Miller and O’Brein (1957) on “Prediction of plant disease epidemics”, Waggoner (1960) on “Forecasting epidemics and Ground level climate and disease forecasting”, Krause and Massie (1975) on “Predictive systems—modern approaches to disease control”, Shrum (1978) on “Forecasting of epidemics”. Some recent publications during the last two decades are those of Zadocks and Stein (1979) Epidemiology and plant disease management, Zadocks (1984) “A quarter century of disease warning”, Fry and Fohner (1985) “Construction of predictive models”, Leonard and Fry (1986) “Plant disease epidemiology” Vol. I and II, Campbell and Madden (1990) Introduction to plant disease epidemiology, Madden and Hughes (1995), “Plant diseases incidence: distribution, heterogenicity and temporal analysis”, McCartney (1996), “The influence of environment on the development and control of diseases.” In our country Nagarajan (1983) has critically discussed the epidemiology of plant diseases in a comprehensive book entitled, Plant Disease Epidemiology. Bedi (1986) has reviewed the work on important plant diseases and their forecasting while Mayee and Datar (1988) have reviewed the work on forecasting crop diseases in India till then. Aggarwal and Mehrotra (1991) have compiled information on Phytophthora diseases in India in relation to environmental factors. Forecasting the incidence of plant disease must take into account not only the disease itself but the weather also, which is more or less predictable. Disease-forecasting is made more reliable if the reasons for a particular disease developing under certain conditions and not under others are known. Importance of Disease Forecasting Services

Plant pathologists are interested in forecasting for three major reasons: 1. Disease forecasts are useful to farmers in the field in the practical management of disease of their crop, 2. Research aimed at accurate forecasting helps us to identify or pinpoint the gaps existing in our knowledge and

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3. Modern research on modelling the complexities of epidemics is stimulating and rewarding in the long run. The ultimate goal of plant pathology is to minimize losses due to plant diseases. As pointed out by Main (1977), the farmer must balance three factors in disease management: risk, cost, and benefit. Here, forecasting can be useful if the forecaster suggests that the risk of disease is great, there is big advantage in spraying or using resistant varieties which may be of moderate yield. Forecasting provides a complex tool for determining when and where a given management practice should be applied. SOME IMPORTANT EPIPHYTOTICS OF THE PAST AND PRESENT

The Bible, ancient Hebrew and Sanskrit literature all refer to plant diseases. During each spring in the Roman empire, the elaborate ceremony of Robigalia was carried out to satisfy the ‘Rust God’ and thus stop causing cereal rust epidemics. So disease epidemics have been known to mankind since time immemorial. The great potato famine in Ireland that followed the late blight epiphytotic of 1845 is undoubtedly one of the landmarks of the 19th century. The crucial role played by Phytophthora infestans in that episode is well known to each and every mycologist and plant pathologist. Epidemics of plant diseases have influenced man’s food, health, social customs, economics, and even his ability to wage war. Horsfall and Cowling (1978) in one of their reviews analyzed the impact of plant disease epidemics such as ergotism of rye, late blight of potato, Dutch elm disease, coffee rust, helminthosporiose of rice, peach yellow, chestnut blight, wheat rust and maize leaf blight. The coffee rust problem of Sri Lanka that made her switch over to tea; the banana wilt of central America; downy mildew of grapes of France and parts of Europe, rice tungro outbreak in Indonesia are first a few random samples from the hundreds of plant disease epiphytotics (epidemics) of the world. In most cases, there was lack of epidemiological information to give any protection-oriented recommendation at that time. Nagarajan (1983) had a close look at some of the epidemics that swept parts of India and the resultant socio-economic problems. The first example of an important plant disease epidemic in India was the stem rust of wheat. Quoting Major Sleeman (1839), “Old and respectable landlords in the district of Jabalpur state, that is Sambut 1843 (A.D.1786), wheat crops throughout the district were destroyed by the same calamity, and that the Saugor Government (MP) obliged not only to remit the revenue, but to provide a supply of wheat grains for almost every village by advances from the public treasury”. So wrote Major Sleeman (1839) about the oldest known stem rust epidemic, P. graminis tritici. The second one is of Helminthosporium blight of paddy. In 1942, there was a fall in rice production in Bengal due to the wide spread epidemic created by Helminthosporium oryzae. India which was exporting wheat prior to World War 1, was no more on the list of exporters, her production falling short of demand. The administration as usual, ignored the notes and files. The easily avoidable human suffering got no attention, as life and property everywhere was unsafe due to the war. The acute shortage drove many to starvation and death—the Great Bengal Famine swept across Bengal and Bangladesh (Padmanabhan, 1973). Turning the pages of history reveals that there was an isolated epidemic of H. oryzae during 1918–1919 in the Krishna delta of Andhra Pradesh (Sundararaman,1922). The weather conditions recorded during that epidemic year seem to be similar to those during the 1942 epidemic.

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There are many other epidemics that India witnessed, such as red rot of sugarcane (Collectrotrichum falcatum), downy mildew of pearl millet (Selerospora graminicola), the present apple scab (Venturia inaequalis) epidemic in Himachal Pradesh (H.P.) and Kashmir (J.K.) and bacterial blight (Xanthomonas campestris pv. oryzae ) in the rice belts. EPIPHYTOTIC GROWTH AND ANALYSIS

The interactions of the structural elements of epidemics as influenced over time by factors of the environment and by human interference, are expressed in patterns and rates. The pattern of an epidemic in terms of the numbers of lesions, the emergence of disease tissue, or the number of diseased plants is given by a curve that is called the disease-progress curve. The point of origin and the shape of diseaseprogress curve reveal information about the time of appearance and amount of inoculum, changes in host susceptibility during the growing period, recurrent weather events, and the effectiveness of cultural and control measures. If the epidemic growth curve is plotted it resembles the English alphabet ‘S’ and is called a ‘sigmoid curve’. The shape of the curve depends upon the level of initial inoculum and the growth rate of the disease. For example, a saturation-type curve is characteristic of monocyclic diseases, a sigmoid curve is characteristic of polycyclic diseases, and a bimodal curve is characteristic of diseases affecting organs (blossoms, fruit) of the plant. Knowledge of disease progress curves also allows disease forecasting and selection of the best control strategy for the particular disease and time. Vander Plank (1963) broadly classified disease into two categories, simple interest and compound interest types. Aerial-borne pathogens such as cereal rusts start from a few lesions and by virtue of their heavy sporulation, disease growth is similar to compound interest paid by a bank. It the case of simpleinterest type of disease, growth is discontinuous, fluctuating both on the upper and lower sides. The simple-interest type of disease spends one generation on that crop and the second cycle is completed on the next seasons crop. Meanwhile, the environment and host factors interact and decide the level of disease the on the succeeding crop. Seldom do such diseases follow the sigmoid growth curve to reach epidemic proportions. Wheat smut (Ustilago segetum tritici) and Karnal bunt (Neovossia indica) are good examples of simple-interest type of disease growth. COMPUTER SIMULATION OF EPIDEMICS

The availability of computers has allowed plant pathologists to write programmes that allow simulation of epidemics of several plant diseases. In 1969, the first computer simulation programe called EPIDEM was developed and resulted from modelling each stage of the life cycle of a pathogen as a function of the environment. This (EPIDEM) was designed to simulate early blight of tomato and potato caused by Alternaria solani. Later on computer simulations were written for Micospherella blight of chyrsanthemums (MYCOS), for apple scab caused by Venturia inaequalis (EPIVEN) and for southern corn leaf blight infected by Helminthosporium maydis (EPICORN). BLIGHTCAST (Krause et al., 1975) for late blight of potato caused by P. infestans is a computer programme written in Fortran IV developed in the USA. This was a synthesis of the Hyre and Wallin’s fungicide scheduling systems developed for the northeastern and mid-western US. BLIGHTCAST is based on predicting periods when infection is likely to occur. Spray recommendation were issued in two

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different triggers: after 10 consecutive days when the cumulative rainfall exceeded 30 mm and the 5 day average temperature did not go beyond 25.5oC, or after an accumulation of 18-20 “severity units”. Each day was scored on a 0-4 “severity” scale according to temperature and hours with relative humidity more than 90%. TOM-CAST (Pitbaldo, 1992)—Modern et al. (1978) developed a computer based forecasting system for tomato early blight (FAST). This was modified by Pitbaldo (1992) in Southern Ontario in Canada for use in that region. TOM-CAST, identifies periods when environmental conditions are favourable for disease development and recommends a fungicidal application schedule. TOM-CAST required direct measurement of wetness duration in the field which was read by wetness sensors and data collecting systems such as those used with BLIGHTCAST. PLASMO (Rosa et al.,1993, 1995) —This is a forecaster developed in Northern Italy to control downy mildew of grapes caused by Plasmopara viticola. It is a very sophisticated computer programme which simulates the critical phases in the life cycle of the pathogen using rate factors determined by environmental parameters (temperature, relative humidity, rainfall and leaf-wetness duration). Conventional control of the disease is done by regular spray of fungicides at 7-to-10 day intervals. In addition to the above, some recent disease forecasting/warning simulation models have been developed which include Pyricularia grisea on rice (Calvero et al., 1996), Erysiphe graminis f. sp. tritici on wheat (Friedrich,1995). In a computer simulation of an epidemic, the computer is given data describing the various subcomponents of the epidemic and control practices. The computer then provides continuous information regarding not only the spread and severity of the disease over time, but also with regard to the final crop and economic losses likely to be caused by the disease under the conditions of the epidemic as given to the computer. The computer simulation of epidemics is extremely useful as an educational exercise for students of plant pathology and also for farmers so that they can better understand and appreciate the effect of each epidemic subcomponent on the final size of their crop loss. The computer evaluates not only the current status of the disease but also the effectiveness of applied management measures in controlling the epidemic. Computer simulation of epidemics is used fairly widely in plant diseases forecasting systems. Such systems allow farmers to take appropriate control measures against a disease as soon as the conditions that are likely to lead to disease development are present. SYSTEMS APPROACH IN EPIDEMIOLOGY

We need to develop forecasting procedures for most plant diseases which can handle all significant components of an epidemic – the pathogen, the host, the weather, the time and perhaps space. The first job of a systems analyst at the conceptual stage is to recognize the problem itself, its limit and the objectives. Conceptual models are the first step in model-building and aim at sensibly arranging available information or processing thoughts for a critical analysis. The conceptual models show the sequence of operations and the decision-making level. It also gives an empirical idea of the system and the factors that interact, influencing the system’s behaviour. Joshi et al. (1974) attempted to correlate disease, weather, time and space relationships for rusts of wheat. In field studies to establish the reasons for the non-functional nature of wheat stem rust over northwest India, Joshi and Palmer (1973) took to geophytopathology. Spread of wheat black, brown and yellow rusts from a point source was mapped over a period of time, at Karnal, Haryana. Their primary aim was to demonstrate that even if stray stem rust uredospores survive in the Himalayas, which is otherwise definite, the disease will not spread. Meanwhile, the continued cold weather of North India favours the brown and yellow rusts of wheat. The

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disease gradient from the point source clearly showed that stem rust spread is the poorest between January and March. A well planned and laid out geophytopathological study can therefore reveal many facts that are apparently unclear. As explained earlier, weather is an important factor that influences disease development. Where the host and pathogen coincide, weather is the only variable that influences epidemic development. Therefore, by monitoring relevant weather parameters and relating them to disease development, empirical prediction models can be developed. Everdingen’s Method

Van Everdingen (1926) in the Netherlands, apparently was the first to base his forecasting system for late blight of potato on scientific principles. His forecast was based on what became known as the ‘Four Dutch Rules’. These rules were based on four weather parameters : 1. Minimum temperature (night temperature must not be lower than 10oC (50oF) ). 2. Dew (leaves must remain wet for at least 4 hours the day following a night dew). 3. Cloudiness (the day following the night dew must be cloudy or with no less than 0.8 of the sky covered by clouds). 4. Rainfall (during such periods described above there must be at least 0.1 mm of rainfall). When all these conditions are satisfied under field conditions continuously for a few days, then in the Netherlands, control measures are recommended. Beaumont and Stainland’s Method

Beaumont and Stainland (1934, 1938) modified the Dutch rules for England and evolved Beaumont’s rules based on: 1. A minimum temperature of 10oC (50oF) or more. 2. Relative humidity of 75% or above for 2 consecutive days. The blight could be expected approximately 10 days afterwards. These periods are called Beaumont periods and are the basis of the blight forecasting system in England and Wales. Cook’s Method

Cook (1947, 1949) in the United States used 7-day moving average total rainfall and average mean temperature charts for blight forecasts. Cook (1949) studied the relationship of temperature and rainfall during the preceeding 17 years to blight on potatoes and tomatoes in Eastern Virginia. He found that the critical period for blight development in that area started on 8th May. The cumulative mean rainfall line starting on 8th May was plotted for blight and non-blight years and a median line was designated the critical rainfall line. The critical temperature was taken as 75oF. Less epidemics were likely to follow a two week period when rainfall was continuously above the line and temperature below 75oF. Later Cook included the analyses for 31 years (1917 to 1947).

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Hyre’s Method

Hyre (1954) developed criteria for the northeastern region of the U.S.A. which were essentially the same as Cook’s excepting that a 10-day moving graph method was followed and which according to him was more accurate than the 7-day moving graph method (Cook, 1949). Wallin and Hoyman’s Method

The forecasting system very much successful in the Eastern United States for prediction of blight in advance and development did not seen to be applicable in the Midwest (U.S.A.). Hyre and Horsfall (1951) attributed the difference to variations in the occurrence of primary inoculum from year to year in the reasonably blight-free areas of the Midwest. According to Wallin and Hoyman (1954) a late blight favourable period consists of 10 or more consecutive hours of temperature of 75oF or less and RH 90% or more and if during a given 7-day interval there was at least one such period with the following days, maximum temperature 90oF or less. The more frequent the occurrence of the favourable periods, the greater was the expected blight development. Hyre and Bonde’s (1955) Method

This is a modification of Cook’s (1949) and Hyre’s (1954) method of a 7-day moving graph in which a 10-day total rainfall and a moving 10-day average mean temperature are considered suitable. In this case also the critical rainfall line is calculated on the basis of total crop season as above. In India Choudhuri and Pal (1959) compared Cook’s (1949) and Hyre’s (1954) moving graphs of temperature and relative humidity at Darjeeling and observed that the 7-day moving graph was more accurate. Later correlations were worked out by different methods between weather parameters and the disease appearance for Shimla, Shillong and Ootacamund by Bhattacharya et al., (1982). This forecasting system so developed for the hills is still working well for the country. The system developed by Bhattacharya et al., is given in somewhat greater detail later in this chapter. The need for a warning service for the late blight of potato in the USA was evident long before a cooperative project was established. Moreover, a considerable amount of experience of various groups had already shown that the appearance and spread of late blight and some related fungi could be successfully anticipated, provided that means for watching and reporting progress were available. The event that finally led to the establishment of the regional forecasting project was the severe, widespread and unexpected epidemic of late blight of tomato in 1946. The project was organized to include a warning service and contributory research for three downy mildew diseases, late blight of potato and tomato, blue mould (Peronospora tabacina) of tobacco, and downy mildew (Pseudoperonospora cubensis) of cucurbits. In the whole of North America all the way into Canada, the seasonal programme of these crops is particularly well adapted to observations and predictions on a widespread regional basis. Canadian pathologists cooperate in the warning service. The appearance and spread of late blight of tomato and potato, blue mould and downy mildew of cucurbits are promptly reported to the Plant Disease Epidemics Unit at Beltsville, Maryland. The reports are compiled into a warning letter sent twice a week during the growing season to pathologists in

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the various states, Canadian provinces and other authorized recipients. By watching the occurrence and severity of disease in areas with an earlier growing season and applying their own previous experience, together with the weather forecasts, the key pathologists are able to judge rather accurately whether or not the local crops are likely to be affected, and if so, how soon and how seriously, and can make their forecasts accordingly. Manufactures of control chemicals and equipment also use the warning letters as a guide to allocate their products. Forecasting and warning systems involve considerable expense and should be economically justifiable, as in the following cases: 1. For destructive diseases of important cash or food crops. 2. For diseases which can be reliably predicted early enough for effective and economical control measures to be applied. 3. For diseases against which economical control measures are available. METHODS USED IN PLANT-DISEASE FORECASTING

Disease forecasting allows the prediction of probable outbreaks or increases in intensity of disease and, therefore, allows us to determine whether, when and where a particular management practice should be applied. It is also useful to farmers in the practical management of crop disease. Most frequently, farmers need forecasts of plant disease development to decide whether to spray a crop right away or to wait for several more days before they spray, since, if they can wait, they can reduce the amounts of chemicals and labour used without increasing the risk of losing their crop. In developing a plant disease forecast, one must take into account several factors of the particular pathogen, host and environment. The methods used in disease forecasting mostly fall into 5 main groups based on: 1. Weather conditions during the intercrop months particularly those affecting the survival of inoculum. 2. Weather conditions during the crop. 3. The amount of disease in the young crop. 4. The number of propagules of the pathogen in the air, soil or planting material 5. Changes in the host physiology. Weather-based Predictions

When the importance of weather conditions in determining the host-pathogen interaction was realized, prediction based on weather conditions were frequently used in forecasting diseases. According to Shanta (1960), the severity and subsequent spread of tikka disease of groundnut caused by Cercospora personata (now Cercosporidium personatum ) and C. arachidicola depend upon the prevalence of high humidity and low temperature during crop season. According to Jensen and Boyle (1966), the chances of rapid development of an epidemic of Cercospora leaf spot of peanut are optimum when the foliage remains wet for a period greater than 10 hours or above and minimum temperature is 21oC or higher for the next 24 hours. Padmanabhan (1965) examined the meteorological factors associated with the epiphytotics of Helminthosporium oryzae involved in the ‘Great Bengal Famine’ of 1942. It was revealed that heavy

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rainfall in September accompanied by a favourable temperature of 25 to 30oC, followed by cloudy weather in subsequent months resulting in low solar radiation were the critical conditions for outbreak of brown leaf spot of rice. Padmanabhan (1965) used weather analysis for predicting outbreaks of blast of rice (earlier Pyricularia oryzae, now P. grisea). Nycto-temperatures ranging from 20-24oC in association with 90% relative humidity or above lasting for a week during any susceptible phase of crop brought an epidemic of blast. Nagarajan et al. (1980) correlated the epidemics of wheat rusts of India with the prevalence of certain environmental conditions at certain places in the country during crop season. According to them, epidemic of leaf rust (Puccinia recondita) of wheat is likely to develop if ; 1. Rust infections are observed in Uttar Pradesh and Bihar between 15th and 20th January at 5-6 different locations separated from each other by a minimum distance of 25 ± 5 km; 2. The number of rainy days during January to mid April over northwest India is at least twice the normal number that is 12 days, and 3. The weekly mean maximum temperature during March to mid April over northwest India, is within 1oC of the normal temperature (26oC). If the first and the second conditions exist fully, then a severe epidemic of leaf rust is likely to occur. If they exist partially, isolated outbreaks of the disease are possible. If the mean maximum temperatures are usually low, stripe rust is likely to become a serious problem throughout northwest India. Similarly Payak et al., (1970) found that the brown stripe downy mildew of maize caused by Sclerophthora rayssiae var. zeae was severe in areas where mean precipitation was more than 190 cm. In areas of low rainfall, the disease would develop under conditions of continuous rain, cloudiness and low temperatures of 24oC or less for a few days. Saxena and Lal (1984) developed a linear equation based on sunshine hours and rainfall for prediction of Erwinia stalk rot of maize. Saharan and Singh (1980) found that the latent period of Melampsora lini varied greatly and the variation can be predicted by relative humidity. Mayee (1986) developed a forecasting system based on temperature for groundnut rust epidemics in central India. Mayee and Ekbote (1983) noted that the inoculum of rust was available throughout the year as also the suscept. However, highest infection rates occurred only during rainy season. Factors such as mean temperature, relative humidity, and number of rainy days can predict not only the sub-phases of infection chains but also the disease development . Gupta (1992, 1995) listed various forecasting procedures for apple scab, a disease where sufficient work has been done on forecasting. In many countries, electronic scab warning instruments have been developed for monitoring the ambient temperature, duration of leaf wetness and relative humidity above 90 percent for predicting infection periods. Inoculum-based Prediction

Forecasting of many diseases is based on the estimates of amount of initial inoculum likely to infect the host plant at the beginning of a crop season. In many cases there is a positive correlation between the amount of primary inoculum in vicinity of host plant and severity of infection. To quote Shrum (1978) “the forecast based on inoculum potential contains a hidden assumption that the weather will continue to be favourable for disease”. If weather changes, the forecast may be inappropriate and the degree of error will be proportional to the degree of change. Plant disease forecasting can be based on the amount of inoculum available in air, soil or planting material. Utility of inoculum potential-based forecasting can however be found for a number of soil-borne diseases.

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The severity of apple scab caused by Venturia inaequalis is related to the amount of perithecia surviving in dead leaves and the subsequent amount of ascospores liberated from them. The forecasting of apple scab epidemic is mostly based on temperature and moisture conditions, affecting the discharge of ascospores from perithecia at full-bloom stage of the crop. The discharge of ascospores takes place when perithecia are thoroughly wetted and the infection takes place 30 hours after leaf wetness at 5°C, 14 hours at 10°C or 9.5 hours 15°C. A system for predicting blister blight of tea caused by Exobasidium vexans has been developed which is based on the number of spores in the tea-garden in the air and the duration of surface wetness on the leaves. It was found empirically, by observation that the number of basidiospores of the pathogen in the air at a given time was related to the percentage of shoots which were infected. The duration of sunshine is negatively correlated with the duration of surface wetness of the leaves. Therefore, the duration of sunshine was used as a measure of surface wetness indirectly. A prediction equation for the development of the epiphytotic has been developed. In India, very limited efforts have been made to develop predictions based on inoculum. Murlidharan and Rao (1981) developed a rice blast warning with the help of trap plots. Shukla and Anjaneyulu (1992) attempted to correlate the rice tungro development with vector population and initial infection. Bedi and Saini (1979) showed that the severity of early blight of tomato caused by Alternaria solani was positively correlated with the concentration of inoculum sprayed on the crop. In India the plant disease forecasting service is in its incipient stage. For forecasting rust epidemics, a number of rain samplers have been set in stations where facilities exist for their handling. The rain analysis gives precise data on the advent of the inoculum in a particular locality. Based on this information as well as data collected by survey teams from time to time, a large number of wind trajectories have been prepared. The air-borne inoculum and its deposition from the southern hills has been traced by wind trajectories and weather satellite cloud images. If ground level conditions are favourable for infection, the early arrival of inoculum can lead to severe rust epidemics (Nagarajan and Singh, 1975). In the case of brown stripe downy mildew caused by Sclerophthora rayssiae var. zeae, Payak et al. (1970) have observed that the disease is severe in areas in India where the average rainfall is 100-200 cm or more. In areas of low average rainfall, the increase in disease severity may occur only if there is a continuous spell of heavy showers and cloudiness for a few days accompanied by lowered temperature (25°C or less). Brown leaf spot of rice is caused by Helminthosporium oryzae. Certain abnormal trends have always been associated with a severe outbreak of Helminthosporium disease. For instance, according to Sundararaman (1922), usually heavy rains at the time of ear formation followed by flooding and illdrained conditions had favoured the epiphytotic of 1918-19 in the deltaic tracts of the Krishna and Godavari. Padmanabhan (1963) studied the meteorological factors associated with the epiphytotic of 1942 in Bengal. A comparative examination of the meteorological data of 1942 with those of the non-epiphytotic years of 1943 and 1944 revealed that in 1942 there was heavy rainfall in September accompanied by a favourable temperature variation of 25–30°C, followed by continuous cloudy weather during October and November, a higher minima than usual in November as well as low solar radiation in this month. Aeroscope studies carried out by Chandwani et al. (1963) had shown that it was precisely under these conditions that an abnormally large number of spores were reported on the slides.

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In the case of bacterial blight of paddy, our knowledge of the relationship of the environment to the disease appears to be based on field observations rather then on critical studies under controlled conditions (Srivastava, 1972). The bacterial blight of paddy develops readily above 25°C and symptoms may not appear below 20°C. Goto et al. (1955) have reported that a combination of rainy weather, strong winds and a temperature of 22–26°C favour the outbreak of the disease. Ramalingam (1970) has discussed the results obtained in Andhra Pradesh in the development of rice blast. He mentioned that a temperature range of 25-27 oC, relative humidity of 86-90%, mild winds and darkness (midnight to sunrise) were critical for the development of epiphytotic. Rice crop is most susceptible to blast at three stages of crop growth, viz. seedling stage beyond 21 days, post-transplanting stage, that is, 25–50th day after transplanting and at flower emergence. When these three stages coincide with favourable environmental factors, blast outbreaks occur. It has been determined as a result of a study made at the Central Rice Research Institute, Cuttack, Orissa, that a minimum temperature of 20–24°C and a relative humidity above 90% occuring in the morning hours are very favourable for an outbreak. When such conditions continue to prevail for two days or more during the susceptible stages of the crop, as indicated above, a blast outbreak is expected. Thus with the help of meteorological data, the outbreak of blast can be forecast (Padmanabhan, 1950). Govindswamy (1970) stressed the need for recording the number of low temperature days (20°C and less) in forecasting blast of rice. Host Physiology-based Prediction

According to Grainger (1968), the physiology of the host plant keeps changing in response to environmental conditions to which it is exposed during its growth. The study of physiology of plant is important for forecasting plant-disease epidemics. Grainger’s (1968) studies with various host-pathogen systems have explained that: 1. Pathogens attack their host plant at a certain period of its growth. For example, Phytophthora infestans, may cause severe infection of either sprouts on tubers or crop at its later stage of growth. Potato plants during the intermediate stage of their growth do not show much susceptibility to the pathogen. 2. The host plant growing under normal conditions usually has sufficient nitrogen and mineral matter for the requirement of the attacking fungus, but at certain stages of its growth there is not sufficient spare carbohydrates for the fungal pathogens needed for their growth and reproduction. At this stage of its growth, the host plant is generally less susceptible to the attacking fungus. 3. Leachates from various plant tissues are known to exert an influence on growth, reproduction and other activities of microorganisms in the environment of a plant. According to Tukey (1970), if the quality of leachates from roots and other plant tissues can be determined or regulated in relation to prevailing weather conditions, it may be possible to forecast the occurrence of many diseases caused by soil-borne and air-borne pathogens. EXAMPLES OF PLANT DISEASE FORECASTING SYSTEMS

Here we shall discuss some Indian attempts at plant disease forecasting.

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Predicting Rust Epidemic in India

Nagarajan and Singh (1975) formulated ‘Indian Stem Rust Rules’ for Puccinia graminis tritici. These rules explain satisfactorily whether the primary inoculum from South India would travel tens of hundreds of kilometers before its deposition in central India. The stem rust prediction methods proposed by them consist of the following steps. 1. Check if there were any rains during November in central India coupled with southerly winds. 2. Check the urediniospore content of the rain. 3. Check if the Indian stem rust rules are satisfied. If a tropical cyclone occurred during November (Rule ‘a’ of the Indian stem Rust) disease would appear near the area where cyclone dissipated. If the inoculum arrives early, the disease can develop into severe proportions. If rainfall satisfying the weather conditions of Rule ‘b’ or ‘c’ occurred during January or February, only traces of the disease would appear; that is too late in the crop season to be of serious threat. 4. Check the weather over the area of urediniospore deposition. 5. Check the viability of the transported spores, that is, if the trajectory for 700/850 m bar passed near the south foci within 120 hours, spores would be viable. 6. Check if the ground level conditions following the deposition by rain, are favourable for infection or not. If all the conditions are satisfied, the first detectable infection could occur in 25 to 30 days at locations close to where the cyclone dissipated. Once infections have occurred in central India, their further spread can be predicted following the same rules with the deletion of step ‘a’ and ‘c’. Further spread occurs to the east and north of India from these established foci. More the number of foci, greater are the chances of epidemic development. Based on this Nagarajan et al. (1978) developed an empirical model, given below to predict if brown rust if wheat can become severe or not. 1. At least five to six infection sites should be observed at a minimum distance of 25 ± 5 km around 15–20 January during the survey of wheat crop in Uttar Pradesh and North Bihar. 2. Amount and number of rainy days during January to mid-April over Northwest India should be twice the normal. 3. Over northwest India weekly mean maximum temperature during March and mid-April should be normal ± 1oC. If the first and second steps are satisfied, then a severe epidemic will occur. But if all of them are partly satisfied then chances are that isolated epidemics can occur. On the other hand, if the temperature is low then in addition to brown rust, yellow rust also would appear aggravating the situation. Predicting Late Blight of Potato in India

Forecasts of potato blight have been made ever since the disaster of 1845. Bhattacharya et al. (1982) had developed methods/rules for late blight of potato under India conditions. According to Bhattacharya et al. (1982), under Indian hill conditions, rainfall is the primary factor for the generation of conducive condition for late blight. Bhattacharya et al. (1982) worked out a correlation between the weather data and the actual data of appearance of the disease under Simla (15 years), Shillong (7 years) and Ootacammund (5 years) conditions. It was established that:

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1. If the day moving precipitation (30 mm for Simla, 28.9 mm for Ootacammund and 38.5 mm for Shillong which were observed to be the critical rainfall lines) associated with a mean temperature of 23.9oC or less continues for 7 consecutive days the potato late blight would appear with in 3 weeks and 2. If hourly temperatures range within 18-20oC and are associated with high RH (80% or more) for 18 continuous hours for 2 consecutive days, the potato late blight will appear within a week. Accordingly a warning service for spraying fungicides on potato crop against late blight has been initiated in the Northern India hills and Nilgiris. Establishment of disease-forecasting services in India is a must for important diseases of vegetables, fruits and food crops. In India, priorities have to be decided on type of diseases that need to be considered for forecasting. For the development of reliable forecasting systems extensive laboratory and field studies are required for understanding the behaviour of the pathogen and the host populations under varying environmental conditions. Forecasting of disease epidemics is still in its infancy in India. There is a great scope and necessity to develop this branch which is, as pointed out in the text, is not only useful to farmers but also intellectually stimulating. No forecasting system at present in use is perfect. With the advent of instruments and gadgets for analysing various environmental variables, forecasting has become more accurate. The problem remains of significant factors which are not measured. Both macroand micro-climate are important and adjustments here to be made to monitor and predict the disease or epiphytotic depending on the disease and the host-pathogen combination.

REFERENCES Aggarwal, A. and R.S Mehrotra (1991), “Environmental studies on certain important Phytophthora species in India”, In: Recent Advances in Environment and Biotechnology (New Forntiers of Plant Pathology), Vol. 2 B.P. Singh, H. N. Verma and K.M. Srivastava (Eds.) Today and Tomorrow’s Printers and Publishers, New Delhi, pp. 27–36. Beaumont, A and L.N. Stainland (1934), Tenth Ann. Rep., Scale-Hayne Agr. College, Abbot, Devon, England for the year ending, Sept. 30, 1933, pp. 39. Beaumont, A. and W.E.H. Hodson (1930), Sixth Ann. Rept., Seale–Hayne Agr. College, Devon, England, p. 28. Beaumont, A. and L.N. Stainland (1937), Ann. Rept., Seale-Hayne Agr. College, Devon, England, p. 48. Beaumont, A. and L.N. Stainland (1938), Fourteenth Annual Report, Scale–Hayne Agric. College, Devon, England, for the year ending September 30, 1 : 937. Bedi, P.S. (1986), “Plant Disease Forecasting”, in: Vistas in Plant Pathology, A.Varma and J.P.Varma, (Eds.), Malhotra Publishing House, New Delhi, pp. 249–261. Bhattacharya, S.K., D.S. Singh and R.N. Khanna. (1980), “Epidemiology and forecasting of Phytophthora infestans inciting potato late blight”, in: Proceedings of the workshop on Phytophthora diseases of tropical cultivated plants, K.K.N. Nambiar (Ed.), Central Plantation Crops Research Institute, Kasaragod, Kerala, pp. 99–101. Bhattacharya, S.K., D. S. Singh and R. N. Khanna (1982), “Epidemiology and forecasting of Phytophthora infestans inciting potato late blight”, in: Phytophthora diseases of tropical cultivated plants, K.K. N. Nambiar, (Ed.) 94–101. Central Plantations Crops Research Institute, Kasaragod, Kerala. Bourke, P.M.A. (1953), “Potato Blight and the weather in Ireland (Tech. Note No. 15) in 1953,” Irish Meteorol. Serv., Dublin, p. 37. Bourke, P.M.A. (1970), “Use of weather information in the prediction of plant disease epiphytotics”, Ann. Rev. Phytopath., 8: 345–370.

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Campbell, C.L. and L.V. Madden (1990), Introduction to Plant Disease Epidemiology, Wiley, New York. Chandwani, G.H., M.S. Balkrishanan and S.Y. Padmanabhan (1963), “Helminthosporium disease of rice V : A study of the spore population of Helminthosporium oryzae over rice fields”, J. Indian Bot. Soc., 42(1) : 1–14. Choudhuri, H.C. and S.C. Pal (1959), “Forecasting late blight of potatoes in the hills of West Bengal”, Am. Potato 36: 284–287. Cook, H.T. (1947), “1947 results–late blight forecasting”, Food Packer, pp. 63–64. Cook, H.T. (1949), “Forecasting late blight epiphytotics of potatoes and tomatoes”, J. Agr. Res., 78: 545–563. Cox, A. E. and E.C. Large (1960), “Potato blight epidemics throughout the world”, Agr. Hanb. No. 174, Dept. Agr. Washington, D.C. , p. 230. De Weille, G. A (1965), “The epidemiology of plant disease as considered within the scope of agrometeorology”, Agr. Meterol., 2: 1–15. Friedrick, S. (1995), “Modelling infection probability of powdery mildew in winter wheat by meteorological input variables”, Z. Pflanzenker. Pflunzenschutz., 102: 354. Fry, W. W. and G. B. Foher (1985), “Construction of predictive models. I. Forecasting of disease development”, in: Advances in Plant Pathology, Vol. 3: Mathematical Modelling of Crop Disease, C. A. Galliga, (Ed.), Academic Press, London, pp. 151. Gaumann, E. (1950), “Principles of Plant Infection,” Hafner Lockwood, London, pp 543 (Translated by W.B. Brierly). Goto, K., Y. Inoue, R. Fukatsu and K. Ohata (1955), “Field observations in the outbreak and fluctuations of severity and bacterial leaf blight of rice plant”, Bull. Tokai-Kinke Agr. Sta., 2:53–68. Gupta, G. K. (1985), “Recent trends in forecasting and control of Apple scab (Venturia inaequalis) ”, Pesticides, 19: 19. Gupta, G.K. (1992), “Apple Scab”, in: Plant Diseases of International Importance, Vol. III Diseases of Fruit Crops, J. Kumar, H.S. Chaube, U.S. Singh and A. N. Mukhopadhyay (Eds.), Prentice Hall, Englewood Cliffs. pp.1–31. Hyre, R.A. (1954), “Progress in forecasting late blight of potato and tomato”, Plant Dis. Rept,. 38: 243–253. Hyre, R.A. and R. Bonde (1955), “ Forecasting late blight of potato in Northern Maine,” Ann. Potato J., 32: 119–125. Jensen, R.E. and L.W. Boyle (1966), “A technique for forecasting leaf-spot on peanuts”, Pl. Dis. Reptr. , 49: 976–978. Joshi, L.M. and L. T. Palmer (1973), “Epidemiology of stem and leaf rusts of wheat in Northern India”, Plant Dis. Reptr., 57: 8–12. Kranz, J. (1973), “Comparison of Epidemics”, Ann. Rev. Phytopath., 12: 355–37. Kranz, J. (1974), “Comparison of Epidemics”, Ann. Rev. Phytopath., 12: 355–374. Krause, R.A. and L.B. Massie (1975), “Predictive systems: Modern approaches to disease control”, Ann. Rev. Phytopath., 13: 31–47. Leonard, K.M. and W.E. Fry (Eds.) (1986), Plant Disease Epidemiology, Vol. 1: Population Dynamics and Management, Macmillan, New York. Leonard, K.M. and W.K. Fry (Eds.) (1989), Plant Disease Epidemiology, Vol. 2: Genetics Resistance and Management, McGraw Hill, New York. Madden, L.V. and G. Hughes (1995), “Plant disease incidence: Distribution, heterogeneity and temporal analysis”, Ann. Rev. Phytopathol., 33: 529–5654. Madden, L.V., S.P. Pennypacker, A.A. MacNab (1978), “FAST, a forecast system for Alternaria on tomato”, Phytopathology, 66 (8): 1354. Magee, C. D. and A. Y Ekbote (1983), “Life cycle and micro-epidemiology and Puccini arachides Speg. rust of ground nut in Maharasthra”, India J. Plant Path., 1: 62– 67. Main, C. E. (1977), “Crop Destruction – the Raison D’etre of plant pathology”, in Plant Disease, Vol. I, J.G. Horsfall and E. B. Cowling (Eds.), Academic Press, New York, pp. 55–78.

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Mayee, C. D. (1988), “Epidemiology and management of groundnut rust”, in: Vistas in Plant Pathology, A. Varma and J.P. Verma (Eds.), Malhotra Publication House, New Delhi, pp. 305–320. Mayee, C.D. and V.V. Datar (1988), “Forecasting plant diseases in India”, in: Perspective in Mycology and Plants Pathology, V.P. Agnihotri, A.K. Sarbhory, (Eds.), Malhotra Publishing House, New Delhi, pp. 324–333. McCartny, A. (1996) “The influence of environment on the development and control of disease”, In: In Environmentally Safe Approaches to Crop disease Control, N.A. Rechcigl and J.E. Rechcigl (Eds.) CRC Lewis Publishing, Boca Raton, pp. 1–31. Menzies, J.D. (1963), “The direct assay of plant pathogen populations in soil”, Ann. Rev. Phytopath., 1: 127. Miller, P.R. (1967), “Plant disease epidemics and forecasting”, In Papers Presented at the FAO Symp. Crop Losses, Rome, 1967 9-37: FAO. 330 pp. Miller, P.R. and M.J. O’Brien (1952), “Plant disease forecasting”, Bot. Rev., 18: 547–601. Miller, P.R. and M.J. O’Brien (1957), “Prediction of plant disease epidemics”, Ann. Rev. Microbiol., 11: 77–110. Nagarajan, S. (1983), Plant Disease Epidemiology, Oxford and IBH Publishing Co. New Delhi pp. 267. Nagarajan, S., L.M. Joshi, K. D. Srivastava and D.V. Singh, (1990), “Epidemiology of brown and yellow rusts of wheat in North India”, Cereal Rust Bull., 7: 1979– 1980: pp 15–20. Nagarajan, S., L.M. Joshi, K.D. Srivastava and D.V. Singh (1978), “Epidemiology of brown and yellow rusts of wheat over north India III. Impact of varietal change”, Plant Dis. Reptr., 62: 694–698. Nagarajan, S. and H. Singh (1975), “Indian stem rust rules on epidemiological concept on the spread of stem rust of wheat”, Plant Dis. Reptr., 59: 133-136. Nilsson, H.E. (1995), “Remote Sensing and image analysis in plant pathology”, Ann. Rev. Phytopath., 33: 489-527. Padmanabhan, S.Y. (1963), “Helminthosporium disease of rice – VII: A study of the meteorological factor associated with the epiphytotic of 1942 in Bengal”, Oryza, 1(2): 101–110. Padmanabhan, S.Y. (1965), “Studies on forecasting outbreaks of blasts diseases of rice (Influence of meteorological factors on blast incidence at Cuttack)”, Proc. Indian Acad. Sci., 62 (B): 117-129. Pitbaldo, R.E. (1992), “The Development and Implementation of TOM-CAST”, Ontario Ministry of Agriculture and Food, Toronto, Canada, 1992. Rosa, M., R. Genesio, B. Gozzini, G. Maracchi and S. Orlandini (1993), “ PLASMO: a computer programme for grapevine downy mildew development forecasting,” Comput. Electron Agric., 9: 205. Rosa, M., B. Gozzini, S. Orlandini, and L. Seghi (1995), “A computer programme to improve the control of grapevine downy mildew”, Comput. Electron. Agric., 12: 311. Saharan, G.S. and B.M. Singh (1980), “Influence of environmental factors in the latent period of Melampsora lini under field conditions”, Indian Phytopath., 33: 270–274. Saxena, S.C. and S. Lal (1984) “Use of meteorological factors in prediction of Erwinia stalk rot of Maize,” Tropical Pest Management 30: 82-83. Shrum, R.D. (1978), “Forecasting of epidemics”, in: Plant Disease – An Advance Treatise, J.G. Horsfall and E.B. Cowling (Eds.), Academic Press, New York, San Francisco and London, II: pp. 223–238. Sleeman, W.H. (1839), “ Extracts from Major Sleeman’s diary”, Trans. Agric. Soc., India. 7: 79–87. Srivastava, D.N. (1972), “Bacterial blight of rice”, Indian Phytopath., 25: 1–16. Van Everdingen, E. (1926), “ The relation between weather conditions and potato blight,” Tijdschr Plantenzilekten., 32: 129–140. (R.A. M. 5 : 627, 1926). Van Everdingen, E. (1933), “The Dutch warning service for outbreaks of potato blight”, Proc. Pacific Sci. Cong. , 3: 1757–1759. Vanderplank, J.E (1963), “Plant Diseases: Epidemics and Control”, Academic Press, New York, pp. 349. Waggoner, P.E. (1960), “Forecasting epidemics”, in: Plant Pathology, J.G. Horsfall and A.E. Dimond (Eds.) Academic Press, New York and London, III: 291–312. Wallin, J.R. and W. M.G. Hoyman (1954), “Forecasting potato late blight in North- Dakota”, N. Dak. Agr. Exp. Stn. Biomonth Bull., 16: 226–231. Zadocks, J. C. (1984), “A quarter century of disease warning”, Plant Dis., 68: 352–355. Zadocks, J.C. and R.D. Schein, (1979), Epidemiology and Plant Disease Management, Oxford Univ. Press, London and New York.

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QUESTIONS 1. How does plant disease forecasting help to control disease? 2. What are the environmental conditions necessary for the development of the epiphytotics of the following in India : i. Potato blight ii. Blast of rice. iii. Downy mildew of bajra. iv. Rusts of wheat 3. Give at least two examples of forecasts based on i. amount of initial inoculum ii. weather conditions iii. host physiology 4. What are BLIGHTCAST, CERCOS, MYCOS, EPICORN, EPIVEN, & EPIDEM? 5. Discuss in brief the contributions of the following towards the epidemiology of diseases. i. K.C. Mehta ii. S. Nagarajan iii. Van Everdingen iv. A. Beumont v. H.T. Cook vi S.K. Bhattacharya

vii S.Y. Padmanabhan

11 Management of Plant Diseases

Although these are several reviews, articles and books on management of plant diseases, a recent article entitled, “Advances in plant health management in the twentieth century” by Cook (2000) needs special mention. The principal methods of controlling plant diseases are: (1) avoidance, (2) exclusion, (3) eradication, (4) protection, and (5) immunization. In order to eliminate all the impediments to maximum production of food, it is imperative to control the spread of plant pathogens by cultural methods, chemical methods and by breeding disease-resistant varieties. CULTURAL METHODS

Stevens (1960) had discussed the cultural methods of disease control. According to him, these measures involve agricultural cropping, harvesting and storage, tillage, crop rotation, soil management, growing of resistant varieties, planning of land use, and other related practices. In this book, the breeding of resistant varieties had been dealt with separately. Avoidance of the Pathogen

Many plant diseases can be prevented by a proper selection of the land or field, choice of time of sowing, selection of varieties, seed and planting stock, and by modifications of cultural practices. The aim of these measures is to enable the host to avoid contact with the pathogen or to ensure that the susceptible stage of the plant and favourable conditions for the pathogen should not coincide. Proper Selection of Geographical Area

Many fungal and bacterial diseases are more severe in wet areas than in dry areas. Crops which are susceptible to these diseases, if grown in wet areas, are likely to be affected by plant pathogens. Tolyposporium penicillariae, the pathogen responsible for the smut disease of bajra and the ergot of bajra caused by Claviceps fusiformis (C. microcephala), are more severe in wet areas in regions where rain occurs for long durations during the flowering stage of the crop.

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Selection of Field The selection of a suitable area or field for cultivation is very important from the point of view of better yields as well as protection of the crop from the ravages of pathogens. In the case of many soil-borne pathogens, if the same field is chosen for cultivating a specific crop, there is every likelihood of the disease appearing in severe form due to the build-up of the inoculum potential of the pathogen. Hence, it is advisable not to grow the crop in the same field, where in the previous year, it was infected with a pathogen capable of surviving in the soil. Colletotrichum falcatum can persist in the soil for a couple of months. Even this short duration is sufficient for the development of disease in the case of sugarcane, if it is successively cultivated in the same field. It follows that a number of soil-borne diseases can be prevented if the same crop is not grown in the same field year after year. The examples are wilt of arhar, green ear disease of bajra, late blight of potato, stem gall of coriander, powdery mildew of wheat, smut of bajra, ergot of bajra, ear cockle of wheat, root knot of vegetable crops, and bacterial wilt of solanaceous plants. The drainage conditions of the field are also important. Low lying, waterlogged fields favour such diseases as red rot of sugarcane and downy mildew of bajra. In the case of fruit orchards, the selection of a suitable site is very important. If apple trees are planted in areas where oak trees were previously grown, there are chances of collar rot due to Rosellinia sp. becoming severe. Choice of Time of Sowing Pathogens are able to infect susceptible plants only under certain environmental conditions. For example, downy mildews require prolonged high moisture for infection. If the crop is grown in such a way that it is not attacked by pathogens during its most susceptible period, disease incidence can be considerable reduced. Rhizoctonia root rot of gram is severe if gram is sown immediately after the rains. This is due to the fact that the pathogen develops rapidly under high temperature and moisture conditions. A slight delay in sowing is helpful in this case. In the same way, Puccinia graminis tritici does more damage to a wheat crop sown late. Early seeding also induces losses due to bunt of wheat. Disease-Escaping Varieties Certain varieties of crops escape damage because of their growth characters. These varieties escape the onslaught of the pathogen and resist the attack due to their inherent characteristics, for example, early maturing varieties of wheat or pea escape damage due to Puccinia graminis tritici and Erysiphe polygoni, respectively. Selection of Seed and Planting Stock In India seed potatoes are invariably imported in the southern states from the Simla hills for control of viral and bacterial diseases. In the USA, seed growing areas have been shifted to the dry Pacific regions for crops such as cabbage, turnip, beans, and peas for obtaining disease-free seeds and indirectly controlling such diseases as black leg and black rot of cabbage and turnip, and anthracnose of beans, peas etc. In the case of foot rot and leaf rot of Piper betle caused by Phytophthora parasitica var. piperina and rhizome rot of ginger (Zingiber officinale) caused by Pythium myriotylum, it is advisable to obtain seeds from disease-free areas where the disease has not occurred due to certain environmental factors. Since many plants propagate by vegetative parts, the selection of disease-free planting material forms a very important control measure. The planting of disease-free fields is often an important control measure for certain diseases such as red rot of sugar cane, black scurf of potato, or various viral diseases of potato. Tuber indexing has been resorted to for obtaining virus-free seeds and protecting crops against the attack of various viral diseases of potato.

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Loose smut of wheat, bunchy top and Panama disease of banana, and whip smut of sugarcane can be effectively kept under check if seeds are properly selected before sowing or planting. Modification of Cultural Practices The distance between plants, time and amount of irrigation, quantity and quality of fertilizers or organic manures, time and method of planting, mixed cropping method, depth of sowing, and so on are some cultural practices which influence the incidence and severity of certain diseases. The necessary changes in these practices can help to reduce disease incidence and loss of the crop. Tillage is an effective method of control of pathogens harboured in crop residue, but it leads to loss of soil organic matter and increased soil erosion. Tillage provides a temporary improvement in aeration, accelerates drying, and redistributes substrates. These environmental and physical changes can predispose pathogens to antagonistic effects of the microbiota. Early and deep ploughing kills cotton roots and favours saprophytes rather that Phymatotrichum omnivorum. If the plant debris is buried deeper by ploughing, the pathogen is at a disadvantage as compared to other saprophytes in soil. Brooks and Dawson (1968) observed less take all in England when wheat was grown in a no-till system. Faulty mechanical sowing makes plants susceptible to infection. This may also result in an unequal and uneven distribution of seeds. Overcrowding may result in damping-off of seedlings. The depth of sowing has an important effect on some pathogens notably those which attack seedlings. By delaying the emergence of seedlings, deep sowing may help to increase the resistance of a susceptible crop to a pathogen. This is true in many diseases caused by Fusarium and Rhizoctonia species and in the smut diseases in which seedlings are attacked. Good control of blind seed is obtained by planting infected seeds at least 1.25 cm deep and with complete soil coverage. Burying the infected seeds prevents the emergence (above the soil line) of the apothecia of the fungus Geotinia temulenta formed on them which is responsible for bringing seed disease of perennial rye grass. The dissemination of the ascospores is prevented by the procedure. The incidence of wheat bunt caused by Tilletia foetida and T. caries and flag smut (Urocystis tritici) of wheat is higher in crops sown deep. Mixed cropping is of special significance in Asian countries. This practice helps to effectively check the spread of infectious diseases. Ploughing under the surface refuse before sowing controls Sclerotium rolfsii on peanuts. This deprives the fungus of a food base since it can grow only on or near the soil surface. The effect, therefore, is very much like that of crop rotation. Deep ploughing of soils that contain diseased potato tubers can effectively reduce the amount of inoculum of Phytophthora infestans. The root rot of cotton caused by Rhizoctonia bataticola and the blight of pulse crops caused by Phyllosticta phaseolina have been successfully controlled in Punjab by mixed cropping. Mixed cultivation of cotton and ‘moth’ (Phaseolus aconitifolius) reduces the incidence of root rot of the former crop (caused by Rhizoctonia solani and R. bataticola). Mixed crops, such as wheat and barley, wheat and gram, arhar and jowar, cotton and moth reduce the economic losses from diseases. Management of soil water offers a means to manage the associated microbiota-directly, through water potential, and indirectly, through gas exchange of the soil. Common scab of potato can be controlled by keeping the soil water potential near the tubers higher than – 0.4 bar during tuber formation. The dryland foot rot of wheat caused by Fusarium roseum ‘Culmorum’ can be controlled by irrigation, partly because the water reduces stress in the host and also because the germlings of Fusarium lyse or convert into new chlamydospores at water potentials above – 10 to – 15 bars.

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Flooding helps to rid the soil of F.oxysporum f. sp. cubense (Stover, 1962), responsible for Panama disease of banana and sclerotia of Sclerotinia sclerotiorum (Stover and Moore, 1953). In Israel, the diseases of tomato caused by Stemphylium botryosum (Pleospora herbarum) and Xanthomonas campestris pv. vesicatoria were increased by overhead sprinkling, whereas Leveillula taurica, a powdery mildew which thrived in dry conditions, was considerably reduced. In the presence of excess free water, bacterial cells and fungal spores are disseminated easily. Sprinkler irrigation spreads bacterial cells and fungal spores in the same manner as raindrop splashes. In diseases where there is heavy sporulation of the pathogen or exudation of bacterial cells on the leaf surface, sprinkler irrigation should be avoided. Pathogens, such as Pythium and Phytophthora species, are dependent on high soil moisture content. Cereal rusts are more common in heavily irrigated crops or those grown in very wet soil. Dhingra and Sinclair (1975) observed a rapid deterioration of sclerotia of Macrophomina phaseolina in warm wet soil and proposed that disease could be controlled by keeping the soil wet for 23 weeks when the temperature was about 30°C. Naturally, a proper control of irrigation practices helps to control these diseases. Exclusion of Inoculum

These methods aim at preventing new pathogens and diseases from reaching an uninfected area and avoiding contact between the pathogen and the crop of field. Seed certification, crop inspection, growing crops in regions unfavourable for the pathogen, and quarantine measures are some means of preventing the spread of pathogens. Seed Treatment Seeds, tubers, grafts, bulbs, and other propagating material can be given heat, gas or chemical treatment to keep then free of pathogens. Inspection and Certification The crops grown exclusively for seeds are inspected periodically for the presence of diseases that are disseminated by seeds, and necessary precautions are taken to remove the diseased plants. The crop is then certified as disease-free. This practice helps to prevent the inter and intra-regional spread of seed-borne diseases. Quarantine Regulation A quarantine can be defined as a legal restriction on the movement of agricultural commodities for the purpose of exclusion, prevention or delay in the spread of plant pests and diseases in uninfected areas. In most agriculturally advanced countries, plant quarantine legislation has been placed on the statute book to restrict the movement of diseased plant material or of fungi, bacteria, mycoplasma, viruses or nematodes that cause diseases in plants. Quarantine measures are of three types: (1) domestic, (2) internal, and (3) total embargoes. The quarantine laws were first enacted in the USA in 1912, and are known as Federal Quarantine Acts. In India, the Destructive Insect and Pest Act was passed in 1914 and subsequently supplemented by other provisions. Such quarantine laws were first enacted in France in 1660, in Denmark in 1903. They aimed at a rapid destruction or eradication of barberry, which has been known since early times to harbour black rust. Some of the examples of diseases, which have been introduced into other countries, are listed in Table 11.1.

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TABLE 11.1 Disease Late blight of potato caused by Phytophthora infestans Powdery mildew of grapes caused by Uncinula necator Blister rust of pine caused by Cronartium ribicola Downy mildew of grapevine caused by Plasmopara viticola Chestnut blight caused by Cryphonectria (Endothia) parasitica Citrus canker caused by Xanthomonas campestris pv. citri Bunchy top of banana (viral) Paddy blast caused by Pyricularia grisea (oryzae)

Year 1830

Introduced in Europe

Introduced from S. America

1845

England

USA

1910

USA

Europe

1878

France

USA

1904

USA

Asia (Mediterranean region)

1907 1940 1918

USA India India

Asia Sri Lanka South East Asia

Examples of plant diseases introduced in India before and after the enforcement of quarantine are given in Table 11.2. TABLE 11.2 Disease Leaf rust of coffee caused by Hemileia vestatrix Late blight of potato caused by Phytophthora infestans Flag smut of wheat caused by Urocystis tritici Downy mildew of grapes caused by Plasmopara viticola Downy mildew of cucurbits caused by Erysiphe cichoracearum Downy mildew of maize caused by Sclerospora phillipinensis Paddy blast caused by Pyricularia grisea (oryzae) Powdery mildew of rubber caused by Oidium heveae Black shank of tobacco caused by Phytophthora nicotianae Crown gall of apples and pears (Agrobacterium tumefaciens) Bunchy top of bananas (viral disease) Wart disease of potato caused by Synchytrium endobioticum Onion smut caused by Urocystis cepulae Golden nematode of potato caused by Heterodera rostochiensis

Year 1879 1883 1906 1910 1910 1912 1918 1938 1938 1940 1940 1953 1958 1961

Introduced from Sri Lanka Europe Australia Europe Sri Lanka Java Southeast Asia Malaya Dutch east England Sri Lanka Netherlands Europe Europe

The DIP Act in India was passed in 1914 and has been revised eight times from 1930 to 1956 and corrected up to 1967; but it still does not fulfil the needs under the prevailing circumstances. In India there are 16 quarantine stations operating under the Directorate of Plant Protection and Quarantine— eight at seaports, six at airports and two on land frontiers (Hussainiwala in Ferozepur and Sukhiapokri in Darjeeling district). There are a number of lacunae in the DIP Act, and as a result many serious plant pathogens have already been introduced in the country. These are now established and are taking a

Management of Plant Diseases 233

heavy toll of crops in certain areas. Golden nematode (Heterodera rostochiensis) and wart (Synchytrium endobioticum) diseases of potato have been introduced in India from the European countries and are now well established in the Nilgiri Hills and Darjeeling. Fortunately, timely action had prevented the spread of wart disease to other parts of India. Synchytrium endobioticum was first described by Ganguly and Paul in 1953 and golden nematode of potato (Heterodera rostochiensis) was reported by Jones for the first time in India in 1961. Bunchy top disease of banana was introduced into India around 1940 (Verghese, 1945). By 1943 it had spread to a few areas in Kottayam and it has now affected an area of about 3000 square miles in Kerala. The disease has also spread to Tamil Nadu, Orissa, West Bengal, and Assam. In India, domestic quarantine measures exist for two insect pests, Fluted Scale (Icerva purcheri) and San Jose Scale (Quadraspidiotus permiciosus) and for three diseases, wart (Synchytrium endobioticum) of potato, bunchy top (virus) and mosaic (virus) of banana with a view to preventing the spread of these pests. Raychaudhuri (1973) had discussed seed pathology in relation to plant quarantines and the seed industry in India. Joshi (1974) had dissertated on the regulatory control of plant disease weeds and pests in India. Plant quarantine measures have also been discussed by Lambat and Ram Nath (1974). Ram Nath and Lambat (1971) had listed fungi recorded on imported seeds and other plant materials in India. The problem of plant disease is global and not national. Hence, in 1951 in Rome, an International Plant Protection Convention was drawn up which at present has about 50 signatory nations. Briefly, each contracting government agrees to make provisions for: 1. An official protection organization with the specific basis of inspecting growing crops and the produce derived from them and issuing phytosanitary certificates. 2. The distribution of information regarding pests and diseases both within the country and to the FAO so that a world reporting service is established. 3. Research and investigation in the field of plant protection on a cooperative basis for diseases which have international effects. Within the framework of the international cooperation there are six regional groups. (a) European Plant Protection Organization (EPPO). This was formed prior to the treaty in Rome. (b) Inter-African Phytosanitary Commission (for South Africa, South of Sahara). (c) Plant Protection Committee for South East Asia and the Pacific region. (d) Organizmo International Regional de Sanidad Agroperciane (Central America and Mexico). (e) Convenio Intermericano de Protection Agricola (South America). (f) Near East Plant Protection Commission. Management of the pathogens under quarantine regulations fall under the following heads–(1) Seed certification, (2) notification of plant diseases, (3) prevention and removing or roguing of diseased plants or seeds containing the pathogen, and (4) plant and field sanitation for reduction of inoculum. Eradication of the Pathogen

These measures primarily aim at breaking the “infection chain” by removing the foci of infection and starving the pathogen. A knowledge of the life cycles of the pathogens, method of perennation, host range, and habit of growth are essential prerequisites for devising suitable measures to prevent the

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disease. Eradication involves eliminating the pathogen from infested areas, and the magnitude of the operation involved may vary considerably. Eradication is attempted through roguing, sanitation, crop rotation, eradication of alternate and collateral hosts, heat and chemical treatment of diseased plants, soil treatments, biological control, and so on. Roguing This practice entails careful removal and destruction of infected plants or plant parts form the fields at an early stage by removing the “foci” of infection and preventing wide dissemination of the pathogen. Roguing is employed in such diseases as loose smut of wheat, loose and covered smut of barley, sorghum and maize, red rot of sugarcane, green ear disease of bajra, ergot of bajra, wilt of arhar, and many viral diseases, such as yellow vein mosaic of lady’s finger, “Katte” disease of cardamom, Panama disease of banana, Loranthus growing on trees, etc. Eradication of Alternate or Collateral Hosts Many plant pathogens complete their life cycles on two hosts; one of them is generally a wild plant. The destruction of such a wild host plant breaks the chain in the completion of the life cycle of the pathogen. Barberry eradication in temperate countries helps to control the black stem rust of wheat. Many plant pathogens have several alternate hosts on which they pass their life cycles. Pyricularia grisea (P. oryzae) can infect and survive on several wild species of rice and other grasses, such as Panicum repens, Digitaria marginata and Setaria intermedia. The destruction and removal of collateral hosts is an important control measure of the disease. The yellow vein mosaic of lady’s finger (Abelmoschus esculentus) persists on wild hosts (Hibiscus tetraphyllus) in nature and the systematic removal of the wild plants helps to control the disease. Similarly, effective control has been obtained in Barbados, West Indies, by the eradication and systematic removal of maize from the neighbourhood, as the latter acts as a collateral host for the perpetuation of the virus infection chain. Similarly removal of Panicum antedotale or Cenchrus ciliaris for pearl millet fields contributes to lowering of ergot disease. Crop Rotation Very often, continuous cultivation of the same crop or related crops leads to the perpetuation of a pathogenic soil-borne fungus and the gradual increase in the intensity of the disease. Soils generally suffer from what we call soil sickness. When the same crop is raised year after year in the same field, the soil-borne pathogens of that crop easily perennate in the soil and increase their population. After a certain period of time, the soil becomes so heavily infested with the pathogen that it no longer supports the growth of the crop. Such soils are called “sick soils”. The interposition of 2 or 3 non-susceptible crops between the cultivation of the susceptible crops in the same field is effective in reducing the soil sickness and populations of soil-borne pathogens. Crop rotation helps to control many soil-borne diseases, such as wheat mosaic, wilt of arhar, wilt of linseed, finger and toe disease of crucifers, wilt of pea, wilt of gram, red rot and wilt of sugarcane, ergot and smut of bajra, bunt of wheat, leaf smut and bunt of rice, flag smut of wheat, molya disease (a nematode disease) of wheat and barley and root rot of vegetable crops. This method is also important from other aspects. If the same crop is sown in the same area year after year, there are chances that a particular essential nutrient in the soil will be exhausted. There can also be the accumulation of toxic substances and organic acids if a specific crop is continually cultivated in the same area. Crop rotation is generally recommended with botanically-unrelated crops as these will have different nutritional requirements and tend to be affected by different pathogens and pests.

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Sanitation Like plant sanitation, field sanitation is also essential for the control of many plant diseases. Pathogens survive or perennate through dormant structures in plant organs in the soil. The collection and destruction of these structures form an important control measure. It is common practice to burn diseased stubble for plant disease control. The effectiveness of fire for controlling plant disease has been reviewed by Hardison (1976). In fact, sanitary conditions are helpful in almost all infectious diseases. Powdery mildew of wheat, barley, peas, downy mildew of peas and maize, red rot of sugarcane, Panama disease of banana, Verticillium wilt of cotton and wilt of arhar, Koleroga of Areca palms, foot rot and leaf rot of Piper betle, brown rot of stone fruits, late blight of potato, angular leaf spot of cotton, etc. are some examples where sanitary precautions help in minimizing losses. The spread of certain diseases, for example, tobacco mosaic virus, would not be rapid if the workers who smoke were to wash their hands before handling the plants. Heat or Chemical Treatment of Diseased Plants The pathogen present in the plant can be killed or inactivated by applying heat or chemicals. Hot water treatments have generally been most successful and are used to eliminate Ustilago segetum (nuda) from wheat and barley seed, Alternaria brassicae from cabbage seeds, Puccinia menthae from mint runners, the nematode Dictylenchus dipsaci from Narcissus bulbs and ratoon stunt and other diseases from sugarcane setts. The most important application of hot air treatments is to eliminate viruses from vagetatively propagated material. Kunkel (1936) started this practice in the USA and Kassanis (1949, 1954) in England. Thermotherapy is a time old practice that is still in vogue in the control of several crop diseases. Thermal inactivation points of sugarcane and its pathogens are taken advantage of freeing infected stocks of many shoot, ratoon stunting smut diseases and red rot of sugarcane. Four types of heat treatment are used for administering thermotherapy to sugarcane setts, (1) Hot air treatment (HAT) at 54°C for 8 hours (2) Moist hot air treatment (MHAT) at 54°C for 4 hours (3) Hot water treatment (HWT) at 50°C for 2 hours, and (4) Aerated steam treatment (AST) at 50°C for 1 hour. Treating the seeds with fungicides, especially organomercurials, is a common practice to remove the superficially-borne inoculum, and there are now systemic fungicides available which can eliminate deepseated fungal infections. Tyner (1953) found that soaking barley seeds for 48 hours in a 0.2% Chloranil (Spergon) solution at 22.2° to 25°C satisfactorily controls the loose smut fungus, Ustilago nuda, on barley. Thiram soak has been successfully utilized to eliminate Septoria appiicola from celery seed, and Mycosphaerella pinodes and Ascochyta pisi from pea seed. The systemic fungicides, Vitavax and Plantavax, have been successfully used as seed dressing for the control loose smut (U. segetum ) and rust (Puccinia graminis tritici) of wheat, respectively. Soil Treatments Soil treatment involves the use of chemicals, heat energy, flooding, fallowing, and so on. Plant pathogens can be eradicated from the soil by dry heat, steam or hot water, flooding and fumigation with chemicals. Heat and chemical treatments have been successfully used for the eradication of plant pathogens from green house soils as well as soils in plant beds. The treatment of soils in large fields has not been so effective. Nematodes can be eradicated from the soil by fumigating it with DD mixture or with other nematicide. PCNB has been found to be effective in eradicating sclerotiaproducing soil pathogens, carbon disulphide in eliminating Armillaria mellea and formaldehyde in controlling onion smut disease. Newhall (1955) had reviewed the literature on the subject of soil disinfestation by heat, flooding and fumigation. Munnecke and VanGundy (1979) had reviewed movements of fumigants in soil, dosage responses, and differential effects.

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Biological Eradication of Plant Pathogens More than seventy five years ago, a paper by Sanford (1926) on potato scab caused by the actinomycete Streptomyces scabies, brought the rather sudden realization that other soil microorganisms must exert a natural biological control even on root disease pathogens. The term biological control clearly implies control of a disease through some biological agency, and the term ‘biological agency’, means a living micro-organism or macro-organism other than the diseased or damaged plant acting as host and the pathogen or pest causing the disease or damage. According to Garrett (1965) “Biological control of plant disease may be defined as any condition or practice whereby survival or activity of a pathogen is reduced through the agency of any other living organism (except man himself), with the result that there is a reduction in the incidence of the disease caused by the pathogen”. There is an enormous amount of literature on biological control of plant pathogens. Some recent books and review articles are those of Adams (1990), Hornby (1990), Nelson (1991), Stirling (1991), Tjamos et al. (1992), Mukhopadhyay (1994, 1996), Dwivedi (1993), Agarwal (1991), and Bagyaraj and Govindan (1996). There is a recent article by Mehrotra et al. (1996) on fungal control agents. Mehrotra (1997) has reviewed the work done on Trichoderma—taxonomy, ecology and biocontrol. Several examples of the biological control of plant pathogens can be given. Millard and Taylor (1927) reported the control of scab in potatoes grown in sterilized soil and inoculated with Streptomyces scabies, through simultaneous inoculation of the soil with S. praecox, a vigorous saprophytic species. It was as early as 1932 that Weindling showed that Trichoderma viride, a common saprophytic fungus, is able to parasitize the mycelia of other fungi. Later, he showed that the lethal action of T. viride is due to the secretion of an antibiotic substance which he called “gliotoxin”. Still later, Brian and McGowan (1945), isolated another antibiotic substance from T. viride which they named “viridin”. These discoveries raised hopes of controlling plant diseases by biological means. In 1951 Bliss in the USA, who was working on root diseases of citrus caused by Armillaria mellea opened a new approach. He suggested that if the soil is fumigated with carbon disulphide, the diseaseinciting fungus is killed not directly by the fumigant but indirectly by the dominance of T. viride, the population of which increases after fumigation. Garrett (1958) tested the effect of fumigation and the antibiotic effect of T. viride and assessed the relative parts played by these two components. He found that 30% of Armillaria mellea was killed due to the dominance of T. viride, while the rest was directly killed by the fumigant. He also pointed out that in the thicker plant roots, T. viride may be instrumental in the process of killing than the fumigant which might not be able to reach easily. The most conspicuously successful biological control is by Rishbeth in England. Rishbeth has been studying a root disease of pines (Pinus sylvestris and P. nigra) caused by basidiomycetous fungus Heterobasidion (Fomes) annosus. The disease spreads through infection by basidiospores produced in the large fructifications. The spores infect the left-over stumps when the trees are felled. Rishbeth at first tried to control the disease by painting these stumps with fungicidal paints, such as creosote. This brought only partial control because the stump tissue cracked with progressive drying, causing fissures through which the fungal spores of F. annosus could enter and cause infections. Rishbeth’s research associate, Meredith (1960), made an important observation that along with H. (Fomes) annosus there were a few other fungi which were of the nature of weak parasites that colonized the cut surfaces of stumps. Rishbeth (1963) had pointed out that he originally selected Peniophora gigantea for this purpose because it is the most common and effective competitor of H. (Fomes) annosus in the natural colonization of pine stump surfaces. It decomposes lignin and cellulose vigorously and the fungus estab-

Management of Plant Diseases 237

lishes itself in the stump quickly and from there into the stump roots. From these stump root infections, the pathogen invades the roots of other healthy trees. Rishbeth showed that if paste of oidia of P.gigantea is applied on the stump, it parasitizes it rapidly and kills the tissues, making it easy for saprophytes to colonize and decompose the stump and making the tissues unsuitable for an attack by H. (Fomes) annosus which is a specialized parasite. Rishbeth determined that a dose of 1 ¥ 104 oidia of Peniophora gigantea allowed a satisfactory margin of safety for effective biological control of the largest spore inoculum of H. annosus for a pine stump of 16 cm wood diameter. Dehydrated tablets of oidia have been produced for distribution and use in controlling H. annosus. Although a large number of microorganisms have been shown to be antagonistic to the growth of a large number of plant pathogens in the laboratory, only a few have been found effective and practicable in field conditions. According to Agrios (1997) only six microorganisms have been registered so far. These are Gliocladium virens sold as GlioGard for control of seedling disease, Trichoderma harzianum sold as F-Stop for control of several soil-borne fungi, T. harzianum/ T. polysporum sold as BINAB-T for the control of wood decays, Agrobacterium radiobacter K-84, sold as Gallex or Galltrol for controlling crown gall disease caused by Agrobacterium tumefaciens, Pseudomonas fluorescens sold as Dagger G for use against Pythium and Rhizoctonia species, and Bacillus subtilis sold as Kodiak and utilized as seed treatment. Pseudomonas rhizobacteria primarily of P. fluorescens, P. putida, P. cepacia and P. aureofaciens applied to seeds, seed pieces, and roots of plants have resulted in less damping off, less soft rot, and an increase in the growth of the crop. Different mechanisms have been proposed to explain disease suppression by Pseudomonas spp. They include: 1. Production of siderophores (iron chelating compounds) which remove iron from the soil environment and make it unavailable to pathogens. 2. Antibiosis-various antibiotics have been reported to be produced by members of genus Pseudomonas. 3. Induction of resistance, and 4. Competitive root colonization. Biological control of chestnut blight caused by Cryphonectria (Endothia) parasitica is controlled naturally in Italy and artificially in France, through inoculation of cankers, caused by the normal pathogenic strains of the fungus, with hypovirulent strains. The hypovirulent strains carry viruses like doublestranded RNAs (ds RNAs). The disease was first encountered in the USA in 1904 and despite attempts to eradicate it, spread to destroy most of the native chestnuts in the eastern states. When in 1938 the disease was first recorded in Italy it was feared that European chestnut trees would suffer a similar fate. The initial outbreak was severe but later it was found that many infected trees begin to show spontaneous signs of recovery with healing. When the strains of the pathogen were isolated from such healing cankers, it was found that these strains were less virulent than the original pathogen. Furthermore, if these hypovirulent strains were coinoculated with highly virulent strains, the resulting cankers also healed. Most strains could be converted to the hypovirulent culture. Some transmissible factor moved from the hypovirulent strains to the virulent strains to make it hypovirulent strains. The agents responsible were shown to be cytoplasmic and subsequently identified as double-stranded RNA molecules. It seems that hypovirulent strains are due to a type of viral infection. The natural transfer of hypovirulents of C. parasitica suggests that the disease might be managed by introducing hypovirulent isolates into the pathogen population. In Europe this has happened to some extent natu-

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rally. But the situation is quite different in USA. MacDonald and Fulbright (1991) have discussed aspects of the use and limitations of hypovirulence. In the USA due to larger genetic variations between Cryphonectria in that region, for the transfer to occur, hyphae must fuse and this often is limited by natural compatibility barriers. To overcome this difficulty there is now interest in engineering replicating forms of the ds RNA into a range of C. parasitica strains representing different compatibility types. In this way more efficient spread of the hypovirulence should occur and the disease epidemic could be restricted. Mycorrhizal fungi as biocontrol agents Mycorrhizal fungi have received considerable attention in recent years. Mycorrhizal fungi group better in infertile soils. The role of mycorrhizal fungi in the biological control of fungal root pathogens is well known (Reid, 1990, Chandra 1992). Several mechanisms have been postulated. These are: (1) creating a mechanical barrier for the pathogen to penetrate, (2) inducing thickening of cell walls through lignification and production of other polysaccharides, (3) stimulation of host roots to accumulate metabolites, (4) stimulating flavanolic wall infusions, (5) increasing concentrations of dihydroxy phenols in roots, (6) producing antibacterial and antifungal antibiotics, and (7) stimulating microbial activity in the root zone. Implementation of biological control is fraught with constraints, and successful application depends on a thorough knowledge of the ecology and biology of both the target pathogen and the biological control agent. Paramount to efficacious employment of biological control is knowledge of the specific mechanism of microbial antagonism involved. Documented biological control mechanisms for plant pathogens include competition, hyperparasitism, predation, and the production of extracellular metabolites such as antibiotics, hydrogen cyanide (HCN), and siderophores. Recently Miller et al. (1996) discovered a novel antimicrobial metabolite, a biosurfactant. Stanghellini and Miller (1997) have recently discussed the biosurfactants, their identity and potential applicability in the biological control of zoosporic plant pathogens. The intentional use of synthetic surfactants for the control of zoosporic pathogens was pioneered by Tomlinson and Faithfull (1980). During their studies on the control of big vein of hydroponically grown lettuce, which is caused by a virus vectored by zoospores of Olpidium brassicae, they observed that disease control was achieved following the addition of a benzimidazole fungicide (Bavistin, BASF) to the recirculating medium. It was subsequently discovered that the inert ingredients in the fungicide formulation exhibited lytic activity against zoospores of Olpidium. It was suspected that the inert ingredients contained a synthetic surfactant. Further studies demonstrated the lytic effects of various surfactants. Biosurfactants of diverse chemical nature are produced by a variety of microorganisms. For example, some isolated from the following genera are reported to produce surfactants: Pseudomanas, Bacillus, Arthrobacter, Rhodococcus, Actinobacter and Corynebacterium. Several fungal genera including Candida and Torulopsis are reported to produce surfactants. Perhaps the best studied of the biosurfactants is a class of glycolipids, the rhamnolipids, which are produced and secreted by Pseudomonas spp. Rhamnolipids as biocides have been demonstated by Stanghellini and Miller (1997). Biosurfactants produced by Pseudomonas aeruginosa were tested against a range of zoospores of Pythium aphanidermatum, Phytophthora capsici, and Plasmopara lactucae-radicis. At concentrations ranging from 5 to 30 mg/ml they caused cessation of motility and lysis of the entire zoospore population in less than a minute. Thus the possibility of utilizing bacteria or fungal species producing biosurfactants for biological control of plant pathogens on the aerial parts is a distinct possibility.

Management of Plant Diseases 239

Soil Amendments Many root diseases such as potato scab have been controlled by ploughing organic material into the soil, which started the chain of these investigations. Several others have been successfully controlled in this way, such as Phymatotrichum root rot of cotton, “take all” (Gaeumannomyces graminis), several Rhizoctonia and Fusarium diseases. In the case of Phymatotrichum root rot of cotton, it was shown by Mitchell et al. (1941), Clark (1942) and King et al. (1934) that fresh organic materials stimulate the germination of sclerotia and this exhausts the inoculum. In the case of Gaeumannomyces (Ophioblus) graminis, it has been suggested that organic amendments, rich in carbon and deficient in nitrogen, control the “take all” disease of wheat. This is explained as follows: As a result of organic amendments with a high C/N ratio, there is a tremendous increase in the microbial number and activity. There is considerable liberation of CO2 by soil saprophytes, which suppresses the pathogenic activity of this fungus. In the process of survival also, low nitrogen content in the soil reduces the longevity of the fungus. The organic amendments increase the activity of saprophytic organisms in a flush which results in the abundant liberation of CO2 as a result of the respiratory activity of the micro-organisms. The pathogens sensitive to CO2 are inhibited. Due to the rapid multiplication of micro-organisms in the soil, the available nitrogen in the soil is rapidly utilized by the fast-growing saprophytes. This results in acute nitrogen scarcity which adversely affects the growth of the pathogens. In their studies on the control of bean root rot with barley amendments, Snyder et al. (1959) established that additions of nitrogen nullified control. It is summarized that Fusarium solani cannot develop without nitrogen which is required for germination and penetration (Cook and Schroth, 1965; Toussoun et al., 1960). Further studies by Maurer and Baker (1965) show that C/N ratios greater than 25/1 significantly suppress symptoms of bean root rot. The germination of the chlamydospores is poor and the hyphae produced are also depauperate. This is perhaps due to the locking up of available soil nitrogen in the cells of microorganisms, which decompose the organic material. This is thought to deprive the chlamydospores of the pathogen of even the minimal amount of soluble nitrogen required for germination and host infection. The pathogen and the disease develop rapidly, if nitrogen is increased. It has been shown that the inability of the conidia to germinate in aqueous extracts of soil solution with cellulose (C/N ratio higher than 25) and barley (C/N ratio 55) can be overcome by as small a quantity of nitrogen as 5 ppm. All these factors show that nitrogen limits pathogenesis. Extensive data on the behaviour of Rhizoctonia solani have been provided by Papavizas and Davey (1960, 1961), Davey and Papavizas (1960) for the control of Rhizoctonia disease of snap bean (Phaseolus vulgaris) by the use of organic amendments. Blair (1943), in studies with Rossy Chlodny slides demonstrated that the growth of Rhizoctonia solani in unsterilized soil is checked by the incorporation of 1% finely divided organic matter, such as dried grass, alfalfa or wheat straw. Concerning the possible mechanism of this biological control, Davey and Papavizas (1963) have produced data that largely confirm the explanation originally given by Blair (1943) based upon the carbon dioxide content of the soil atmosphere and the scarcity of available nitrogen in the soil solution. Both effects are produced by rapid decomposition of nitrogen-deficient organic matter in the soil. Davey and Papavizas (1963) showed that maximum inhibition of competitive saprophytic colonization of buck wheat stem segments by Rhizoctonia solani occurred in soils receiving amendments adjusted to C/N ratios from 100–40. A lower degree of inhibition was provided by amendments with either higher (400200) or lower (20,10 and 5) C/N ratios. This result can be explained by postulating that nitrogen exerts two distinct and opposite effects upon the activity of R. solani, the first being predominant down to C/N ratio 40 and the second coming into action at C/N ratios of 20 and below.

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The first or inhibitory effect can be attributed, as Davey and Papavizas (1960) have pointed out, to the action of nitrogen in increasing the rate of decomposition of the organic material, and hence in promoting a more rapid out put of carbon dioxode. This increase in carbon dioxide output can be expected to closely follow the trend in total microbial number from C/N ratios 400 down to 5. At C/N ratio 20, however, the second or stimulatory effect (with respect to Rhizoctonia solani) of nitrogen comes into force and the total depressing effect of the organic amendment upon the fungus is suddenly reduced sharply. This can be ascribed to the presence of the available nitrogen that can be directly absorbed by the mycelium of Rhizoctonia solani, thus removing the disability of nitrogen starvation suffered by the fungus in the presence of organic amendment adjusted to higher C/N ratios. This change is precisely where we might have predicted it to have occurred, that is, in the region of C/N ratio of 20. The C/N ratios of fallow soils and of microbial substances is generally around 20. Mitchell and Alexander (1962) reported the control of root rot of bean by manuring soils naturally infested with F. solani f. phaseoli with chitin at the rate of 1020 kg/hectare. The number of actinomycetes was greatly increased following the addition of chitin, but not of bacteria, while the number of fungi was somewhat depressed. Pursuing the hypothesis that the addition of chitin and related substances to the soil promotes the heterolysis of chitin-walled fungi, Mitchell and Alexander (1963) reported the presence of a polysaccharide laminarin in the hyphae walls of F. solani f. phaseoli but not in Pythium debaryanum. Mitchell (1963) reported that soil amendment with either chitin or laminarin at 1020 kg/hectare reduces incidence of bean root rot and also of radish wilt due to Fusarium oxysporum f. conglutinans. But none of these amendments can control diseases caused by pathogens, which do not contain chitin in their walls, such as Pythium debaryanum and Agrobacterium tumefaciens. Phytophthora root rot of avocado was controlled by Zentmyer (1963) by amending the soils with alfalfa meal—a material of low C/N ratio. Similarly, Mehrotra and Tiwari (1976) could successfully control the root rot of Piper betle by organic amendments, and antagonists growing on organic amendments in the soil. Amendment with corn straw gave best control. Corn straw supplemented with nitrogen (NH4NO3) gave still better control. The effect of different antagonists growing on corn straw and til (Sesmum indicum) oil cake amendments showed that Trichoderma viride gave best control. Mehrotra and Claudius (1972) tried to control the root rot and wilt diseases of Lens culinaris by organic amendments of the soil and also by introducing antagonists in the soil along with the organic amendments. The wilt of lentil caused by Fusarium oxysporum f. lentis was checked with amendments of wheat straw, wheat straw plus ammonium nitrate, corn straw and sugarcane straw. The successful antagonists were T. viride and Streptomyces gougeroti. Biological control in potato diseases of underground parts has been studied by Shrivastava and Saksena (1968) at Sagar. In soil amendments, the Rhizoctonia disease was controlled by nitrogen-rich amendments, such as the tops of bean plants. Carbon-rich amendments such as cellulose and sugarcane straw did not control the disease at all. This could be explained on the basis of enhanced activity of Rhizoctonia solani which readily decomposes cellulose. The wilt disease due to Fusarium solani could not be controlled in these experiments by organic amendments. One noteworthy example of biological control with green manuring is that of potato scab with a soybean cover crop in southern California (Weinhold and Bowman, 1968). B. subtilis multiplied equally well in pots of soil amended with either green soybean or green barley tissue, although the bacterium multiplies in much greater amounts on soybean than on barley. There are many instances where attempts have been made to infest the seeds or propagules with organisms with a view to reducing root disease. The Russian workers are specially advanced in this line

Management of Plant Diseases 241

of work. Some workers have however, emphasized the inconsistent effects of seed inoculation with respect to biological control. Hyperparasitism Certain fungi and bacteria are parasitic on plant pathogens. The genus Cinccinobolus includes species of fungi, which attack members of the family, Erysiphaceae. Cinccinobolus seldom reduces the attacks of the powdery mildews in nature because it rarely occurs in large quantities. Cinccinobolus can be cultivated on artificial media, however, and some good results have been obtained by artificial inoculation. Several workers have tried to utilize Eudarluca australis (pycnidial stage known as Darluca filum) for controlling rusts. The fungus does control the disease under certain natural climatic conditions over which man has little control. Extra inoculum of the hyperparasites will not be of much use as the weather, unless very favourable to the development of hyperparasite is unlikely to permit any marked control of the disease. There are several other fungal species which attack rusts, such as Tubercularia spp. attacks aecia and spermogonia of several rusts and Verticillium hemileiae on Hemileia vastatrix. Many fungi are known to parasitize phytopathogenic nematodes. In 1934, Rozsypal and Schmidt reported a Protomycopsis chytridiale which attacks Heterodera schachtii. In 1933, H. Goffart recognized that the eggs and embryos of Heterodera avenae could be parasitized by an ascomycete, Cylindrocarpon radicicola, which is known to attack the underground parts of various cultivated plants. Hyphomycetes as nematode predators are numerous, and are frequently met with in nature. Drechsler (1937) described 40 species of fungi which attack nematodes. The important ones are Dactylaria, Acrostalgamus, Dactylella and Arthrobotrys. Several attempts have been made to utilize fungi for the control of nematodes, such as Meloidogynes which attacks pineapple in Hawaii, for nematodes parasitic on animals in France, and for controlling the golden nematode of the potato in England. Some encouraging results were obtained but practical utilization of these fungi in agriculture is still a remote possibility. Bacteriophages In 1917 d’Herelle presented evidence of a transmissible lytic principle that acted on the shiga bacillus, showing that bacteria have their infective diseases too. A number of phages have now been discovered for many phytopathogenic becteria such as Agrobacterium tumefaciens, Erwinia aroidea, E.carotovora, E.atroseptica, Pseudomonas angulata, P. coronafaciens, P. glycinea, P. lachrymans, P. phaseolicola, P. pisi, P. syringae, P. tabaci, P. xanthoclona, Xanthomonas campestris pv. citri, X. campestris pv. malvacearum, X. campestris pv. phaseoli, X. campestris pv. pruni, P. campestris pv. solanacearum, X. campestris pv. oryzae, etc. but not much success has been obtained in controlling these diseases in nature with the help of their respective phages. Also, no plant disease carried by a bacterium has been cured yet by treatment with phage after the diseases has developed. Entomogenous Fungi There are several fungi, which are capable of growing in the bodies of insects thus destroying them. Scientists are looking for ways of protecting crops against insect pests. Late in 1940, with the introduction of DDT, it was felt that a method had been found to destroy insects affecting crops. DDT and scores of other insecticides that were introduced exterminated not only all the insects but also the predators, including fish in water (the insect enemies), with the result that in some cases more damage than any tangible benefit has occurred. Plant pathologists can use micro-organisms, which are immune or at least not affected by pesticides for biologically controlling insects. One important fungus which has been utilized is Entomophthora. About 100 species are known, mostly as insect

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parasites. While some appear to be rather specific in their host range, others are capable of attacking a wide range of hosts. Some species have been grown in culture. One of them is Entomophthora coronata, a fungus which has been referred to by various names, for example, Delocroixia coronata, Conidiobolus villosus, C. coronata (Sreenivasan and Thirumalachar, 1964; Sreenivasan et al., 1964). This fungus is a parasite of aphids and termites but is also a common saprophyte of plant detritus. CHEMICAL METHODS

Plant diseases play an important role in determining the amount and cost of food. Plant pathology must alleviate the food problem by devising new control measures and improving the older ones. The annual loss to world crops as a result of disease has been estimated at 25,000 million dollars; of this, a major part is due to fungal pathogens. Surface deposits of fungicides can control many fungal diseases if properly applied, but it appears that more effective control can be achieved with systemic fungicides which can penetrate and move into the plant. There is an enormous amount of literature available on chemicals used for plant disease control. Some important Indian literature is that of Vyas (1983) “Systemic Fungicides”, Grover (1986) “Chemical control of plant diseases in India—an overview” and Nene and Thapaliyal (1993) “Fungicides in plant disease control”. Role of fungicides in modernizing agriculture in India by Mehta (1971); Chemical Control of Plant Disease: An Exciting Future by Sbragia (1975); and acquired resistance to fungicides by Dekker (1976). A comprehensive and up-to-date reference source available in the field of fungicides is Antifungal Compounds, in two volumes edited by Siegel and Sisler (1977), Thompson (1993) “Antifungal Chemicals, Book IV: Fungicides”. The use of chemical sprays, dusts or seed treatment for protecting plants from the ravages of pathogens is not an innovation of the 20 th century. The early agriculturists did try to control plant diseases by utilizing chemicals and fungicides. The first recorded mention of plant disease control is in the writings of the Greek poet, Homer (1000 B.C.), who mentions sulphur, which is still in use. Also, the Roman patriot, Cato (200 B.C.), mentioned the fumigation of trees with bitumen and sulphur. The first landmark in the control of fungal diseases of plants was the discovery by Anton de Bary that the causal agents of many plant diseases are fungi. The development of fungicides rapidly followed this discovery. It is about this time that Louis Pasteur discovered the bacterial parasite of the silk worm. He conclusively proved the real nature of fermentation and put an end forever to the doctrine of spontaneous generation. It is after this period that Pierre Alexis Millardet (1885) showed that the downy mildew of grapes could be controlled by mixtures of copper sulphate and lime. Copper sulphate had been in use even before that but it was only in 1885 that its toxicity was reduced by mixing it with lime. The Bordeaux mixture was used both in Europe and the USA for the control of many diseases. Substances used as late as the middle of the 19th century had offensive or caustic properties, acid or bitter taste and a pungent odour. These were regarded as good qualities in a fungicide. The Bordeaux mixture was followed by lime sulphurs in the late 19th century, and formalin, coper carbonate dusts and organomercurials (1913) as seed treatments. This gave way to an era of organic fungicides—Thiram, Chloranil, Dichlone were developed from the mid-1930’s onwards, and Captan, one of the most successful ones, was developed only in 1952. The emphasis then shifted to the oxides of copper. Many groups of chemical compounds are now available with the plant pathologists for plant disease control. These include heterocyclic nitrogen compounds, quinones, phenols and antibiotics. There is increasing

Management of Plant Diseases 243

interest in the chemotheraphy of plants, and systemic fungicides are of current interest to workers in this field. Before we discuss the various chemicals for plant disease control it becomes essential that we define a fungicide. A fungicide is a chemical agent that kills or inhibits the development of the fungus spore or mycelium. The fungicides used on plants may be classified as protectants, eradicants and therapeutants on the basis of their uptake by and mobility within plant. Protectants

As the name suggests, protectant fungicides are prophylactic in their behaviour. Protectant fungicides, which may be applied to seeds, plant surfaces or the soil cannot penetrate plant tissues in efficient amounts. Therefore, they act outside the plant prior to infection by the pathogen. Eradicants

These chemicals eradicate the dormant or active pathogen from the host. They can remain effective on or in the host for some time and function as protectants also. Chemicals can be protectants as well as eradicants. Therapeutants

A therapeutant is an agent that inhibits the development of a disease syndrome in a plant when applied subsequent to invasion by a pathogen. Therapy can be by physical means, such as solar energy treatment or hot water treatment of smutted wheat grains, but more often it is by chemical means and is then called chemotherapy. Usually the chemotherapeutants are systemic in their action, that is, they enter the plants and affect deep-seated infection. To be successful, fungicidal sprays must have some constant characteristics. To be effective against rain and dew they must be nearly insoluble in water, but at the same time, they must be soluble enough to function as fungicides. Tenacity or adhesiveness is another quality needed to enable them to stick well and last for a longer period. Further, they should not be toxic to the host parts (phytotoxic) so that they can be used safely. To meet all these requirements, various modifiers are mixed with the fungicides; these are known as stickers, spreaders and safners. The fungicides are dealt with below under the following categories: 1. Inorganic copper compounds 2. Inorganic mercury compounds 3. Sulphur sprays 4. Organic sulphur compounds 5. Quinone and phenolic fungicides 6. Heterocyclic nitrogen compounds 7. Benzene compounds 8. Organomercurials 9. Systemic fungicides

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10. 11. 12. 13.

Plant Pathology

Organophosphate fungicides Antibiotics Soil fumigants Oils

Inorganic Copper Compounds Bordeaux Mixture In 1882, Millardet, investigating downy mildew of the vine (Plasmopara viticola), a disease then recently introduced in France from the USA, observed that along the roadside at Medoc in Gironde, certain vines bore leaves while others had been defoliated by the disease. Millardet believed that the foliage on some of the vines had persisted because of the dabbing of the leaves with verdigris or with a mixture of lime and copper sulphate. Following this slender clue, Millardet in collaboration with Gayon, a chemist, was able to announce in 1885 the successful use of a lime-coppersulphate mixture as a fungicide against Plasmopara. This mixture has the reaction product of copper sulphate and calcium hydroxide. This discovery has doubtless been the greatest boon to plant protection since about fifty million kg of copper sulphate is commonly used in the USA for this purpose every year. The formulae for Bordeaux mixture varies. The original mixture contained 15% copper sulphate and 8% lime (CaO). The proportion of these now varies according to the crop and the disease. Higher concentrations are useful in the case of woody and tough tissues, while lower concentrations are used for succulent and softer parts. The following formula (5:5:50) is regarded as a good standard type. Copper sulphate (blue stone) 5 lb (2.268 kg) Stone or hydrated lime 5 lb (2.268 kg) Water 50 gallons The mixture is prepared by mixing CuSO 4 solution with limestone solution. The two solutions are prepared separately in wooden or earthen vessels and the diluted solution of CuSO 4 is slowly poured into a concentrated lime solution in a tub with constant stirring. The resulting mixture is neutral or slightly alkaline and is not injurious. The mixture is used almost immediately after preparation and deteriorates if left for some time. However, the mixture may be stabilized by adding sugar or jaggery at the rate of 0.5 lb in 50 gallons of the mixture. The mixture stabilized in this manner will not deteriorate for several days under normal conditions. Alternatively a stock solution of the two ingredients can be prepared separately and stored in wooden vessels. These can be mixed when actually required. The chemistry of Bordeaux mixture is complex and is not fully understood. The expected reaction is:

CuSO4 + Ca (OH)2 = Cu(OH)2 + CaSO4 It is not cupric hydroxide as would be expected by a straightforward reaction of one molecule of copper sulphate with one molecule of lime. The blue hydrogel contains tribasic copper sulphate [Cu4SO4(OH)6] with absorbed lime, free lime, and calcium sulphate. With absorbed lime, substances of various composition are formed, depending upon the ratio of lime to copper sulphate, their total concentration and the period of time that has elapsed after mixing. Eventually the blue hydrogel contains a series of compounds in transition rather than one final product. Cupric hydroxide is the active principle which is soluble and is toxic to spores and sporelings. The Bordeaux mixture is a suspension, which is fairly tenacious, nearly insoluble and spreads quite well. It may be detrimental or beneficial to different hosts. Beneficial effects have been observed on grape, potato and other crops. Greener foliage is often the outstanding result and it also has insect repellent properties.

Management of Plant Diseases 245

The Bordeaux mixture is sometimes phytotoxic to tender leaves and other parts and burns them as well as fruits and other aerial parts. Sometimes blemishes occur, as on apple fruit. The fungicidal action is due to the formation of soluble copper, which is toxic to spores or sporelings in drops of atmospheric water. The factor that brings about the solubility of toxic ions is still not fully understood. There are three possibilities: (a) Carbon dioxide and ammonia in meteoric water. (b) Secretions by the fungus either before or after spore germination. (c) Secretion from the host plant. Or all these processes might be instrumental in the solubility of copper. The Bordeaux mixture is specific against downy mildews, such as late blight of potatoes, koleroga of areca nuts, coffee rust, various leaf spots diseases, blights, anthracnoses, and so on. It has remained the standard fungicide against downy mildews of grapes and other hosts since its inception in 1882. Bordeaux paste Bordeaux paste consists of the same ingredients as those of Bordeaux mixture but it is in the form of a paste. It is mainly used for tree wound dressings to prevent fungal pathogen attacks such as in the control of stem bleeding disease of coconut. It is generally prepared by adding 1 lb of copper sulphate and 1 lb of lime in 1 gallon of water. Burgundy Mixture Burgundy mixture, which is a modification of the Bordeaux mixture, is often used for tender foliage, since it is less phytotoxic. In this, lime is substituted by sodium carbonate, and hence, is some times called ‘soda Bordeaux’. Copper sulphate 10 lb (4.536 kg) Sodium carbonate 12.2 lb (5.666 kg) Water 50 gallons Burgundy mixture does not produce blemishes. It should be mentioned that neither burgundy mixture nor other substitutes of Bordeaux mixture have been widely accepted. Cheshunt Compound Bewley suggested this compound in 1921. It contains two parts of copper sulphate and 11 parts of ammonium carbonate. The two substances are well powdered and thoroughly mixed and the dry mixture is stored in an airtight receptacle for 24 fours before being used. If it is left in an exposed condition it gradually loses ammonia and becomes less effective. When the spray solution is to be prepared, one ounce of the mixture is dissolved in a little hot water and two gallons of the solution is made by adding cold water. Cheshunt compound is recommended for the control of ‘damping off’ diseases as a soil drench in nursery beds. Chaubattia Paste This paste was developed by Singh (1942, 1943) at the Government Fruit Research Station, Chaubattia in the Almora district of Uttar Pradesh (now Uttaranchal) in India. The paste is prepared by mixing copper carbonate (800 g) and red lead (800 g) in one litre of lanolin or raw linseed oil. This paste was developed as a wound dressing fungicide to be applied to pruned parts of pears, apples and peaches for the control of diseases, such as stem-black (Coniothecium chomatosporum), stem-brown (Botryosphaeria ribis), pink disease (Corticium salmonicolar), stem canker (Monochaetia mali) of apples and pears, and collar rot of apples, peaches, apricots and plums (Rosellinia sp.). Inorganic Mercury Compounds Many inorganic mercury compounds are highly effective as fungicides and bactericides. Mercuric chloride (HgCl2) and mercurous chloride (Hg2Cl2) are used as

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1:1000 dilutions for soaking the seed, rhizome and corms of vegetables and flowering plants to control certain bacterial diseases. Club root disease of crucifers can be controlled by the treatment of Brassica seedlings against the pathogen. The use of inorganic mercury compounds is limited because they are highly toxic to animals and humans. Inorganic Sulphur Compounds The use of sulphur as a fungicide dates back to the Greeks but its effective use has been discovered only in the 19th century. Sulphur is effective against powdery mildews on many plants, but is also effective against certain rusts, leaf blights and fruit rots. Self-boiled Lime Sulphur This is made by using 8 lb of sulphur flour and 8 lb of burnt lime with 50 gallons of water. A small amount of water is added to the lime and when well shaken, sulphur is added and the ingredients are mixed vigorously. After boiling has ceased, the remainder of the water is added. This material is less toxic than the lime sulphur described above. Even this fungicide is now sparingly used and is being replaced by wettable table sulphurs. Lime sulphur has some eradicant properties also and is active against apple scab and powdery mildew. Wettable Sulphurs These are very finely divided (micronized) elemental sulphurs, also called floatation sulphurs, which are made from hydrogen sulphide gas from the coke industry. These have come into extensive use because they cause less foliage injury. Wettable sulphurs are used against apple scabs, powdery mildews of grape and other crops, ornamentals and vegetables. In green houses it is used against rusts also. Strangely enough, the mode of action of sulphur as a fungicide is still unclear. Originally it was assumed that sulphur acted directly on the protoplasm of the fungus spore or mycelium and destroyed it. Later it was suggested that sulphur acted in the form of H2S. Still others thought that it acted as sulphur dioxide. The most recent evidence suggests that sulphur acts as a hydrogen acceptor and thus interferes with the normal hydrogenation and dehydrogenation reactions of the cell. Organic Sulphur Compounds During the second world was there was a dearth of metallic compounds of Cu and Hg which gave impetus to the development of organic compounds. Tisdale and Flenner (1942) first demonstrated the fungicidal possibilities of carbamates in 1931 in the laboratories of E.I. Du Pont Company, USA but commercial production started a decade later. It was in 1934 that the patenting of dithiocarbamates was announded by Tisdale and Williams who patented ‘Thiram’ and the use of this group of materials as insecticides and fungicides. Since then volumes of work have been done on the chemistry of thiocarbamates. Carbamic acid is the starting point. It is derived from carbonic acid by the replacement of a hydroxyl group by an amino group.

** Dithiocarbamic acid is derived by the replacement of two oxygen atoms by sulphur, giving the hypothetical dithiocarbamic acid. From dithiocarbamic acid the following important fungicides have been derived.

Management of Plant Diseases 247

Thiram (Tetramethyl thiuram disulphide) TMTD is a condensation product of dithiocarbamic acid (2 molecules in which the H atoms have been substituted by methyl groups). This was used as an activator in the production of synthetic rubber.

S

S CH3

H3 C N—C—S—S—C—N H3 C

CH3 Thiram

Thiram is being sold under a variety of other trade names, such as ‘Arasan’, ‘Tundas’, ‘Terson’, ‘Tulisan’, and so on. It is a leading seed treatment for wheat, rice, gram, peas, mustard, linseed, sorghum, and onion. Thiram is also used in the control of foliage diseases and acts as an insecticide. Among the soil-borne pathogens controlled by it are Pythium, Rhizoctonia, Fusarium and Protomyces. As a foliage spray, Thiram was not as successful as its metallic compounds. For this reason, several compounds have been developed which are as follows: Ferbam (Ferric dimethyl dithiocarbmate) In Ferbam, three molecules of dithiocarbamic acid react with one atom of iron. Its trade names are Fermate, Ferbam, Karbam or Coromet. Ferbam is compatible with most pesticides, but its use with copper, lime or mercury compounds tends to reduce fungicidal efficiency. Ferbam has been successfully used as a protectant fungicide against a wide variety of fungus diseases of fruits and vegetables. As a foliage spray it is also used to correct iron deficiency.

Ziram (Zinc dimethyl dithiocarbamate) The trade names are Cuman, Zerlate, Karbam white, Corozate, and so on. In India, this fungicide is available under the trade name of Cuman. It is used against many foliar diseases in field and orchard crops.

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Plant Pathology

Nabam (Disodium ethylene bisdithiocarbamate) This is sold as Dithane D-14, Parzate liquid or Dithane A-40. Nabam has been found effective against many leaf blights, such as early and late blight of potatoes and seedling blights caused by Pythium, Rhizoctonia and Fusarium species. A similar compound of zinc, Zineb was later developed.

H

S

H2C—N—C—S—Na H2C—N—C—S—Na H S Nabam

Zineb (Zinc ethylene bisdithiocarbamate) Zineb is derived by the substitution of sodium in Nabam with zinc. It is called Dithane Z-78, Parzate C or Lonacol. Zineb is mainly used for foliar sprays against diseases, such as early and late blight of potatoes and tomatoes, blast of rice, leaf blight of rice, ripe rot of chillies and downy mildew of maize.

H

S

H H H

C—N—C—S Zn C—N—C—S

H H

S

Zineb

Maneb (Manganese ethylene bisdithiocarbamate) This is called Dithane M-45. Maneb is excellent for the control of foliage and fruit diseases of many vegetables, flowers, trees, turf and some fruits. In many diseases Maneb has been found more effective than Zineb. It is especially used in vegetable crops as a spray material. Some of the diseases controlled by Zineb are bean anthracnose caused by Colletotrichum lindemuthianum, downy mildew and anthracnose of cucurbits, fruit rot of chillies, citrus greasy spot caused by Cercospora spp., lettuce downy mildew, maize leaf blight, blight of tea caused by Exobasidium vexans, and leaf spots and blights caused by Alternaria, Phytophthora and Cercospora on crops, such as potato, tomato, cabbage, carrot, beet, and cauliflower. In all these dithiocarbamates, the usual dose is 1 kg in 500 litres of water. These carbamate compounds are very widely used both as dust and sprays for a very wide variety of crop diseases, such as Alternaria blights of potato and tomato, Colletotrichum anthracnose of tomato, cucurbit, bean, several rusts of Uromyces spp., several downy mildews of vegetables by Peronospora, Plasmopara and many others like brown rot and scabs.

Management of Plant Diseases 249

H

S

H H H

C—N—C—S Mn C—N—C—S

H H

S

Maneb

Two important clues to the toxic action of the dialkyldithiocarbamates and thiuram disulphides are their formation of metal chelates and their polymodal dosage response. The first property is well known and is commonly used in analytical chemistry. The second property was first reported by Dimond et al. (1941) for Thiram. This latter effect is also called ‘inversion’ and quenching is described in detail and explained by Goksoyr (1955). The zone of inversion where increasing dosage actually depresses toxicity has been particularly hard to explain. The dimethyldithiocarbamate ion combines with minute traces of copper in the medium to give the highly toxic 1:1 or half chelate. Cu+ This is the toxic species. But as the amount of copper in the medium is constant, the addition of more dithiocarbamate forms the less toxic 1:2 or full chelate, and toxicity falls.

S 2 Cu.

CH3—N—C S

As more dithiocarbamate is added, the intrinsic toxicity of the dimethyl dithiocarbamyl ion exerts itself, acting perhaps by adding essential metallic micronutrients. Thus, toxicity increases again. The toxicity of ethylene bisdithiocarbamates is believed to be due to the production of isothiocynate which reacts with SH groups. Quinone and Phenolic Fungicides Though not as important as the carbamates, quinone and phenolic compounds are quite important as fungicides in certain respects. Chloranil or Spergon (Tetrachloro-p-benzoquinone) It was reported in 1940 by Cunningham and Sharvelle to give protection to lima bean seedlings from damping off. Chloranil is mainly used in seed treatment. It is sold as Spergon and is used in seed and bulb treatment of legume flowers and vegetables. It is also used as soil drench.

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Plant Pathology

Dichlone or Phygon (2,3 dichloro-1,4 naphthaquinone) This is a later development. Though it is used for seed treatment, it is also useful as a foliage spray against many plant diseases and is apt to cause dermatitis. It has been reported four to eight times more effective than chloranil in the protection of legume and cotton seed.

There are two possible mechanisms by which Chloranil and Dichlone act – bringing the quinone nucleus to – SH and NH2 groups in the fungus cell and disturbances in the electron transport system. Phenols Phenols have shown exceptional qualities as bactericides, but as fungicides their use has been limited. Phenols have been chiefly applied to textiles and woods as preservative fungicides, but have also been used as soil fungicides and in seed treatments. Derivatives of cresol and O-phenyl phenol have been mainly used as preservatives. The bisphenols are a particularly interesting group of phenolic fungicides. Heterocyclic Nitrogen Compounds These compounds have a number of valuable fungicidal properties. Their principal uses are as foliage protectants and eradicants of fruits and vegetables. Glyodin (2heptadecy 1-2 imidazoline acetate) and Captan are the two important fungicides of this group. Captan Captan is the common name adopted for N-trichloromethyl thio-4-cyclohexene-1,2 dicarboximide. Kittleson (1952) prepared this compound and so it is some times called Kittleson’s killer.

Captan is commercially sold under different names, such as Captan 50 W and Esso fungicide 406, but it is also claimed that it acts systemically. Hochstein and Cox (1956) suggested that Captan competes with co-carboxylase (thiamine pyrophosphate) for sites on coenzyme-free carboxylase in the decarboxylation of pyruvate, and thereby interferes with the process of decarboxylation. Another theory of Captans action was presented by Lukens and Sisler (1958). They found that Captan interacts with – SH compounds, such as cystein and glutathione, to give thiophosgene. Although a fairly broad spectrum

Management of Plant Diseases 251

fungicide, it is not generally effective against rusts, powdery mildews and downy mildews. Owens and Blaak (1960 a,b) showed that Captan inhibits many oxidative enzymes and considered that its basic toxic action was an interaction of the intact Captan molecule with free sulphydryl groups. Captan is used as a spray, dust or for seed dressing. It has been used against diseases of different types of crops, but its use in fruit disease control and as a seed-dressing fungicide has found popularity all over the world. Captafol or Difolatan [N-(1, 1, 2, 2-tetrachloro ethyl sulfenyl)-cis 4-cyclohexene 1, 2dicarboximide] Captafol or Difolatan has properties similar to Captan. It is good fungicide for the control of early and late blight of potatoes and tomatoes.

O

O Cl Cl

C

C

N—S—C—C—H C O Difolatan

Cl Cl

C17H35

Cl N—S—C—Cl

C O Folpet

Cl

O

C N

NH2

H2C

CH2

O

C

CH3

Glyodin

Folpet (N-trichloromethylthio phthalimide) It is an analogue of Captan. Folpet is effective against Sphaerotheca pannosa. It is sold under the trade names of Phaltan, Orthophaltan, and so no. Glyodin (2-heptadecyl-2-imidazoline acetate) West and Wolf (1955) suggested that the biological activity of Glyodin depended on the interference, in the biosynthesis, of those purines which are components of nucleic acids. It is effective against Venturia inaequalis, Coccomyces hiemelis, Diplocarpon rosae, and Podosphaera leucotricha. Benzene Compounds Many benzene compounds are toxic to micro-organisms and several have been developed as fungicides. These have been discussed by Corden (1969). Dinitro-O-cresol is contained in the formulation called Kerenite or Elgetol. It is used as a dormant spray for the control of many diseases of fruit trees and ornamental plants and for the treatment of wounds in trees.

Chloroneb The fungicide, Chloroneb, 1,4-dichloro-2,5-dimethoxy benzene, was introduced by Ryker (1965) for the control of seedling diseases of cotton. It has been found active against R. solani, S. rolfsii and Phytophthora cinnamomi. This is a systemic fungicide and is unusual in that its activity does not follow clear-cut taxonomic divisions. This fungicide has been used for the most part as a seed or as in-furrow soil treatment at planting time for systemic control of treatment of seedling diseases of cotton, beans, sugarbeets and soybeans.

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Plant Pathology

Pentachloronitrobenzene (PCNB) This is sold as Quintozene, PCNB, Terrachlor, Brassicol, and so no. The chlorinated nitrobenzenes were introduced as fungicides in 1935 by Brown. They control many soil-borne diseases. It is used primarily against Rhizoctonia and Plasmodiophora. PCNB also has nematicidal properties.

Cl

Cl NO2

Cl Cl

Cl

Pentachloronitrobenzene

Diseases caused by Rhizoctonia, Sclerotium, and others are controlled by PCNB. It is not effective against pythiaceous fungi. Dexon This is p-(dimethylamino)-benzene-diazosodium sulfonate and is used against soil-borne phycomycetous fungi causing root rot and damping off diseases of plants. Zentmyer and Gilpatrick (1960) found that Dexon had a curative effect on avocado seedlings afflicted with Phytophthora cinnamomi when applied in the root region.

Chlorothalonil (tetrachloro-isophthalonitrile) Sold as Daconil, Bravo and Termil this is a popular fungicide. It is a broad-spectrum contact fungicide belonging to the phthalimide group. It is used for the control of early and late blights, leaf spots, powdery mildews, downy mildews, apple scab, coffee berry disease, rusts (yellow and black), sigatoka of banana, and leaf spots of ground nuts. Dinocap (Karathane) Dinocap or Karathane is a mixture of isomers of 2-(1-methyl-heptyl), 4-6 dinitrophenyl crotonate. Dinocap is an excellent substitute for sulphur for the control of powdery mildews and is highly specific against them. It is also effective against mites. Actually it was developed as a miticide and is now extensively used against powdery mildews.

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Non-aromatic Organic Compounds This group includes Dodine (n-dodecylguanidine acetate) used for the control of apple scab and several other diseases caused chiefly by Ascomycetes and Fungi Imperfecti. Due to its toxicity to some crop plants, its use is limited. The mechanism of action of Dodine is due to its binding with sterols in the cell membrane, with the result that the membrane structure is altered. Organomercurials Organomercurials developed due to efforts directed towards finding less toxic substitutes for inorganic mercurials. The first important commercial product was ‘Uspulum’ of the Bayer Company of Germany in 1915. Organomercurials are very widely used for seed treatment. They are derivatives in which the mercury atom is attached directly by one or both valency bonds to carbon atoms. The general structure of these derivatives is R.Hg.X where R represents a hydrocarbon with or without substituted groups, X represents an acid radical, such as chloride, nitrate, acetate, benzoate, gluconate. R is usually an ethyl or phenyl group. Some of the important compounds are Ceresan M (ethyl mercury p-toluene sulphanamide), Panogen (methyl mercury dicyandiamide), new improved Ceresan (ethyl mercury phosphate), Ceresan wet, Agallol or Aretan (methoxy ethyl mercury chloride), Ceresan dry (phenyl mercury acetate) and Agrosan G.N. (phenyl mercury acetate and ethyl mercury chloride mixture). Many other trade names are used for compounds which are in wide use. The chemicals can be used either as dips or dusts. Organomercurial fungicides are mainly used to control externally seedborne diseases since these compounds are able to eradicate the inoculum from the seed. Their use in the control of externally seed-borne cereal smuts has been widespread. Tubers and bulbs are also similarly treated with dusts against adhering organisms, such as potato scab and black scurf. The hazards which organomercurials pose are too well known. Because of their persistence they get into the food chain. Several countries have now banned the use of these fungicides. India has also banned the use of organomercurials (Nene and Thapaliyal,1993). Systemic Fungicides A new aspect was added to the control of fungal pathogens of plants when the oxathiin group of chemicals was developed in 1963. These compounds are systemic in their action and are receiving the active attention of pathologists. Most systemic compounds are absorbed by the roots and transported acropetally to the xylem. They are usually not transported downwards through the phloem tissue. Major advances in plant disease control will now be made from systemic fungicide-compounds, which penetrate and move from their site of application to other parts of the plant. A systemic fungicide could eradicate established infection and protect the new parts of the plant as they are produced and it would not be subject to loss by weathering. The idea of controlling disease by systemic fungicides is not new. Much of the earlier work has been done with antibiotics in view of their selective action against

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Plant Pathology

bacterial pathogens in animal tissues: sulfonalinides were investigated for the same reason. Systemic insecticides have been in use for several years, and after much effort, effective systemic fungicides are now becoming available. Vyas (1984) has ably dealt with systemic fungicides. They can be applied in solution as spray on the foliage but application to foliage is less likely to be effective against the pathogens responsible for root diseases. Several systemic fungicides have been tested as seed-dressing to eliminate seed infection. Systemic fungicides have not been very successful in the case of trees and bushes. From the practical point of view, the systemics offer unique possibilities in the tropics, since the active spray residues are not subjected to tropical weathering, thus permitting fewer applications. Action of Systemic Fungicides Very little is known about the mechanism of systemic fungicides. The following are the possibilities: 1. Inactivation of the enzymes and toxins of the pathogen. 2. Selective accumulation of the fungicide due to greater permeability of the fungus cell wall. 3. Damage to the membranes of the fungal hyphae and inhibition of structures, such as appressoria, cushion formation, emergence of germ tubes, and formation of haustoria. 4. Inhibition of fungal enzymes or their destruction. Systemic fungicides are more specific in their action than non-systemic fungicides. The study by Horsfall and Zentmyer at the Connecticut Agricultural Research Station of 8hydroquinonline as a systemic chemotherapeutant against Dutch elm disease was the first of its kind. Since 1964, however, a perplexing variety of successful systemic fungicides has appeared in quick succession on the agricultural scene. The following are some of the systemic fungicides used in plant disease control. Benomyl This is also marketed as Benlate. This is methyl-N (butyl carbamoyl)-2 benzimidazole carbamate. Benlate was discovered by E.I. du Pont de Nemours and Co. Inc. in 1968. The benzimidazoles have been very effective in the control of Mycosphaerella on banana and peanut. The systemic activity of Benomyl, the most striking fungicide at present, was first reported by Delp and Klopping (1968). Benomyl hydrolyses in acidic solution to methyl benzimidazole carbamate (MBC). MBC has been shown to be the principal fungitoxicant of Benomyl, which is systemic in plant tissue.

Benomyl has been found effective against a wide range of pathogens. It has a high degree of mite-ovicide activity. It is effective against Cercospora leaf spot of sugar beet, rice blast, apple scab, powdery mildew on cucurbits, cereals, and legumes. It is ineffective against Oomycetous pathogens and dark-coloured fungi such as Alternaria and Helminthosporium, some basidiomycetes, and bacteria. Application of Benomyl induced a remission of symptoms of spike disease of sandal caused by MLOs. Many soil-borne pathogens can be controlled, provided the roots are confined to the Benomyl-treated soil.

Management of Plant Diseases 255

Another benzimidazole derivative is Thiabendazole, a thiazolyl compound originally developed as an antihelminthic drug by E.Merck and Co. Inc. In 1964 Starol and Allard described its systemic fungitoxicity for the first time. Another derivative is Bavistin. This is 2-(methoxy-carbamoyl)benzimidazole which is effective against Ascomycetes and Fungi imperfecti which are considered to be hard to control. It is ineffective against phycomycetous fungi. Bavistin is translocated within the plant and has both prophylactic and curative actions. The actions of Benomyl and MBC are usually the same with most fungi. DNA synthesis is severely affected before other systems are affected (Clemons and Sisler, 1971). A study of synchronized cells shows that the inhibition of DNA synthesis occurs after a disruption in mitosis. Doublets formed contain only a single compact nucleus (Hammerschlag and Sisler, 1973). Benomyl can affect respiration, RNA and protein synthesis. Secondary actions can be attributed to butyl isocynate a co-product of MBC in the hydrolysis of Benomyl. Terrazole Terrazole-5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole is toxic to phycomycetous fungi, such as Pythium and Phytophthora. It has a systemic action in plants. Little is known about its fungitoxic action. Like most other systemic fungicides the serious disadvantage of the commercially-used present-day fungicides is that their repeated use results in the selection of resistant fungal strains.

Thiophanates Thiophanates seem to occupy an exceptional position since they are not heterocyclic compounds, but instead disubstituted benzene derivatives. In 1962, Aelbers (1971) related the development of these bisthioureido benzene derivatives to the discovery by Sijpestein and Pluijgers of the systemic protectant action of phenylthiourea. It was found that both methyl and ethyl thiophanate are broad spectra fungicides which have each the same antifungal spectrum as Benomyl, though it is at a somewhat lower level of activity. Thiophanate (methoxy carbonyl thioureido benzene) is sold as Topsin M or Cercobin M.

The fungitoxicity as well as systemic action of methyl and ethyl thiophanates may at least in part be attributed to their easy transformation into MBC and its ethyl analogue, respectively.

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S

O

SH

O

NH—C—NH—C—OCH3

NH—C—NH—C—OCH3

NH—C—NH—C—OCH3

NH—C—NH—C—OCH3

S

O

S

O

Thio form

Methyl thiophanate

- H2S N

O C—NHC—OCH3

N MBC

O

N

C—NH—C—OCH3 N—C—NH—C—OCH3 S

O

Thiocarbamyl analogue of benomyl

‘pp’ 675 A pyrimidine derivative coded ‘pp’ 675 was discovered by the Jealott’s Hill Research Station of Imperial Chemical Industries Ltd., and found to give outstanding results as a soil treatment for the control of powdery mildew (Sphaerotheca fuliginea) on the leaves and stems of cucurbits. Chemically it is 5-butyl-2 dimethylamino-4hydroxy-6 methylpyrimidine.

Oxathiins In 1966, Von Schmelling and Kulka of Uniroyal announced the development of carboxin, 2,3-dihydro-5 carboxy anilido-6 methyl-1, 4, oxathiin, the effects of which were discussed by Edgington et al., in the same year. The oxathiins are systemic fungicides which are effective only against Basidiomycetous fungi, such as rusts, smuts, Rhizoctonia solani (Corticium solani). Vitavax (2,3 dihydro-5-carboxyanilido-6-methyl 1-1,4, oxathiin) has been used to free barley seed from Ustilago nuda (loose smut) and Ustilago segetum var. tritici (U. tritici) from infected wheat seeds. Plantavax, the sulphone derivative of Vitavax, is effective against rusts. Seed or soil treatment has given good control. The oxathiins are taken up by sensitive and insensitive fungi alike, with most of the fungicides accumulating in the ribosomal and soluble fractions of disrupted cells. Although synthesis of protein and nucleic acids is strongly interrupted, the site of action is the inhibition of succinate dehydrogenase in the mitochondrial fraction. Oxathiin compounds adversely affect nitrogen metabolism of the pathogens (Grover and Chopra, 1972). The hexose monophosphate shunt appears to be an important respiration pathway in sensitive fungi.

Management of Plant Diseases 257

H2C H2 C

O

CH

H2

CH

H2

S

O

S

CH3 C— —NH— O O

Oxathiin

Carboxin (Vitavax)

Calixin Among the recently-developed systemic fungicides is Calixin with excellent prophylactic and curative action against powdery mildew of cereals (Erysiphe graminis), Sigatoka disease of banana (Mycosphaerella musicola), and other ascomycetous pathogens. Its chemical name is N-tridecyl-2,6dimethyl morpholine, commonly known as Tridemorph.

CH3 C13H27 æN

N CH3

Calixin (Tridemorph)

Acyalanines These are acyalanides. In 1973, Ciba Geigy in Basle, Switzerland, discovered a new class of chemicals which showed promise with regard to curative activity and systemicity against Oomycetous pathogens. The most important acylalanine fungicides is Metalaxyl. It is sold as Ridomil for use in the soil. It is also sold as Apron for use as seed dressing. Studies on the mode of action of Metalaxyl on fungi has been reported by Staub et al. (1980). Metalaxyl interferes with the development of the pathogens only after these pathogens penetrate their hosts. Metalaxyl interferes with RNA synthesis. It has also been reported that Metalaxyl fungicide elicites the production of phytoalexin in the plants which imparts resistance. Organophosphate Fungicides Historically, organophosphorus compounds are important because the first practically applied systemic fungicide Triamiphos, is a member of the group. The fungicides in this group are Kitazin, Ediphenphos (Hinosan), Pyrazophos (Aufgon, Euramil), and Triamiphos (Wepsin). Kitazin Kitazin, a systemic fungicide developed by Kumiai chemical Industry Tokyo, Japan. The first fungicide of this group was S-benzyl diethyl phosphorothioate (Kitazin). Later another compound Kitazin P was developed which is now used for the control of blast disease of rice. Kitazin inhibits the incorporation of C-glucosamine into the cell wall fraction of Pyricularia grisea. Chitin biosynthesis is interfered with this systemic fungicide which was earlier developed as an insecticide. Triazoles Triazoles include several excellent fungicides such as RH-124 (INDAR), Triadimenol (BAYTON), Triadimefon (BAYLETON), Biloxazole (BAYCOR), Diclobutrazole (VIGIL), Tricyclazole and several others. These fungicides show great protective and curative activity against a broad range of foliar, root and seeding diseases such as leaf spots, blights, powdery mildews, rusts, smuts and others. Morpholines Morpholines are heterocyclic ring compounds, which have yielded derivatives showing systemic movements in plants. Two compounds have reached commercial use. Morpholines interfere

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with the function of cell membranes and also in some cases with respiration. Triademorph (Calixin) a morpholine derivative was developed under the code name BAS 220 05F (formerly BAS 220 F) by BASF A6, West Germany. It is marketed under the trade name Calixin. Triademorph is active against representatives of all taxonomic groups of fungi except Peronosporales. Phosphites Two phosphite compounds, Aluminium–tris (aluminium ethyl phosphite) and LS 73 IO38 (sodium ethyl phosphite) have been developed by May and Baker Ltd., UK as first systemic fungicides based on organometallic compound effective against Oomycetous fungi. However only Aluminium tris has reached commercial production. Aluminium tris is not toxic to fungi in vitro but gives excellent prevention and has curative activity in fields for the control of diseases caused by Oomycetous fungi. Aluminium – tris (Aliette) This has been introduced under the code name LS 74-783 by Societe Rhone-Poulenc, France and is being marketed by May and Baker Ltd., UK under the trade name Aliette. It possesses the following structural formula.

O

H5C2 æ O P H

O

Aluminium-tris

There are quite a number of other systemic fungicides such as fungicides belonging to Piperazines (heterocyclic nitrogenous compounds) such as Triforine (Saprol). Triforine controls cereal, vegetable, fruit, and flower diseases such as powdery mildews, rusts, scabs, rots and post-harvest rots by foliar and soil applications. Pyrimidine and Pyridine derivatives such as Triarimol, Pyroxychlor Triarimol is a broadspectrum fungicide which is particularly effective for the control of scab and powdery mildew of apple and pear. Triarimol inhibits sterol synthesis in fungi. Pyroxychlor is a fungicide based on the pyridine nucleus. The compound is of great interest because it was the first compound effective against phycomycetous fungi and capable of controlling root diseases by foliar application because of its downward translocation. Then there are the Acetamides (acetic acid amides) such as DPX-3217 (Curzate) effective against Oomycetes, aliphatics such as prothiocarbs (Previcur) effectively inhibiting Oomycetes such as Pytium and Phytophthora, Peronospora and Bremia spp. Antibiotics Antibiotics are substances which are produced by micro-organisms and which act against micro-organisms. Most antibiotics known uptil now are products of actinomycetes and some are from fungi and bacteria. The chemical nature of antibiotics is complex and they are not, as a rule, related to each other. A large number of antibiotics have been tried for plant disease control but only a few have been successfully utilized. The important antibiotics for the control of plant diseases are Streptomycin, Tetracyclines, Griseofulvin, Cycloheximide and Aureofungin. The use of antibiotics is a comparatively new method of plant disease control, but antibiosis or antagonistic phenomena among micro-organisms and their relation to plant diseases have been recognized for more than 75 years. Gliotoxin, the first antibiotic to be used for plant disease control, was isolated and purified by a plant pathologist even before the discovery of Penicillin.

Management of Plant Diseases 259

Bajaj and Ghosh (1975) in an article on antifungal antibiotics have made the following observation: “The advent of antibiotic era had raised the hope that the antibiotics would provide us with much awaited chemotherapeutants effective in plant disease control. As far as fungal diseases are concerned, this expectation has not been fulfilled to a significant level. This fact will be evident when the plethora of reports on new antibiotics effective in vitro against numerous phytopathogenic fungi, that had been pouring in for the past three decades, is compared with the meagre number of antibiotics which have found any real application in plant disease control under field conditions till today”. Zaumeyer (1958) reviewed the antibiotics used for controlling plant diseases. Misato (1977) discussed in detail the development of agricultural antibiotics. Streptomycin It is produced by the actinomycete Streptomyces griseus. Streptomycin controls both gram positive and gram negative plant pathogenic bacteria. Streptomycin or streptomycin sulphate is sold as Agrimycin, Phytomycin, ortho-streptomycin, and so on, and when used as a spray shows activity against a broad range of bacterial pathogens, such as Xanthomonas campestris pv. citri, the cause of citrus canker, Xanthomonas campestris pv. juglandis of walnut blight, Xanthomonas campestris pv. vesicatoria of bacterial leaf spot of tomato and pepper, Pseudomonas syringae pv. phaseolicola of hollow blight of French bean, and Erwinia amylovora of orchard trees to be effectively controlled with an antibiotic under commercial conditions. This was first reported by Murneek (1952) of Missouri. In 1953, Goodman obtained 100% control of fire blight of apples by spraying streptomycin either in combination with oxytetracycline (Terramycin) or alone at concentrations of 100 to 500 ppm. Streptomycin is also used as a dip for potato seed pieces against various bacterial rots of tubers and as a seed disinfectant in bacterial pathogens of beans, cotton, crucifers, cereals, and so on. Although streptomycin is an antibacterial antibiotic, certain fungal diseases (caused by Oomycetes) are also suppressed by it. Saksena (1977) has reported that Phytophthora parasitica var. piperina, responsible for foot rot and leaf rot of Piper betle, can be controlled when cuttings are dipped in a solution of streptomycin sulphate. Streptomycin has been used to control early infection of hop downy mildew (caused by Pseudoperonospora humuli). It has been found effective to some extent against late blight of tomatoes and potatoes; even as little as 4 mg/ml of nutrient solution is sufficient to check P. infestans completely on tomatoes. In greenhouse and field tests, it has been found that the seed piece decays caused by Erwinia atroseptica and Pseudomonas fluorescens could frequently be controlled by immersing infected, freshly cut potato seed pieces in water solution of this antibiotic or a mixture of this antibiotic and oxytetracycline hydrochloride. The activity of streptomycin against bacterial plant diseases may be explained as being due to a direct action of the antibiotic on the parasite. It is more difficult to understand its effect against diseases caused by fungi, mainly Oomycetes such as late blight of tomatoes and potatoes and hop downy mildew (Pseudoperonospora humuli). A mixture of streptomycin and 8hydroxyquinoline sulphate as a combination has been successfully chemotherapeutic for black leg disease of geraniums. Cycloheximide This is commonly referred to as ‘Actidione’ –the trade name of Upjohn Co. and is obtained as a by-product in streptomycin manufacture. Cycloheximide (actidione), a glutarimide antibiotic, is produced by different species of Streptomyces, including S. griseus and S. nouresi. A demonstration by Kerridge (1958), showing that the cyclohexomide is a potent inhibitor of protein synthesis in Saccharomyces carlsbergensis, was followed by similar observations involving other organisms by

260

Plant Pathology

various workers. It is active against a wide range of fungi and yeast but is inactive against bacteria. Cycloheximide is the common name for b-[2-(3,5 dimethyl-2-oxcylohexyl)-2-hydroxyethyl] glutarimide. It has been reported to control cherry leaf spot caused by Coccomyces hiemalis at 2 ppm concentration. It is also used to control powdery mildew of beans caused by Erysiphe polygoni. This has also been utilized for the control of covered smut of oats caused by Ustilago hordei, bunt of wheat caused by Tilletia spp., brown rot of peach caused by Sclerotinia fructicola, and the post-harvest rots caused by Rhizopus and Botrytis spp. However, its use is limited, as it is extremely phytotoxic. Cycloheximide is sold as Actidione, Actispray, Actidione PM, Actidione RZ, etc. Griseofulvin This is produced by Penicilium griseofulvum, P. patulum, P. nigricans and P. jancyewski, is toxic to several plant pathogenic fungi, such as members of powdery mildews, Botrytis fabae, Alternaria solani, and some rusts. It is active against chitinous-walled fungi. Griseofulvin has been successfully used for the control of tulip fire caused by Botrytis tulipae. Root application of griseofulvin was found to be quite effective against certain powdery mildews, Botrytis diseases and bean rust (Uromyces appendiculatus). The systemic control of several powdery mildews, which are rather resistant to most fungicides, is interesting. Brian (1960) concluded that systemic control is not very practical since it is usually found to be no better or worse than other fungicides already in general use. In 1960, Brian postulated a hypothesis that Griseofulvin interfered with the biosynthesis of chitinous elements of fungal cell walls, which could also explain the relative non-toxicity of this chemical to animals and higher plants. Subsequent studies have shown that this antibiotic does not inhibit the synthesis of protein, chitin and lipids, nor does it alter the rates of respiration, glycolysis or cell well synthesis in fungal cells. Byrde (1959) reported that Griseofulvin (0.1%) is effective as a foliar spray for the control of apple rot caused by Sclerotinia fructigena. Monilia disease of apples caused by S. mali was successfully controlled in Japan by Griseofulvin. Blasticidins It is produced by Streptomyces griseochromogenes. In Japan it has been utilized for the control of blast disease of rice (Pyricularia grisea). It is active against both bacteria and fungi but is selective in its activity against the latter. The antibiotic inhibits the rice blast fungus at concentrations of 5 to10 mg/ml. This antibiotic interferes with protein synthesis. Aureofungin Aureofungin is a new broad-spectrum antifungal antibiotic produced in submerged culture by Streptoverticillium cinnamomeum var. terricola Thirum. (Thirumalacher et al., 1964). Chemical characterization indicated that it belongs to a new aromatic group heptanes and is, therefore, distinct. Its remarkable property is that it is absorbed when applied as a spray or given to the roots as a solution. Thus, it is translocated and can be detected in other parts of the plant. This has been detected both by bio-assay methods as well as by spectrophotometry. The most useful property of the antibiotic is its high activity against a large number of phytopathogens and its absorption and translocation in living plants. Some of the examples of effective control of plant diseases incited by pathogenic fungi are: Citrus gummosis incited by Phytophthora species, identical with P. citrophthora, which is a devastating disease in several parts of India and which has been controlled by Aureofungin and powdery mildew of apples incited by Podosphaera leucotricha which is completely controlled by spraying 100 ppm antibiotic spray solution (Agarwal et al., 1970). Systemic translocation and the arresting of Dutch elm disease has been reported by Dr. Benedict, University of Windsor, Canada.

Management of Plant Diseases 261

Spraying Aureofungin controls downy mildew, powdery mildew, and anthracnose of grapes. Seed treatment of rice effectively controls Helminthosporium oryzae. Similarly, Pyricularia grisea of rice and ragi are effectively controlled by Aureofungin. Diplodia rot of mango and Alternaria rot of tomato, Sclerotinia rot of peach, Pythium rot of cucurbits (Sharma and Wahab, 1970-71) have been controlled by Aureofungin. Penicillium rot of apples has been controlled by Aureofungin. (Naik and Joshi, 197374). Stripe disease of barley caused by H. gramineum has been controlled by Aureofungin (Dharamvir and Raychaudhuri, 1968). Interestingly Aureofungin has also been reported to possess plant growth stimulatory activity. Tetracyclines Tetracyclines are antibiotics produced by a number of species of Streptomyces. Terramycin (oxytetracyline), Aureomycin (chlorotetracycline) and Actinomycin (tetracycline) have been used for plant disease control. Oxytetracyline has been used as a soil-drench or as root dip for controlling crown gall and has been utilized for the control of fire blight of apples and pears in conjugation with Streptomycin. Tetracycline has been found to be effective against plant diseases caused by mycoplasmas. An injection of a concentrated solution of Tetracycline or oxytetracycline into tree trunks offers possibilities of curing peach-x, pear decline, coconut palm yellows and citrus greening. Tetracyclines when, injected into trees infected with mollicutes or fastidious prokaryotes stop the development of the disease and induce remission of symptoms. Agrimycin –100 This is 15% streptomycin sulphate and 1-5% Terramycin. Agrimycin –500 is 1.755% Streptomycin sulphate, 0.176% Terramycin and 42.4% metallic copper. It has been utilized for the control of bacterial diseases, such as Erwinia amylovora, halo-blight caused by Pseudomonas syringae pv. phaseolicola, citrus canker caused by Xanthomonas campestris pv. citri, seedling blight, leaf spot and black arm disease of cotton caused by Xanthomonas campestris pv. malvacearum and soft rot, and black leg of potato caused by Erwinia carotovora. Nystatin Nystatin is also known as Mycostatin or Fungicidin. It is a polyene (tetraene) antifungal antibiotic produced by Streptomyces noursei. It is manufactured for clinical use but has also been used to a limited extent for plant disease control. This has been found efficacious in controlling anthracnose of beans and downy mildew of cucumber, and as seed treatment against stripe disease of barley (Dharamvir and Raychaudhuri, 1968). Ananthanaryana and Seshadri (1965) reported that it can be utilized as post-harvest dip against peach brown rot and anthracnose of banana. Bulbiformin Bulbiformin is an antifungal polypeptide antibiotic produced by Bacillus subtilis and was reported by Vasudeva et al. in the year 1958. The production of this antibiotic was demonstrated in sterilized soil amended with molasses, groundnut cake, sweet clover roots, and so on. Inoculation of amended soil with B. subtilis controlled the wilt disease caused by F. udum on pigeon pea. Singh et al. (1965) demonstrated the production of bulbiformin on seed coats, spermatosphere and rhizosphere by bacterized pigeon pea seeds sown in sterilized and unsterilized soil. The antibiotic was absorbed, and because it is systemic, considerably reduced the incidence of wilt of pigeon pea. Soil Fumigants The most promising method of controlling nematodes in the field has been through the use of chemicals called nematicides. Some of these, including chloropicrin, methyl bromide, mylone, vapam and vorlex, give off gases after being applied to the soil. They are general purpose preplant fumigants and are effective against a wide range of soil micro-organisms, including, in addition to

262

Plant Pathology

nematodes, many fungi, insects and weeds. These are also highly volatile fumigants, such as formalin and carbon-disulphide. Formalin Various chemical treatments of the soil have been developed, one of the oldest being the formaldehyde drench. Formaldehyde corrodes the hands and is somewhat erratic in effectiveness. For the control of damping off and seedling blights, a 37-40% solution in water is used. Excellent control of onion smut has been obtained in the USA by a solution of formaldehyde (1-128 parts) applied at seeding time at a rate of 200 gallons per acre with the help of machines especially devised for the purpose. It is an effective fungicide in seed beds to prevent damping off fungi. Carbon disulphide (CS2) Carbon disulphide is perhaps the earliest chemical used as a soil fumigant for checking nematodes. Carbon disulphide is used in citrus orchards against Armillaria mellea. It is used like formalin. This substance was originally employed in 1872 by Thenard against phylloxera of the vine (Phylloxera vitifoliae). Carbon disulphide is cheap, volatile and is an insecticide also. Chloropicrin This is useful both as a fungicide and a larvicide. It is injected into the soil at a depth of 3 to 6 inch in holes 9-12 inch apart. The soil is covered with impervious plastic sheeting for 48 hours. Vapam Other fumigants produced in recent years include methamsodium (Vapam: sodium Nmethyldithiocarbamate), a colourless liquid which decomposes rapidly in moist soil to release a fumigating gas.

Vapam is not volatile but it decomposes in soil to yield methyl isothiocyanate (CH3—N==C==S). In addition to methyl isothiocyanate, Vapam also yields carbon disulphide, hydrogen sulphide, methylamine and other products when it is mixed with the soil. Vapam has been used as a nematicide and selective weedicide. But it has been found to be quite effective in the control of wilt of cotton, damping off of papaya, root rot of beet (Sclerotium rolfsii). Methyl Bromide (CH3Br) Methyl bromide has also been used as a soil nematicide, but it is too phytotoxic to be used in fields with a standing crop. It should be applied in the soil as a fumigant a few weeks before the crop is sown or planted. The use of this sterilizing gas has been restricted to the treatment of nursery beds, vegetables and fruit production, and ornamentals. Methyl bromide has a low boiling point (4.5°C) and requires a plastic cover for effective confinement of the gas or for application with deep injectors. This chemical is scheduled for withdrawal from use as it affects the ozone layer in the earth atmosphere. DD Mixture Dichloropropene and dichloropropane (DD mixture) and Ethylene dibromide (EDB) are two effective nematicides, which came into the market soon after World War II and are used extensively for nematode control. Dazomet Dazomet (tetrahydro 3,5-dimethyl-2H-1,3,5-thiazene-2-thione) is a wettable powder used at the rate of about 440 kg/ha. It is applied with a fertilizer spreader or by spraying a suspension in water, followed by thorough mixing and ‘sealing in’ with water. In the soil, Dazomet releases methyl

Management of Plant Diseases 263

isothiocyanate and is effective for the control of nematodes, wireworms and some soil-borne fungi, such as Pythium, Rhizoctonia, Fusarium, Verticillium, and Colletotrichum. Oils Various oils are capable of controlling several plant diseases. The effectiveness of oils in the control of plant diseases has been reviewed by Calpouzos (1966). Shigatoka disease of bananas caused by Mycosphaerella musicola has been controlled by oils, which, because of their advantages have almost completely replaced conventional fungicides. Oil sprays are now widely used for the control of M. musicola. Nearly 50 million litres are estimated to have been used in Latin America and the Caribbean in 1964 and perhaps another 7.6 million litres in other banana producing areas. The advantages of oils are: 1. superior disease control, 2. effectiveness at very low dosages of even less than one gallon per acre, 3. excellent spreading and sticking properties on leaf surfaces, 4. low cost, and 5. little or no toxicity to man and animals. There are several disadvantages also. These are: 1. Effective against a limited number of plant diseases, and 2. Phytotoxicity may occur on sensitive plant species. The oils involved in disease control are mainly mineral oils from petroleum, and to a much lesser extent, glyceride oils from plants, and synthetic oils. It is not exactly known how mineral oils control plant diseases. Light viscosity oils atomized into very fine droplets of 50-1000 mm in diameter seem to be most effective and can apparently stop the growth of the fungi after it has become established inside the leaf. According to Calpouzos (1966), any hydrophobic fluid of low volatility, such as silicon oil, which is chemically inert, will control M. musicola. The therapeutic action of oils is perhaps due to the oily physical barrier which interferes with gas exchanges in the leaf and alters its physiology so as to stop the development of the fungi. There is evidence that anti-transpirants may reduce disease by, for example, inhibiting stomatal penetration. It is also likely, but not certain, that oils exert their therapeutic action not directly on the pathogen but rather on the physiology of the host. M. musicola is encouraged by fairly high light intensities which no doubt favour photosynthesis and high sugar concentration. Some high-sugar pathogens, such as powdery mildews and rusts are apparently susceptible to oils. Oils have been used successfully in the control of greasy spot of citrus cased by Cercospora citrigrisae. Oil alone controls angular leaf spot of tung tree (Aleuites fordii) caused by Mycosphaerella aleuritides. Oil sprays have also been successfully utilized for the control of Septoria apii on celery, Puccinia graminis tritici on wheat and several powdery mildews, such as Sphaerotheca pannosa on rose. Blister rust has been affected by application of fuel oil. Some synthetic oily fluids (polybutenes) are also effective against Peronospora tabacina responsible for blue mould of tobacco. According to Castellani and Matta (1964), mineral oil alone misted on grapes, effecively controlled downy mildew caused by Plasmopara viticola. Ocana and Hansen (1960) tested a non-volatile mineral oil against Phytophthora palmivora which attacks the pods of the cacao tree. Commerially acceptable control of the disease did not occur but it did retard the development of lesions. Thus the use of oils in the control of plant diseases is an interesting recent development. Oils obtained from seeds of several plants such as sunflower, olive, corn, and soybean gave excellent control of powdery mildew of apple when applied 1 day before to 1 day after inoculation.

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Safeners, Spreaders and Stickers

Safeners A safener is defined as a chemical that reduces the phytotoxiciy of another chemical. Copper sulphate is phytotoxic, but with the addition of lime its toxicity is reduced. Lime is, therefore, a safener. Lime is used universally with chemicals to prevent the formation of, or to neutralize arsenic acid, which is phytotoxic to sensitive foliage. Glyceride oils act as good safeners for copper sprays by providing a film around each particle, but they reduce the fungicidal value of the product. Spreaders Materials which are added to the spray mixture to establish improved contact between the fungicides and the sprayed surface, are known as spreaders. These can be classified as water soluble and water-insoluble. Mineral oils, glyceride oils, and terpene oils are water-insoluble spreaders. Soap has long been known as a spreader. Sulphated alcohols, sopamines, resinates and petroleum sulphonic acids are some of the common spreaders available in the market. Spreaders reduce the surface tension and improve contact between the fluid and the sprayed surface so that the fluid does not collect into large drops and run off easily. Stickers Materials added to spray or dust, which improve its adherence to plant surfaces are known as stickers. Flour, starch, gum arabic, and dextrins are commonly used as stickers. Bentonite clays when added to dusts improve the latter’s sticking quality. Oils, oil emulsions, and hydrocarbon oils have also been employed as stickers. Carriers When the fungicide is used as a dust, the active ingredient is generally diluted with a finely divided powder called a carrier. Acquired Resistance to Fungicides

Georgopoulos and Zaracovitis (1967) reviewed the tolerance of fungi to organic fungicides. Dekker (1972, 1976) had reviewed acquired resistance to fungicides. Ashida (1965) had reviewed adaptations of fungi to metal toxicants. Development of fungal resistance to fungicides had been discussed by Georgopoulos (1977) in Vol. 2 of Antifungal Compounds. The term ‘acquired resistance’ is used when, in a population which is normally sensitive to a biocide, forms arise that are less sensitive to this biocide. This resistance may have a genetical basis and then as a rule be stable, or it may be caused by a physiological adaptation and disappear again rapidly when the organism is no longer exposed to the toxicant. This temporarily reduced sensitivity is called tolerance and the former type is called resistance. The high specificity of action of a toxicant is both a strength and weakness. Many organisms are resistant to selective organic insecticides. The dithiocarbamates and inorganic compounds are non-specific toxicants and do not penetrate the plant tissues. The development of resistance in micro-organisms due to these toxicants is unusual (Dekker, 1972). However, with the introduction of selective systemic fungicides, there was a significant development of resistance. In practice, sometimes resistance is so great that the use of the compound concerned has to be severely restricted or even abandoned in the treatment of certain diseases. Resistance to many of these new systemic fungicides could be readily induced in the laboratory by treatment with ultraviolet irradition or other mutagenic agents. Dimethrimol was introduced in Holland in 1968 to control powdery mildew in cucumbers. During the following two years, this control measure was found to be less effective. Even after the removal of the

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fungicide, tolerant fungi were reported in 1971 (Bent et al., 1971). Georgopoulos and Dovas (1973) reported that the Benomyl was very effective in the control of heart rot in cyclamen. Within a short period, a strain of Botrytis cinerea developed that could tolerate 1000 ppm, while the wild type was controlled at 0.5 ppm. The tolerant strains showed cross resistance to Thiabendazole, Fuberidazole and Thiophanate methyl. Benomyl protected sugar beets in 1970 and 1971 at comparatively low levels. However, in July of 1972, resistance was noted and subsequently a large acreage of beets was lost. Dekker (1972), Wolfe (1971) and Vargas (1973) have suggested a number of ways to reduce fungicide tolerance. Among these is the avoidance of the use of one fungicide on an exclusive basis. Systemics with different modes of action should be alternated along with the protectant fungicide. An economically feasible level of fungicide dose should be used, phytotoxicity should be avoided and toxic residues should be avoided. Resistance to the fungicide Metalaxyl has also been observed. Metalaxyl fungicide is used for controlling Oomycetous pathogens. The continuous and prolonged use of a selective, systemic fungicide may result in loss of effectiveness, which may be incorrectly diagnosed as fungicide tolerance. Origin of Fungicide-Resistant Strains

It is not clear how fungicide resistant strains of fungi arise as little work has been done on their genetics. The disappearance of resistance in the absence of the fungicide could be due to sub-lethal concentrations of the latter. This results in the formation of a substance or substances which confer resistance which could otherwise not be formed. Perhaps, adaptive enzymes, the formation of which are induced by the fungicide, are involved. Stable resistant strains perhaps arise by mutations,which occur either by chance, or in the case of certain fungicides, could be brought about by the fungicide itself. It is significant that the exposure of Fusarium caeruleum (potato dry rot) to tetrachloronitrobenzene (TCNB) resulted in the appearance of TCNB-resistant mutants and that variants of Botrytis allii (onion neck rot) were produced after the fungus was treated with the vapour of Pentachloronitrobenzene. Some aromatic hydrocarbons are said to produce gene mutation in fungi. The fungicide resistance of some fungi, including a few which attack plants, has been shown by genetical analysis to be of chromosomal origin, as in Hypomyces solani f. sp. cucurbitae (seedling blight and fruit rot of cucurbits), where resistance to TCNB is determined by three independently-inherited genes (Georgopoulos, 1962, 1963). However, there is little critical evidence that fungicides can induce mutations in fungi which bring about resistance to fungicides. Mechanism of Resistance to Fungicides

The development of resistance to a fungicide may be due to changes in the fungal cell that inhibit the fungicide, to a greater or lesser extent, from reaching the site of action. Such changes include a decreased permeability of the protoplast membrane to the fungicide, or an increased detoxification before the site of action is reached. If the fungicide is able to reach this site, resistance may be due to a decreased affinity between the chemical and the reactive site, to circumvention of the blocked site by operation of an alternate pathway or to compensation for the effects of inhibition. Dekker (1976) had discussed examples of various types of resistance to fungicides in detail.

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BREEDING FOR DISEASE RESISTANCE

The promise of resistance to pests and diseases has attracted the attention of plant breeders ever since the demonstration near the turn of the century that resistance could be selected and that it is often simply inherited. The built-in protective resistance effective throughout a crop plant’s life offers a compelling alternative to fungicides and insecticides. In the early years, minimizing the use of protective chemicals was desirable because it saved money but today there is an added advantage of reducing their hazard against those pests and diseases such as cereal rusts, soil-borne smuts and certain nematode diseases. There is considerable literature available on the breeding and inheritance of disease resistance in plants. Hansen (1934) had given a list of papers published on the inheritance of disease resistance in plants up to 1934. The literature on the genetics of diseases resistance in vegetables has been reviewed by Walker (1965, 1969) and on field crops by Ausemus (1943) and Dickson (1956). Inheritance of resistance to viral diseases has been reviewed by Holmes (1954), resistance to rusts by Hooker (1967), and to nematodes by Hare (1965). Some more review articles are those of Hooker and Saxena (1971), Roane (1972) on trends in breeding for disease resistance in crops, Hooker (1974) on cytoplasmic susceptibility in plant disease and Sadasivan (1975). Alien germplasm as a source of resistance to disease has been discussed by Knott and Dvorak (1976). An interesting article is by Browning et al., (1977) on managing host genes: epidemiological and genetic concepts. The genetics of resistance to plant viruses has been reviewed by Fraser (1990). Multiple disease resistance in grain legumes has been discussed by Nene (1988). Molecular genetics of plant disease resistance has been recently discussed by Staskawicz et al. (1995). The use of disease-resistant varieties for controlling plant diseases has been termed the “painless method” because it does not cost the farmer anything. In an underdeveloped country like India, it is all the more important since we cannot pay for the heavy costs of spraying and dusting crops on a large scale. The use of resistant cultivars and hybrids has several other advantages. It eliminates the hazard to human health and wildlife which is caused by large scale use of dangerous fungicides and pesticides. It also reduces pollution which results from the use of poisonous chemicals and their residues. Resistant crop varieties check epidemics of pathogens and pests and thus help to maintain the biological balance in the environment. It has earlier been seen that resistance can be varied in different hosts and parasites. It can be mechanical, such as waxiness, hairiness and toughness of the epidermis or it may be biochemical in which case it is the result of the interaction between the cytoplasm of the host and the parasite. The biochemcial defence mechanism is due to the synthesis of polyphenols in the host that may be toxic to any organism present and to the plant itself. Susceptible and resistant reactions differ principally in the speed and scale of host responses which are more rapid in the latter. As a result, the development of the invading parasite is restricted. Susceptibility and resistance to a parasite are largely inherited characteristics. The laws governing their inheritance are the same as those governing other characteristics of the host and parasite. It has been proved in several cases that the resistance or susceptibility to disease is located in the genes. The breeding of resistant varieties of crops is, therefore, based upon the laws of inheritance. The Darwinian principles of a variety’s struggle for existence and survival of the fittest apply to all the wild varieties and their pathogens. Wild plants have been growing for centuries in the presence of the pathogens and natural selection must have eliminated the most susceptible biotypes and left the most resistant ones. In

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this way regional biological balances exist between the native plants and their pathogens and pests. The balance will tend to persist unless disturbed by new factors such as the introduction of new biotypes of pathogens or hosts. This is likely to occur if barriers such as oceans or high mountains protect the region. Often it is man who disturbs the natural balance by transporting plants and pathogens to new regions, or who, through crop improvement, produces new varieties of crop plants. Many of the serious outbreaks of plant diseases are traceable to the introduction of parasites to which non-resistant crop plants immediately succumb. The following are some of the notable examples: 1. A very superior variety of hops (Humulus lupulus) was cultivated in western Europe. In 1917, downy mildew Pseudoperonospora humuli was observed in England in a locality, which grew on seeds and plants imported from Japan. In 1922, the fungus attacked south eastern England. The pathogen soon spread to Belgium, France, Holland, Germany and other European countries and had to be controlled at heavy cost by spraying. 2. Cryphonectria (Endothia) parasitica, the cause of chestnut canker, does not cause serious damage in the Eastern countries. When it was introduced in the USA in about 1904, it brought heavy losses to the chestnut forests (Castanea dentata) in a few years time. It is estimated that within a period of about 10 years it has caused losses of about 50 million dollars. 3. Taichung Native 1 (T.N. 1) is a high yielding variety but it brought with it the bacterial blight a serious disease of this crop in 1966. T.N.1 is highly susceptible to bacterial blight. There are so many other important diseases, which have been imported into countries which were earlier free from them. For example, Phytophthora infestans was brought in infected tubers from South America to England and other European countries in the 1830s and Plasmopara viticola from America to France in 1870. Failures accompany successes while breeding resistant varieties (Stakman, 1954, and Caldwell, 1966). Plant breeders even now introduce genes for resistance, as they did in 1918. Borlaug (1965) has remarked that the average useful life of a rust-resistant variety is as short as five years in some parts of the world after which it succumbs to a new race of rust. Walker (1959) wrote, “By and large the development of resistant varieties must be looked upon as a continuing programme. The potential variability of most pathogens (including viruses) will not permit any currently successful variety to remain so for an indefinite period”. Several other examples can be cited where resistant varieties did not stay resistant for long, such as Hessian fly on wheat, powdery mildew on barley, Cladosporium leaf mould of tobacco, mosaic virus of tomato, golden nematode and leaf blight of potato. The question arises as to why these varieties were so short-lived and why the long and continued series of efforts failed to give the desired results. The reason is that while the breeder tries to make plants more resistant, nature tries to make pathogens more virulent, and ultimately nature gets the upper hand. It was earlier stated that the characteristics of an organism are governed by the laws of genetics which are common to both the host and parasite. Resistance or susceptibility (both of which are relative expressions) is the result of interaction between these characteristics. It has now been shown that the genotypes of the host as well as the parasite can be varied by segregation – recombination of genes, mutation, heterokaryosis, somatic hybridization, etc. Genetic studies have now been extended to bacteria and viruses where new strains arise due to hybridization, transformation and transduction. The previous conception that the pathogen is a fixed entity which damages the crops year after year without change is no longer acceptable. There is a great array of genotypes of the parasite as there is of the host.

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Types of Resistance

Plant pathologists commonly divide resistance into monogenic, polygenic and oligogenic, according to whether resistance is governed by one gene, many genes or a few genes. Resistance can be described as genetic, mechanical or epidemiological. The general behaviour or the genetic behaviour of disease-resistance is governed in many simple-tocomplex ways. Monogenic resistance (governed by a single gene) is governed by one gene with a 3:1 Mendelian ratio. It can be monohybrid dominant, such as the resistance of oats to Helminthosporium victoriae (Caldwell, 1966), of sorghums to Periconia circinata (Vander Plank, 1968), of cabbage to the pathogen Fusarium oxysporum f. sp. lycopersici (Walker, 1965), of onion to the pathogen, Pyrenochaeta terrestris and to Cercospora leaf spot of cucumber. It can be monohybrid recessive, with resistance as a recessive character, such as powdery mildew of barley, sorghum smut, and yellow rust of wheat (Biffen, 1905). Oligogenic resistance (governed by several genes) is determined by two-to-several genes. Polygenic resistance (governed by many genes) involves many genes which are more difficult to analyze and which are known to be involved in a large number of diseases such as cotton wilt. Extrachromosomal Inheritance Extrachromosomal inheritance of disease resistance is rare but is of great importance. The widespread epiphytotic of southern leaf blight of corn in the USA was due to the sudden appearance and distribution of a new race identified as race T (Smith and Hooker, 1970) of Helminthosporium maydis. Resistance to the new race is mostly extrachromosomal (Hooker et al., 1970). Corn with cms-T cytoplasm is more susceptible to yellow leaf blight caused by Phyllosticta sp. Symptom production in potato virus X in peppers is apparently determined by cytoplasm (Nagaich et al., 1968). Gene Interaction Disease resistance to pathogens is not always simply inherited. In many instances the pattern of inheritance is quite complicated. The resistant phenotype is the ultimate expression of a chain of events in which genes for resistance perhaps control only a few links. Although genes may appear to act as autonomous units, in effect gene products interact and this becomes clear in genetic analysis by the appearance of complementation, modification and epistasis. Modifier Genes A host may carry a gene for strong resistance but this is not expressed because of the presence of an inhibitor gene. In onions, resistance to smudge disease is controlled by a single dominant gene R. In the presence of a partially dominant inhibitor gene, I, the resistance is reduced or completely suppressed, depending on whether the gene I is present in the heterozygous or homozygous state. In wheat rust, resistance is completely suppressed by a dominant inhibitor gene. Inhibitor genes affecting recessive genes for rust resistance are also known to be present in maize. Reversal of Dominance A gene may be dominant for resistance to one race of the pathogen, but recessive for resistance to another race of the same pathogen. This reversal of dominance may be due to dosage effect, that is, one dose of the gene in a heterozygote being sufficient to provide resistance against one race of the pathogen but not against the other, or due to closely-linked genes for resistance, one dominant and the other recessive.

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Vertical and Horizontal Resistance When a variety is more resistant to some races than to others, resistance is called “vertical” “perpendicular” or “specific”. When resistance is such that it is uniformly spread against all races of the pathogen it is called “horizontal”, “lateral”, “generalized”, “non-specific” or “field resistance”. The derivation of this term is mathematical and refers to the horizontal axis of the graph. Horizontal resistance can be demonstrated when a series of pathotypes is tested against a series of pathodemes. Horizontal resistance has been called field resistance against Phytophthora infestans and generalized resistance against Puccinia graminis. Vertical resistance (VR) confers complete but impermanent protection, whereas horizontal resistance (HR) confers incomplete but permanent protection. The concept of vertical and horizontal resistance (VR and HR) was put forward by Van der Plank in 1968. Vertical resistance is conditioned by oligogenes and is effective against only some races of a pathogen, whereas HR, which is polygenic in inheritance is effective against all races of a pathogen. The terminology is a bit confusing. All oligogenes are not necessarily major genes in the sense of being important genes. For example, the R1 gene governing resistance to potato blight merits being called major gene. But the gene R4 is weak and of little importance to potato breeders, and hence, it would be an exaggeration to call the R4 gene a major gene. In the same way, all polygenes are not minor genes in the sense of being relatively less important. The derivation of the term “vertical resistance” is mathematical and refers to the vertical axis of a graph (see Fig. 1.3). Vertical resistance is proved by analysis of variance in which there is significant differential interaction between pathotypes and pathodemes. Vertical resistance is generally (but not necessarily) inherited by means of major genes. It often (but not invariably) produces a quantitative presence or absence effect in that it operates completely or not at all. The epidemiological characteristic of vertical resistance is an apparent reduction in the initial inoculum; as a result, the epidemic is delayed (cf. horizontal resistance which slows down the epidemic). Methods of Selection of Resistant Genotypes

While searching for resistant genotypes, a breeder has to look for the former by one of the following methods: Selection from Existing Crops

This is done in the following ways:

Selection from Crops that Escape Damage in Infected Fields The early breeders used this as one of the prominent methods, but it should be noted that such selected material cannot be usually called a genetically pure stock. As early as 1900, Orton of the US Department of Agriculture began selecting cotton varieties against cotton wilt (Fusarium oxysporum f. sp. vasinfectum). He selected individual plants, which showed resistance to disease in a wilt-infested plot of land, that is, even when they were drastically exposed to the disease. The seed from the selected plants was repeatedly tested for resistance in progeny tests on the infected soil. By this process a number of good yielding wilt resistant varieties were evolved. Flax wilt caused by F.oxysporum f. sp. lini is another good example of selection being done to obtain resistant varieties. Prior to the development of resistant varieties, flax was a migratory crop in the USA, and as it spread westward to fresh lands, the crop, after a few years of cultivation, became “hard on the soil” till the soil became so “flax sick” that wilt destroyed practically every crop. In 1901, it was Bolley

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of North Dakota who first announced that the disease was due to F. lini. A special plot was established in which the disease was allowed to develop freely and flax was grown on it continuously year after year till almost no crop survived except for a few plants. From these surviving plants, seeds were collected and resistant varieties were developed. In North Dakota, a similar plot of Fusarium-infested soil was set up at Minnesota in 1912. These two plots were constantly inoculated with every kind of Fusarium wilt organism from various areas and sources. Screening centres were established for testing all the resistnt varieties, which were evolved from time to time. The wilt disease caused by F. oxysporum f. sp. conglutinans has practically ruined cabbage production in parts of the USA. The disease was investigated by L.R. Jones and others. Some seeds were collected from three highly resistant plants and resistant varieties of Hollander types were developed. Further work at Wisconsin by L.R . Jones showed that temperature plays an important part in the onset of this disease. He found that some varieties became non-resistant at high temperature. He showed that when the soil was kept at 18.3°C or lower, plants grown in infected soil did not develop disease. As the temperature rose, the intensity of disease increased up to about 27°C after which it declined. Resistant varieties were selected on the lines given above. Later on, hybridization was also resorted to, and homozygous resistant varieties were evolved. This was the first experimental proof of the fact that in disease resistance, environment plays a very important part. Curly top of sugar beets, a virus disease, which causes devastating losses has also been controlled by the production of resistant varieties by selection. The curly top resistant varieties were a spectacular success and saved the sugar beet crop in western USA. It should be emphasized here that the selection methods for resistant varieties can succeed only when the population has some resistant biotypes. Pure Line Selection The above method is followed in pure line selection also but seed from selected plants is collected and tried separately. The stock thus produced is genetically pure. Plant Introduction Very often, the genes needed to control a particular disease or race of a pathogen cannot be found in domestic varieties. It becomes necessary to introduce plant materials from other countries, especially from places where the pathogen and the host species may have evolved together and thus provide rich pools of resistance genes (Leppik, 1970). Hybridization Among the existing varieties it is found that a variety may be resistant to a disease but may not possess suitable agricultural characteristics, that is, it may not be high-yielding or the quality of the produce may not be satisfactory. On the other hand, there may be good varieties of high yields but they may be susceptible to disease. A breeder tries to combine the good characteristics of both the varieties to evolve a high yielding and resistant variety. Hybridization: H-High yield h-low yield R-Resistant r-Susceptible HHrr ¥ hhRR (High-yielding (Low-yielding susceptible) resistant) Gametes Hr x hR F1 generation = HhRr Phenotypically high-yielding resistant

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Gametes HR, Hr, hR, hr HR Hr hR hr HR HHRR HHRr HhRR HhRr Hr HHRr HHrr HhRr Hhrr hR HhRR Hhrr hhRR hhRr hr HhRr Hhrr hhRr hhrr (Out of these the first HHRR is high-yielding and resistant and is a pure stock). Selection from Wild Varieties A search has often to be made for resistant genes from the wild varieties, species or related species of the cultivated plants. The best places to find wild varieties resistant to certain pathogens are the primary and secondary gene centres of cultivated plants (Vavilov, 194950; Zhukovsky, 1961). Vavilov (1950) used the term “gene centres” or centres of origin for primary regions of diverse cultivated species and varieties. He considered that these centres contain the greatest wealth of hereditary variants with definite naturally-inbred characteristics. Cultivated plants in these centres were frequently, but not always, accompanied by closely related wild species as possible progenitors of domesticated plants. It soon became necessary to distinguish between primary and secondary gene centres. In the primary centres the cultivated plants evolved from wild progenitors native to these regions. During cultivation, however, these plants were frequently brought to other areas, where they were subjected to different climatic and edaphic conditions. These climatic factors forced the plant population to produce new diverse genetic characteristics. This is how secondary and tertiary centres of diversity were developed outside the primary centres. Zhukovsky and Migushova (1969) have added some new phytogeographic terms. A new term, ‘genofund’ is used by Zhukovsky and Migushova (1969) to indicate the genetic source or gene pool for breeding. Both the host and parasite have long been associated in their centres of origin as reciprocal selective factors in evolution. Every new and more virulent race of parasite necessarily eliminates most of the susceptible individuals in the local host population. The individuals that survive definitely carry genotypes different from these of the bulk of the population. In this way, the survivors perpetuate genotypic resistance. Whenever serious diseases occur in nature, the highest degree of resistance develops among wild species and varieties as a result of natural selection. It has, however, to be remembered that the gene centre of the host plant is also the centre of origin of its specialized pests and pathogens, as well as the new and more virulent idiotypes, biotypes and pathogenic races which occur in the population of the parasites. According to Vavilov (1949-50) there are nine world megacentres of origin of the most important cultivated plants in China, India (with a secondary Indo-Malayan centre), Certral Asia, Australia, the near East, the Mediterranean region, North-east Africa or Abyssinia, Southern Mexico and Central America, and South America (Peru-Equador-Bolivia) with two subcentres (Chile and Brazil-Paraguay). Although breeders have often turned to wild varieties of cultivated plants to find new sources of resistance and have isolated stocks possessing the desired resistance, it is difficult to transfer this resistance because of incompatibility problems between subject species or because of sterility in the hybrids. Breeders have now come to rely on special cytogenetic techniques and embryo culture to facilitate the more difficult crosses. Induced Mutations New gene mutations and polyploids can be induced artificially with the help of abnormal radiations, such as X-rays, ultraviolet rays, etc. and also mutagenic chemicals like colchicine,

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ethyl methane sulphonate. The new mutations, which arise may be useful or useless depending upon chance. The useful ones are selected and bred as per one of the above described methods. Muller established mutation genetics in the late 1920’s and plant breeders immediately realized the possibility of utilizing it. Sigurbjomsson and Micke (1969) have listed 77 cultivars that have been developed. As a direct consequence of induced mutations, 16 of these were cited as having improved disease resistance. Examples of Resistant Varieties Through Breeding The first classical example of hybridization is provided by William A Orton (1907). Failing to get resistance in the edible varieties, he turned to the highly resistant citrus melon, which is commonly used as cattle feed. He synthesized a diseaseresistant variety by hybridization capable of giving good crops despite disease. Breeding Wheat for Disease Resistance

The power and potency of modern plant breeding for the improvement of crops have been amply demonstrated in wheat. Wheat improvement has played a key role in ameliorating world food shortage and also in the green revolution in India. Organized and systematic wheat breeding in India was started by the late Sir Albert Howard and Mrs. Howard in 1904 at the Agriculture Research Institute, Pusa, Bihar. Since then a number of research and breeding institutions have come up where wheat improvement work is being carried out. The major techniques used in Wheat breeding are: (1) introduction, (2) selection, (3) hybridization, and (4) induced genetic changes. Early work on wheat improvement in India was concentrated on developing a suitable strain from the local ones, because the varieties introduced from abroad were mostly found to be late in maturing under Indian conditions. On the other hand, introduction played a key role one in the USA and Canada, because wheat was not a natural crop in these countries. A successful introduction in India was that of Ridley variety from Australia. This variety is stiff-strawed, fairly resistant to rusts and suitable for hilly areas. More recently, quite a few introductions have been made from Mexico and a number of useful varieties have been selected and bred from these introductions. Some of the dwarf varieties introduced from Mexico include Sonara 63, Sonara 64 and Lerma Rozo. From these, red and white kerneled varieties have been developed in India. Norin, the gene for dwarfness, arose in Japan, from where it was sent for testing to the USA after the Second World War. With the help of suitable crosses this gene was transferred to the American Brevor wheat varieties, which were sent to Dr. Norman E Borlaug, who introduced this gene into many Mexican varieties. Selections made by Borlaug, as well as unselected hybrid materials were later introduced in India. Besides introduction hybridization has also played a key role in the development of a number of new varieties, especially the rust-resistant ones. Simple hybridization, back crosses and composite crosses involving various varieties, species and genera have been used for developing suitable varieties. Biffen (1905) was the first to apply the Mendelian factorial analysis in the case of resistance in wheat against stripe rust. He crossed the resistant wheat variety, Rivet (American Club variety), and the susceptible variety, Michigan Bronze. He showed that resistance was inherited as a simple recessive factor independently of other factors and could be combined with other desirable characters. Following the early accomplishments of Orton and Biffen there was a tendency to think that resistance was always governed by a simple monohybrid Mendelian factor which could be manipulated easily. Later work proved otherwise. It was found that resistance in some cases could be dominant and in others recessive, and it may be governed by a numbers of genes which segregate independently, produc-

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ing great variations in progeny. Linkage and crossing over also complicate the inheritance of resistance as in the case of any other character. Black or Stem Rust of Wheat (Puccinia graminis tritici) Much work has been done in other countries on the breeding of wheat varieties resistant to black, brown and yellow rusts and other diseases of the crop. The existence of several races and the evolution of new races of rusts from time to time make breeding work complicated, requiring 12 to 15 years to establish a new variety. The rust resistance of wheat, a classic case, demonstrates most convincingly the importance of gene centres of hosts as sources of genes for disease resistance. The origin of known main gene complexes in cultivated wheats that determine rust resistance can be traced back to the gene centres of wild wheats in the Middle East. The main donor of genes for resistance in present cultivars is Triticum timopheevii from the Transcaucasian mountain region. Other native wheat species and varieties from the same and adjacent areas are immune to various pests and pathogens. They provide large gene pools for resistance breeding, but they are mostly unexplored and remain almost untouched by breeders. In one of his papers Zhukovsky and Migushova (1969) has listed nine highly resistant endemic wheats and close relatives that can be freely crossed with cultivated wheats. 1. Triticum monococcum var. hornemannii (Clem. and Rubio) Koern. (2n=14). Cultivated eikorn from Georgian SSR or Gruziya. 2. T. timopheevii (Zhuk.) Zhuk. (2n=28). Frequently used by breeders as a source of disease and pest resistance. This is the most resistant wheat species; also carries cytoplasmic male sterility. 3. T. militinae Zhik. Et Migush. (2n=28). A spontaneous mutant from T. timopheevii. Highly resistant to rusts, ergot, downy mildew, and Helminthosporium. 4. T. zhukovskyi Men.& Er. (2n=42). A spontaneous hybrid between T. timopheevii x T.monococcum var. hornemannii with a double set of chromosomes. It contains the genes of resistance of its parents. 5. T. persicum Vav. Ex Zhuk.. (=T. carthlium) Nevski (2n=28). An ancient cultivated species from West Gruziya. Highly resistant to rusts and Erysiphe graminis f. tritici. 6. T. timonovum Heslot and Ferray (2n=56). A synthetic auto-octaploid, produced by Heslot and Ferrary by the redoubling of chromosomes from T. timopheevii resistant to common diseases. 7. T. fungicidum Zhuk. (2n=56). Obtained by Zhukovsky by the crossing of T. persicum and T. timopheevii. Resistant to rust, ergot, and downy mildew. 8. Haynatricum Zhuk. A new genus obtained by Zhukovsky’s crossing of Haynaldia villosa (L.) Schur. (2n=14) and T. dicoccum var. farrum Koern (2n=28). Its mechanical resistance to rusts, ergot, and mildew is due to the waxy layer on the whole plant. 9. Aegilops umbellulata Zhuk. (2n=14), from Turkey, described by Zhukovsky. This species can be crossed with soft wheats and its resistance to rusts has been transferred to wheats by Sears (1948). Wheat Varieties

Common bread wheat Durum wheat Einkorn wheat Emmer wheat Pollard wheat

T. vulgare T. durum—hard rich in gluten (macaroni) T. monococcum (one grain in each spikelet) wild type, still cultivated in Spain for fodder. T. dicoccum (two grains). T. turgidum – an old one, not of much value.

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An extensive series of crosses between these varieties and bread wheats were attempted. The results were not encouraging due to a variety of reasons. The hybrids were often sterile and did not produce seeds and there were many undesirable linkages which made progress very difficult. The inheritance of resistance against stem rust was not simple, as was proved by Biffen in the case of yellow rust. Nevertheless, a wheat cross between Marquis (T. vulgare) and Iumillo (T. durum) was obtained which combined bread wheat qualities with disease resistance of durum. This was named Marquillo. This variety did not become popular because of the poor colour of the flour. The existence of a number of physiological races also presented another obstacle to further development. However, there was hope of breeding resistant varieties when some varieties of bread wheats, such as Kanred, Kota and Webster were found to be resistant. Kanred was imported from Crimea, Kota from Russia, and Webster was a selection made in Dakota. Since more and more physiological races were being found, the varieties proved susceptible to one or the other of these races of the pathogen. Hayes and Stakman (1921) tested the important resistant varieties against 21 rust races known at that time. They supplied the following information: Khapli emmer Resistant against all races but high degree of sterility which prevented progress in hybridization. Kanred Resistant to 13 races (out of 21). Kota Resistant to 4 races out of the remaining 8 races. Iumillo (T. durum) Resistant against 2 of the remaining 4 races. In this way, resistance against 19 out of the 21 races existed in these three varieties put together. Some of the important varieties evolved by hybridization were: Marquillo = Marquis ¥ Iumillo (Durum). Thatcher = A double cross; (Marquis ¥ Iumillo) ¥ (Marquis ¥ Kanred). A double cross by H.K. Hayes at Minnesota had the resistance of both Kanred, a winter wheat, and Iumillo, a durum wheat. Thatcher’s performance was spectacular in the rust epidemic of 1955 when race 56 destroyed 100 million bushels of wheat in the northern great plains of the U.S.A. Ceres = Marquis ¥ Kota. Ceres was the first variety with resistance which was released by the North Dakota Agriculture Research Station in 1926. Hope = Marquis ¥ Yaroslav emmer. Webster = A selection variety of vulgare wheat. A definite progress was made. The genes for resistance in the durum and emmer groups were then transferred to bread wheats. Many linkages were broken and the handicap of sterility in wide crosses was overcome. Despite this progress the complex problem of controlling the stem rust still remained. The problem became worse due to other diseases of wheat which presented their own problem of resistance, the important one among them being the “head blight” caused by Fusarium sp. (F. graminarum) which severely attacked Marquis but not other varieties; root rots were abundant in the case of durums. Race 56 eliminated the variety ‘Ceres’. In 1935, a new wide cross, named ‘New thatch’, was successful between Thatcher and Hope. New thatch was composed of four varieties—Marquis, Kanred, Iumillo, and Yaraslov emmer, genes for resistance were combined from two bread wheats, one Yaraslov emmer. The above mentioned hybrids and the durum varieties kept in check the ravages due to rust between 1938 and 1950, when a new race of rust, named 15B, was developed. It eliminated the durum wheats almost completely and damaged some vulgare varieties also. This is an indication of the potential danger of new varieties.

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In India all the three rusts of wheat are prevalent in most of the growing areas. There are also several races and biotypes of each of the three rusts. Systematic work on the breeding of wheat varieties resistant to all the three rusts was started by Pal in collaboration with Mehta in 1934. The breeding work was done in several stages. Strains of wheat resistant to the races of each rust were first obtained by crossing the susceptible Indian varieties with the resistant foreign varieties. The resistant segregates obtained from each set of crosses were used for multiple crosses. The segregates resistant to all the three rusts were obtained. These resistant strains were back crossed with the original Indian varieties to concentrate their economic characters. One of the outcomes of the crosses is the variety NP 809, which has proved resistant to all the rusts in the hill stations of North India and in Uttar Pradesh, Punjab and Himachal Pradesh. Another simple cross between the susceptible but economically important NP 4 and the resistant Japanese variety, Konon, resulted in a promising variety, NP 770, which showed a high degree of resistance to the rusts at high altitudes. A large number of foreign collections of wheat varieties were also tested for resistance to the races of the three rusts and the Australian variety, Ridley, has been found to be very promising. The introduction of Mexican dwarfs in India in 1965 brought about a green revolution and the problem of rust was considered to be almost over. However, the parents of the varieties were bred in Mexico where the main rust is black rust or stem rust of wheat. Therefore, the varieties based on the Mexican genetic stock have requisite resistance only to black stem rust. Nevertheless, some of these varieties did show some degree of resistance to more common rusts (specially leaf rust). During the last four years it has been observed that due to continuous cultivation of only selected varieties over large areas in India, resistance is breaking down. Some of the important Mexican dwarf varieties introduced in India and developed further are: Sonora 64, Lerma Rojo 64A, K 65, Sharbati Sonora, Kalyan Sona, Sonalika, Choti Lerma (S331), Safed Lerma, Hira, Lal Bahadur, HD1918 (Pratap), HD 2009 (Arjun), HD 2122, HD 4530, HD1102, UP 262, and JNK(4-W-184). Breeding Rice for Resistance

In India, rice breeding programmes go back to the early 1920’s when varieties as such GEB 24 were evolved. The greatest reservoir of breeding materials for introducing disease and insect resistance into the new varieties has come from India. This appears to be a direct result of breeding and selection Programmes conducted in a country where the use of agricultural chemicals is minimum and where most of the major insect pests and diseases of rice are rampant. In 1946, the Indian government established the Central Rice Research Institute at Cuttack, Orissa, in an effort to intensify scientific research on rice. In 1950, one of the major steps of this institute was to lead an FAO-sponsored project called the Japonica-Indica Hybridization Programme. Although this project was pursued vigorously, it appears from the reports of the Central Rice Research Institute that very little attention was given to morphological plant type and too much emphasis was placed on the performance of advanced generations of bulked seed. Irrespective of the cause, the performance of the progeny was not outstanding as compared with the local varieties used as controls. Blast disease-resistant rice was bred in 1936 in Tamil Nadu, with the parent material Co 4, which is immune or near immune to the blast disease. Co 4 is a selection from the variety ‘Gobi Anaikomban’. This was crossed with a local variety ‘Korangu samba’ which is highly susceptible to the disease. Co 4,

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though resistant to the disease, is a long duration variety with coarse grains and is not popular among the cultivators. ‘Korangu samba’ though susceptible to the disease, is a high yielder, with many economic properties. Cultures obtained from these crosses were tested for resistance in the field and in the greenhouse and the highly resistant varieties, Co 25 and W 26, were selected from them. In India, as far as blast disease is concerned, the resistant variety, Co 25 (a cross between A dt 10 and Co 4) was released for cultivation in 1948 and Co 29, a cross between Co 13 and a selection from ‘Arupathamkodai’, was released subsequently. They have been grown in the fields for about 25 years. Instances of heavy infection occurring on these varieties are now being reported. Resistance may break down within a short period of time in endemic areas. The release of Co 25 brought into prominence Helminthosporium in the Thanjavur delta, because Co 25 was susceptible to this disease. Several solutions, including the development of multilineal lines and horizontal or field resistance based upon polygenic resistance, have been suggested to overcome the problem created by the variability of the pathogen. Apparently the penetration phase in blast is controlled by polygenes. Work done by Padmanabhan (Padmanabhan, et al. 1974) had shown that it is possible to combine a high degree of resistance to penetration (polygenic) with resistance to the spread of diseases inside the tissue which is controlled by major genes. There is evidence that in some of the moderately resistant varieties, resistance may be conditioned by polygenes. As early as 1954, a programme of breeding for resistance to blast and Helminthosporium disease was initiated by crossing Co 25 (resistant to blast but susceptible to Helminthosporium) and BA 410 (susceptible to blast but resistant to Helminthosporium). Varieties resistant to both were evolved and widely tested in India. Some of the important varieties of rice introduced and developed in India are as follows: IR8, IR20, Jaya, Padma, Hansa, Pankaj and Kavery. Table 11.3 gives a birds-eye view of the disease-resistant varieties used in the IRRI rice-breeding programme to several diseases. TABLE 11.3 Disease-resistant varieties from the world collection used in the IRRI rice-breeding programme Disease Resistant Varieties Blast Zenith, Sigadis, Tadukan, B589 A4-18, Dawn, H105, T172, Nahng Mon S-4, Leuang Yai34 Tungro virus Peta, Sigadis, Pankhari 203, TKM6, HR 21. Bacterial leaf blight Zenith, Sigadis, TKM6, Malagkit Sungson, Tadukan, B589 A4-18 Bacterial leaf streak Zenith, Cp 231-SLO-17, B589 A4-18, Nato Tadukan, RL, Gopher, PI 208443.

The International Rice Research Institute (IRRI) has named IR-28, IR-29, and IR-30 their early maturing rice varieties. All the three varieties are insensitive to changes in photoperiod. Hence IR-28, IR-29 and IR-30 are the only known rice varieties that are highly resistant to grassy stunt virus disease, which is a serious problem in many countries. The varieties inherit this resistance from Oryza nivara, a wild relative of rice. IRRI pathologists report that IR-28 and IR-29 are also resistant to tungro, bacterial blight and blast diseases. IR-30 is resistant to tungro and bacterial blight, but is moderately susceptible to blast.

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IR-32 and IR-34 have been named by the International Rice Research Institute. Both varieties are resistant to blast and insect diseases. The incorporation of disease and insect resistance into improved germ-plasm is a major objective of the IRRI and GFU programme. Several donor parents that have been used as sources of resistance to each major disease and insect are poor plant types (tall with weak stems and droopy leaves). They are crossed with races of improved plant types, such as TN-1, IR-8, IR 26243-8 and IR-24. Semi-dwarf lines with good grain quality and resistance to specific diseases or insects are selected and evaluated for several reasons and inter-crossed to develop lines with multiple resistance. Gurdev S. Khush and Radalpho et al. used several sources of resistance for each pest and disease, except grassy stunt virus, for which Oryza nivara is the only source of resistance. For blast, such donor parents as Gam Pai 15, H-105, Sigadis, Nahng Mon S-4, Dawn, Zenith, Kam Baunagan, and Tetep were used. For bacterial blight, Zenith, Malagkit Sungsong, TKM-6, Sigadis, hase Aikok 43, BJI and DZ 192 were used, and for tungro, Gam Pai 15, TKM-6, Peta, Pankhari 203, Malagkit Sungsong, HR21, Co 13 and Ptb 18 were used. The breeding of blast-resistant varieties is now an important objective in rice-breeding programmes of Japan, the USA, India, Taiwan, Philippines and other countries. The problem of breeding for resistance is complicated by the large physiological races of the fungus. Furthermore, the races of the blast fungus appear to be different in different countries. For example, varieties resistant in India may not be resistant in Taiwan or the Philippines. A comparison of races in different countries has been difficult because various differential varieties have been used in the countries for identification of the races. Attempts have been made to develop a set of differential varieties, which may be used uniformly in all the countries. Resistance to blast has been reported to be controlled by one, two or three genes with resistance partially dominant. Strains resistant in the seedling stage are also resistant to the same races of the blast fungus in the neck-rot stage (Ou and Nukue, 1963), for example, accession 6741 (CD 231 ¥ HD-12), accession 9914 (MO.R-500 ¥ Nato) and H-105 from the USA. Taichung 172 and 176, Chinan 8 from Taiwan and Kataktara from Pakistan are among the varieties resistant to blast at the International Rice Research Institute. Bacterial leaf blight (Xanthomonas campestris pv. oryzae) is one of the most destructive rice diseases in India, Indonesia, the Philippines and other countries of South and South-east Asia. The breeding of resistant varieties appears to be the principal means of control. Out of the 102 varieties tested at the International Rice Research Institute, one variety of rice from the USA, and 13 from Taiwan were resistant. Thirty varieties from 8 countries were rated intermediate in resistance. Resistance was noted in many of the wild species of rice. Accession 6973 and 9797, Sigadis, Kaohsiung 68, Chinan 8, and Taichung 172 and 176 are among the resistant varieties. A single dominant gene, Xo, is reported to control resistance to bacterial leaf blight. Virus and mycoplasmal diseases In 1961, when the International Rice Research Institute was established in the Philippines, it was rightly believed that viral diseases caused damage to rice in Southeast Asia. Since then at least four viral or mycoplasmal diseases have been reported from the Philippines. These are orange leaf, tungro, yellow dwarf and grassy stunt (now thought to be bacterial). There is also evidence that the so-called ‘physiological diseases’ known to occur widely in South-east Asia are caused by viruses. The sources of resistance should be located and genes for resistance incorporated into locally adopted varieties of rice.

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Brown Spot In India, the varieties BAU 10, T. 141, and CH-13 are reported to be resistant. A single dominant gene was reported to control resistance in a study made in India, while polygenic control was reported in the USA. Breeding Sugarcane for Resistance

The breeding of sugarcane has been closely related to the outbreaks of serious diseases in the crop. The sereh disease, thought to be caused by a virus, led to the abandonment of the black Cheribon variety of the noble sugar canes in Java. Another virus disease, mosaic, combined with red rot, almost closed down the sugarcane industry in Lousiana. The disease was later controlled by the breeding of resistant varieties. Fiji disease caused severe damage to sugar cane in Fiji until that crop was brought under cultivation by breeding. Saccharum officinarum is susceptible to mosaic, sereh, streak, red rot and gummosis, although it is generally resistant to smut. S. barberi varieties are generally susceptible to red rot and smut, but moderately resistant to Pythium root rot, mosaic, sereh and streak. S. spontaneum forms are usually resistant to sereh, Pythium root rot, and largely susceptible to smut. S. spontaneum has been widely used in crosses as a source of disease resistance in many present day hybrids. The possibility of breeding for disease resistance was dramatically demonstrated first by a chance hybrid between a noble sugarcane, Black cheribon, and a wild cane of Java which proved to be resistant to sereh disease (Abbott, 1953). The principal sugar cane diseases in India are red rot, smut, mosaic, ratoon-stunting and rust. Wilt and grassy shoot also occur but not much information is available on the sources of resistance to them. Red Rot S. spontaneum has been the chief source of resistance. S. officinarum and S. barberi are susceptible. There are several physiological strains of the red rot organism. To meet the challenge of host-resistance breakdown posed by the emergence of new strains/races of pathogens, a number of genotypes are tested at endemic locations and varieties are available for replacement. Co 7314, Co 5767, BO91, BO99, Co 62198, Co 6907, and Co 7219 are the varieties currently popular for red rot resistance. Smut Varieties of S. barberi and S. spontaneum are largely susceptible but S. officinarum varieties are resistant. Co 449, derived from a cross between POJ. 2878 and Co 331, has in it the complements of S. officinarum, S. barberi and S. spontaneum. It is a fairly high yielder and is resistant to smut. Co 527, derived from a cross between Co 349 and Co 312, is resistant to smut. Co 658 a hybrid between Co 605 and Co 443, is resistant to smut. Co 7704 is a popular smut-resistant variety these days. Ratoon Stunting Little information is available on resistance in India. A number of resistant and high-yielding varieties have been bred in Coimbatore which have become world-famous and are known as Co varieties. In these crosses, resistance and hardiness is incorporated from the wild, grassy S. spontaneum which is crossed with S. officinarum (noble sugar canes) and other species. The P.O.J. varieties of Java are also notable in this regard. In these varieties also, S. spontaneum is one of the parents. P.O.J. varieties are known for their resistance against mosaic.

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Breeding Potato for Resistance

A world-wide research in Solanum species showed that a number of them had natural resistance to Phytophthora infestans. One species which has proved to be an important source of resistance is S. demissum. Although this species is not of economic value, it is possible to cross it with S. tuberosum, and some of the hybrids that are obtained are resistant to the disease. Formerly, it was thought that Solanum demissum contained four major genes for resistance (Black, 1952), but later work showed that there are at least six major genes for late blight resistance in potato (Black, 1960). On this basis the possibility of the occurrence of races of Phytophthora infestans should be 26, that is, 64 races. In Canada, five new R genes from S. demissum have been identified. Another R gene has been identified in S. stoloniferum. At least nine genes have now been identified (Malcolmson and Black, 1966). The unit for selection is a single tuber or the tubers from a single plant. Kufri Kundan is a good example of a hybrid variety that was developed at CPRI, Simla, from a cross between Ekishizazu and Kalahdin, introduced from Japan and the USA, respectively. Sometimes multiple crosses are used to develop suitable varieties. In India, hybridization is done at Simla but the F1 seeds are sent to the regional stations in different parts of the country for performance trials. The variety, Kufri Kaver, released in 1958 was developed from the cross (Solanum curlilobum x S. tuberosum) S. andigena. Kufri Sanduri is a selection from a cross between Kufri Red and Kufri Kundan. There are several resistant varieties now such as Kufri, Jeevan, Kufri Alankar, Kufri Khasi Garo and Kufri Muthu for areas where these varieties give good yield and late blight is of regular occurrence. In 1978, the variety Kufri Badshah was released for the northwestern plains of the country to replace varieties which had started losing resistance to late blight. For the hilly regions of the north and northeast varieties K. Himabim and K. Sherpa were released in 1981. Variety Kufri Jyoti which was resistant to late blight has now become susceptible to certain races of the pathogen. Potato Wart Varieties resistant to wart have been developed in the UK, USA and Holland. All these varieties have been utilized in Simla as sources of resistance, which is also found in several wild species. Several virus diseases infect the potato crop. These are virus X, virus A, virus Y, leafroll, virus S, virus C, and a spindle tuber as well as several soil-borne viruses. In India, breeding work has concentrated on resistance to viruses X and S. In Solanum acaule and S. andigena, a single dominant gene R controls immunity. Yet in another case of selection of Solanum tuberosum from Wisconsin, immunity is reported to be controlled by a gene C in the recessive homozygous condition. The hybrid HC 294, a selection from the cross Kufri red x (Gudstone x taborky) has been reported to be resistant to M. incognita. The most common potato beetle is Epilachna ocellata. A few wild species of Solanum, such as S. garciae, S. malinchense and S. polyadenium are reported to be resistant to this beetle. The main objectives of potato breeding in India are high yield, regional adaptability, quality, and resistance to heat, frost, drought, diseases, and insects. In order to achieve their introduction, selection, hybridization, mutation and polyploidy, breeding has been resorted to. Many varieties have been introduced in India from time to time, among them being over 1500 commercial varieties and 120 wild species of Solanum which are maintained at the Central Potato Research Institute, Simla. This collec-

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tion contains indigenous as well as exotic strains. Varieties, such as Magnesium, Barium and Craigs Defiance are some of the introduced varieties which are commercially grown in India. New varieties are developed by clonal selection and hybridization. The variety, Kufri Red, is selected from the Darjeeling Red Round which was introduced in India many years ago and whose history is lost. In 1975, Kufri Jyoti was introduced as a wart immune and late blight resistance cultivar. Between 1976 and 1988 many wart-immune cultivars were developed and released for cultivation. There are Kufri Muthum, Kufri Sharpa, Kufri Sheetman, Kufri Khasi Garo, Kufri Bahar and Kufri Kumar. Kufri Kanchan with red skin has been found resistant to wart recently. Breeding for Disease Resistance in Tomato

Tomato breeders have made use of several Lycopersicon spp. to breed a single cultivar resistant to several diseases. The following species were utilized in the breeding programme to bring in genes as follows: L. pimpinellifolium – Fusarium, verticillium, bacterial and spotted wilt, bacterial canker, grey leaf spot and leaf mould. L. hirsutum – Septoria leaf spot, tobacco masaic, leaf mould. L. peruvianum – Beet curly top, root knot spotted wilt, Septoria leaf spot. L. chilense – Anthracnose. Breeding for Disease Resistance in Pulses

Such breeding has been confined mostly to blight and wilt diseases of gram and the wilt disease of pigeon pea. Gram blight is caused by Ascochyta (Phyllosticta) rabiei. Studies on blight resistance have been made in India and other countries and sources of resistance are available. Adequate information is not available regarding the races of the fungus although in India there are indications of the occurrence of new races. In Punjab, the variety, C.1234, which was originally found to be blight resistant, was susceptible in later years. Root rot of gram is caused by Rhizoctonia bataticola. A Fusarium wilt disease has also been reported. The inheritance of Fusarium wilt resistance is reported to be controlled by a single pair of genes. The variety, C.24, from Punjab is reported to be resistant to wilt. In Pakistan, the gram variety, C. 612, is reported to be resistant to blight and tolerant to wilt. The most important disease of pigeon pea is wilt caused by Fusarium udum. Resistance is reported in the types, NP- 41, NP-51, and NP-80. Crosses of NP-51 x NP-24 have resulted in the development of four highly wilt-resistant selections, NP (WR) 15, NP (WR) 16, NP (WR) 16, NP (WR) 38. The wild varieties, Atylosea lineata and A. sericea genus related to Cajanus are reported to be sources of wilt resistance and have been utilized in crosses for this purpose. Resistance to wilt is stated to be controlled by a pair of duplicate dominant genes and also by multiple genes. The important diseases of black gram and green gram include leaf spot, incited by Cercospora cruenta and chlorosis. The black gram variety, Kulu Mash. 4, is reported to be resistant to leaf spot, and the green gram selections 24-2 and 24-3 are resistant to chlorosis in Punjab.

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Breeding for Disease Resistance in Groundnut

Leaf spot or Tikka disease (Cercosporidium personatum and Cercospora arachidicola) and wilt or root rot (Rhizoctonia destrens) are important diseases of groundnut in India, the former being the most common. Resistance to leaf spot is independently inherited and a single factor inheritance for resistance to Tikka has been reported in wild species, such as Arachis villosa. Hybrids from the inter-specific crosses have been obtained with resistance to the disease. Varieties resistant to leaf spot and wilt include 5203 of Gwalior, G-0120, G-1032 and G-0607 of Mysore and Exotic-4 of Indore. Jute Breeding for Disease Resistance

The stem rot fungus, Macrophomina phaseolina, can produce seedling blight, stem rot, or root rot in jute. Screening for existing varieties, both local and exotic, and breeding for resistance are in progress at the Jute Research Institute, Barackpore. The standard varieties, D-154 and JRC-918, a selection from the Brazilian material, are sources of resistance for stem rot. Mishra and Mukherjee (1984) had reported Capsularis strains like 8694, 212, 918, and 5854 to be highly resistant to M. phaseolina diseases. Breeding for Disease Resistance in Tobacco

There has been progress in disease resistance in tobacco in the USA. Some of the lessons learnt from the experiences indicate that: 1. Resistance to black shank and bacterial wilt is present in common tobacco. 2. Adequate resistance to many diseases is found only in wild species of Nicotiana. 3. Intra-specific resistance is frequently polygenic, although inter-specific resistance is often simply inherited. 4. Transfer of high resistance or immunity from other usually results in the production of strains undesirable in plant type, yield or quality unless back crosses are made to eliminate the undesirable genes introduced from the alien species. 5. Desirable genes in other species are some times linked with undesirable genes. The black shank disease was first found in the USA in 1916. Florida 301, a resistant shade tobacco variety, was developed around 1930 by crossing and selection within local varieties of ‘Big Cuba’ and ‘Little Cuba’. Florida 301 was later used as the source of resistance in the breeding of other black shank-resistant varieties. Some of the black shank-resistant varieties developed in the USA are RG, Oxford 1,2,3, and 4, Dixie Bright 101 and 244, Vesta and Dixie shade. Resistance to black shank appears to be controlled by multiple factors, varieties differ in the degree of resistance, and N. longiflora and N. plumbagenifolia are highly resistant or immune. Resistant genes from these are being transferred to common tobacco. Breeding work on the resistance to bacterial wilt was started in 1934 by studying 1034 collections of tobacco from Mexico, Central America and South America.

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Breeding for Disease Resistance in Sorghum

Research in breeding for resistance in sorghum has begun in India. Leaf spot disease caused by Helminthosporium turcicum often causes severe damage to sorghum in India. The disease is carried on the seeds or in the soil. This is the same disease that causes leaf blight in maize. Several varieties were found to be resistant in the USA, including two varieties of Sudan grass. Tift Sudan grass was developed from the cross (Sudan grass x Leoti sorgo) x Sudan grass. Only one of the thirty thousand F2 plants of this cross possessed the resistance of Leoti sorgo and the plant characters of Sudan grass. Breeding for Disease Resistance in Cotton

Many diseases attack cotton plants in India, but breeding work has concentrated on two diseases, Fusarium wilt, and Bacterial blight or black arm disease. Breeding for resistance to bacterial wilt was started before 1900. A wilt- resistant variety, Sea Island cotton, Rivers and two upland varieties, Dillon and Dixie, were developed in the USA by selection of resistant plants growing on wilt-infested soil. Seed harvested from the resistant plants was then tested on a progeny row basis. The principles of survival and progeny testing were introduced in cotton breeding. Various studies on the inheritance of disease indicated the presence of two or more dominant genes which control resistance to the wilt disease. Resistant varieties in India include H-420 and Virhar in Gossypium arboreum, KFT, Digvijay and Jayawant in G. herbaceum and Co. 2 in G. hirsutum. The US varieties, such as Coker 100 wilt, Stonewilt and Auburn 56 are nematode- resistant, besides being highly wilt-resistant. Bacterial blight, also called black arm, angular leaf spot, and boll blight, is a bacterial disease caused by Xanthomonas campestris pv. malvacearum. The Indian arboreum and herbaceum varieties are not much affected by the disease but the American hersutum cottons in India are highly susceptible. Extensive studies in resistance have been carried out in Sudan, the USA, India and other countries. In India, resistant varieties have been developed from hirsutum x herbaceum crosses. A lot of work for incorporating resistance to agronomically better and high-yielding but susceptible varieties has been done in India and abroad. Several sources resistant to bacterial blight are available such as 101-102B, BJA-592, P-14-7-128, Raba B-50, HG-9, Tamcot-CAMDE, Tx Bonham. In India, a variety BJR (Bacterial blight Jassid Resistant) has been developed. In India, infestations in cotton due to Jassids (Empoasca devastans) are most severe in hirsutum varieties grown in the irrigated tracts. The arboreum and herbaceum varieties are more resistant owing to the presence of dense hairs. Much has been done on the genetics of the hairiness of cotton, and hairy genes have been identified. INNOVATIVE METHODS OF PLANT DISEASE CONTROL

In a multi-authored book entitled “Innovative methods of plant disease control ” edited by I. Chet (1987) several aspects of innovative methods have been discussed. Mehrotra (1993) has discussed the innovative methods for plant disease control. The first important innovative technique is soil solarization. There are others such as use of siderophores, mycovirus infection and hypovirulenceinduced resistance, utilization of mycorrhizal fungi, genetic engineering for plant disease resistance, pollen management and biological control. Some of these have already been discussed earlier. Here soil

Management of Plant Diseases 283

solarization and pollen management will be discussed in somewhat greater detail. Soil solarization— According to Katan et al., (1976) this technique for control of plant diseases refers to a new approach for soil disinfestation by means of solar energy. Solarization is done by covering the soil with transparent polythene sheets 100 mm thick for 6-8 weeks during summer before planting any crop. At ICRISAT, Pattancheru, Hyderabad, Chauhan et al., (1984) successfully controlled wilt of pigeonpea and chickpea with the help of soil solarization. Sharma et al. (1984) successfully demonstrated reduction in the viability of sclerotia of Sclerotium oryzae when soils were mulched with transparent polythene sheets 0.5 mm thick. Lodha (1989) showed the reduction of the population of Macrophomina phaseolina in arid soils of Rajasthan as a result of solarization. Dwivedi (1993, 1997) studied the soil mycoflora of sugarcane and sugarbeet crops in relation to soil solarization and reported encouraging results. Soil solarization has also been successfully employed to control soil nematodes in India. Pollen Management

Thakur and Williams (1978, 1980) and Thakur et al., (1983) have reported excellent control of ergot in pearl millet through pollen management. Based on the findings that rapid pollination reduces ergot infection, experiments were conducted at ICRISAT Centre at Pattancheru, A.P, where an ergot less susceptible early flowering line was strategically planted as a pollen donor for susceptible hybrids, ergot severity was considerably reduced and grain yields were increased in different hybrids. Genetic manipulation (engineering) has been discussed in Chapter 29 where techniques and applications of biotechnology have been discussed in relation to biotechnology and plant disease control. Integrated Pest Management

IPM (integrated pest management) is a paradigm that is widely used or adopted by all pest control disciplines but whose early definitions and philosophical basis belong to entomologists (Jacobsen, 1997). In phytopathology another term IDM (integrated disease management) is being used and is a concept derived from integrated pest management. Integrated pest management aims at keeping the pest population below the level of economic injury using best combinations of various components of management. FAO has defined IPM as a pest management system that, in the context of associated environment and the population dynamics of the pest species, utilized all suitable techniques and methods, in as compatible a manner as possible and maintained the pest population at levels below those causing injury. Srivastava (1999) has briefly summarized the concept of integrated pest management for sustainable agriculture. This new concept actually describes what the farmers were doing before chemical pesticides came into vogue. In IPM the various components of management are so tailored so as to get cost-effective control without any adverse effect on the environment and ecosystems. The IPM or IDM relies on non-toxic means but does not intend to keep pesticides at bay from the management system, rather advocates minimum or requsite use of pesticides as may be appropriate to make disease management not only effective but non-hazardous, user-and environment friendly. IPM or IDM thus rules out total reliance on pesticides which by and large are the strongest weapons against pathogens or pests. The various components of IDM are host resistance, cultural practices (such as physical treatment of soil by baking the soil by direct exposure to the sun or through soil solarization, removal of infected plants before they contribute to disease severity such as removal of seedlings affected by downy mildew of

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pearl millet caused by Sclerospora gaminicola within 3-4 weeks after sowing followed by gap filling by healthy seedlings). Crop rotation is immensely beneficial in reducing inoculum levels. In the case of flag smut of wheat caused by Urocystis agropyri (syn. U. tritici), manipulation in the dates of sowing is aimed at avoiding the susceptible age with congenial environmental period. For example blast of rice in Haryana can be minimized by planting Basmati varieties by the first week of July and ergot of bajra incidence can be minimized by sowing the crop by June end or the first week of July. Integrated disease management also encourages biological control of plant pathogens. This aspect has been discussed earlier. In India efficiency of several biological control agents has been reported from TANU, Coimbatore, PAU, Ludhiana, G.B. Pant University of Agriculture and Technology., Pantnagar, University of Agricultural Sciences, Bangalore and Kurukshetra University, Kurukshetra, and several potent biocontrol agents reported are species of Trichoderma, Gliocladium, Chaetomium, and certain Pseudomonas. In recent years several botanical pesticides have been reported to control plant pathogens. Neem products/neem extracts have become quite popular as insecticides. The efficacy of neem has also been reported against some fugal pathogens also. Though IPM or IDM relies on nontoxic chemicals for plant disease control, it does not preclude the use of pestcides. It favours minimum and judicious use of chemicals. Of course growing resistant varieties is the best method of controlling any disease and will form an important strategy for managing plant pathogens. This IPM or IDM is a system approach that combines a wide array of crop production and protection practices to minimize the economic losses caused by pests (insects, pests, diseases, nematodes, weeds, rodents, birds etc.). It emphasizes careful monitoring of pests and conservation of their natural enemies. The Indian Council of Agricultural Research (ICAR) established a National Centre of Integrated Pest Management (NCIPM) in 1988 to cater to the emerging plant protection needs of agriculture in the country.

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QUESTIONS 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

What are the cultural methods of plant disease control? What are plant quarantines? When and where was the first quarantine act enacted? What is the D.I.P. act? When was it first enacted? Name some plant diseases, which have been introduced before and after the enforcement of plant quarantines in India. Is quarantine necessary for all types of diseases? Name some plant diseases, which can be controlled by crop rotation. What is seed certification? What is biological control of plant pathogens? Give some suitable examples where it has been successfully used to control disease. What are the different chemical systemic fungicides for plant disease control? How do the fungicides act in the pathogen? What is chemotherapy? What is the mechanism of the action of various groups of fungicides? Name some important antibiotics used for plant disease control. Breeding for disease resistance is the best method of controlling plant diseases. Why? What is integrated Pest/Disease Management (IPM or 1DM). What is solarization? What are the innovative methods of plant disease control? Name a few. Name some plant pathogens of plant quarantine importance to India. What is resistance to fungicides by plant pathogens. What strategy will you employ to overcome this?

12 Plant Diseases Caused By Fungi— Characteristics of Plant Pathogenic Fungi, Classification of Plant Pathogenic Fungi

GENERAL FEATURES OF FUNGI

Although it is not easy to define fungi, biologists currently use the term ‘fungus’ to include nucleated, spore-bearing, achlorophyllous organisms with absorptive nutrition which generally reproduce sexually and asexually, and whose filamentous branched somatic structures are typically surrounded by cell walls containing cellulose or chitin or both. Although fungi are a diverse group of organisms, a feature which they all have in common is their mode of nutrition. Like animals, fungi are heterotrophic organisms, which must consume preformed organic matter. They may live as saprophytes or parasites. Fungi are of great economic importance to man and play an important role in the disintegration of organic matter. They affect us directly by destroying food, fabric, leather and other commercial goods. They are responsible for a large number of diseases of plants, animals and man. Cell Structure

Fungal cells may be minute and a single, uninucleate cell may constitute an entire organism. A single cell of Olpidium or yeast cell falls in this category. Alternatively, fungal cells may be elongated and strand-like and several may be joined to form a thread of cells—a hypha (pl. hyphae). A large number of hyphae collectively form the mycelium. The cell wall of fungi is generally made up of chitin or cellulose. The majority of the fungi have chitinous walls. Occasionally, both chitin and cellulose may occur simultaneously. One cell in the hyphae may be separated from another by a cross wall or a septum. Septa in different groups of fungi have important differences in structure. For example, the septa in the lower fungi are pseudosepta, which are septa that are perforated by so many pores that they are sieve-like. In Ascomycota and some Fungi Imperfecti, the septum is perforated by a single pore. In the Basidiomycota and some members of Fungi Imperfecti, the septum is more complex than that found in the Ascomycota. The septum here is called a dolipore septum.

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Hyphae characteristically grow at the tips and can usually produce lateral branches in their older parts. The mycelium may remain microscopic or develop easily visible organized structures, such as strand- or cord-like rhizomorph compact resting bodies comprising fungal tissues made of prosenchyma or pseudoparenchyma. All growing hyphae are filled up with cytoplasm, are frequently vacuolated and may be aseptate, hence, they form a single multinucleate cell (coenocyte) or are divided into segments by septa. Fungal nuclei are very small. The nuclei have well-developed nuclear membranes. The nuclear envelope consists of a double nuclear membrane and is marked by large gaps. During mitosis the nuclear envelope does not disappear ordinarily, instead it constricts in a dumbell-like fashion and eventually separates into two daughter nuclei. Meiosis conforms more closely to the norm in other organisms than does mitosis. Ribosomes are of 80S and mitochondria occur in the cytoplasm. There is also the endoplasmic reticulum. Storage products occur in the fungal cell. These may be in the form of glycogen or lipids. Golgi apparatuses similar to those in higher plants and animals have been found in some fungi. A feature which is of widespread occurrence is the presence of lomasomes. These are made up of membrane bounded tubules or vesicles. The function of lomasomes is not known. Reproduction

Fungi reproduce both asexually as well as sexually. Asexually reproduction takes place by a variety of methods. This may be by: (1) fragmentation of the soma, (2) fusion of the stomatic cells, (3) budding of the somatic cells or (4) production of spores, such as conidia, sporangiospores – motile or non-motile. Sexual reproduction is of widespread occurrence in fungi. Fertilization can be brought about in a variety of ways. The most common methods are: (1) planogametic copulation, (2) gametangial contact, (3) gametangial copulation, (4) spermatization and (5) somatogamy. Life Cycle

As in other organisms, in fungi too there is generally a cycle of haploid and diploid structures, corresponding to the gametophyte and sporophyte in the green plants. The diploid phase begins with karyogamy and ends with meiosis. In the majority of fungi there is no distinct alternation of generations. Raper (1954) recognized seven basic types of life cycles. However, Burnett (1976) recognizes five basic life cycles in fungi. These are: 1. Asexual, in which sexual reproduction is lacking, namely, Deuteromycetes. 2. Haploid, in which meiosis immediately follows nuclear fusion. This is very commonly seen in fungi or some members of Ascomycota. Here the diploid phase is of minimum duration. 3. Haploid-dikaryotic, which is similar to the second type, excepting that paired, potentially conjugate nuclei persist in close physical association in the same hyphal segment (hence dikaryon). The examples of this are many members of Ascomycota. Binucleate ascogenous hyphae develop just prior to ascus development and such a dikaryon cannot exist independently of the haploid phase. On the other hand, there may be a condition where the meiospores fuse to form a dikaryon so that the fungus is dikaryotic throughout its life cycle, excepting for the moment of fertilization and during the subsequent meiosis. This occurs in yeast (Endomycetales) and more commonly in smuts (Ustilaginales). In the Basidiomycota, the mycelium derived from the germination of a meiospore may persist in the haploid condition as a monokaryon, but once a dikaryon is formed, it shows unrestricted and independent growth and is the ‘long-lived’ phase of the life cycle.

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4. Haploid-diploid, in which the haploid and diploid phases alternate regularly. This is restricted to the order, Blastocladiales. 5. Diploid, in which the haploid phase is restricted to the gametes. The vegetative phase is diploid. Reduction takes place at the time of gametogenesis, such as Pythium, Phytophthora, Saprolegnia spp., in the Oomycetous fungi. CLASSIFICATION OF FUNGI

Whittaker (1969) broke the tradition of a three kingdom system of classification of all living organisms. In doing so he recognized that the classification of all living organisms as Prokaryotes, Animals and Plants (including Fungi) did not reflect their relationships. Whittaker added two more kingdoms in this classification viz. Fungi and Protista. In an attempt to recognize monophyletic groups the organisms once classified as Fungi are now considered in three different groups, the monophyletic kingdom Fungi, Straminopila or Chromista and four protist phyla. The kingdom Fungi includes four phyla—Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota. Kingdom Straminopila or Chromista includes phylum Oomycota,

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Diagrammatic representation of the 5-kingdom system of Whittaker (1969) based on three levels of organization—the prokaryotic (Kingdom Monera), and eukaryotic kingdoms (Protista, Plantae, Fungi and Animalia).

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Hyphochytridiomycota, and Labyrinthulomycota. The four phyla of the Kingdom Protista or Protictista are Plasmodiophoromycota, Dictyosteliomycota, Acrasiomycota and Myxomycota. An outline classification of fungus and fungus-like organisms is given below: FUNGUS LIKE ORGANISMS KINGDOM PROTISTA KINGDOM CHROMISTA OR STRAMINOPILA Phylum Plasmodiophoromycota Phylum Oomycota Phylum Dictyosteliomycota Phylum Hyphochytridimycota Phylum-Acrasiomycota Phylum Labyrinthulomycota Phylum Myxomycota

TRUE FUNGI KINGDOM FUNGI Phylum Chytridiomycota Phylum Zygomycota Phylum Ascomycota Phylum Basidiomycota

Kingdom Protista

It is a kingdom of eucaryotic microorganisms exclusive of the kingdoms Animalia, Fungi, and Plantae, but including Myxomycota and other plasmodial groups (Plasmodiophoromycota, Dictyosteliomycota and, Acrasiomycota). Phylum Plasmodiophoromycota (endoparasitic slime moulds ) The phylum Plasmodiophoromycota contains a single class Plamodiophoromycetes, with a single order Plasmodiophorales and a single family Plasmodiophoraceae. There are 10 genera and a total of 29 recognized species. Plasmodiophorids produce multinucleate unwalled protoplasts typically referred to as plasmodia. These plasmodia are significantly different from Myxomycota as they are incapable of translocational movement, lack the ability to phagocytize food materials and exist wholly within the cells or hyphae of their hosts. Their life cycle involves the production of two different plasmodial phases. Mitotic division of nuclei is called cruciform division. Plasmodiophora and Spongospora are two important genera from the plant pathological stand point. In Plasmodiophora the cysts lie free in the cells of the host, in Spongospora the cysts are in the form of spore balls. Plasmodiophora brassicae causes club root disease of crucifers (also known as finger and toe disease of crucifers). Spongospora subterranea causes powdery scab of potatoes. Phylum Dictyosteliomycota (dictyostelid cellular slime moulds) These are distinguished from most other organisms by the aggregation of their somatic amoebae to form a pseudoplasmodium also designated grex or slug. The component amoebae never fuse but retain their individuality while cooperating as members of a well organized community until the formation of a sorocarp. Dictyostelium discoideum is an important member of this group. Phylum Acrasiomycota (acrasid cellular slime moulds) Acrasids are characterized by cylindrical amoebae exhibiting phagotrophic nutrition. The amoebae are all of the limax type. Dictyostelid and acrasid slime moulds are significantly different in a number of morphological and life cycle details. Phylum Myxomycota (true slime moulds) In the assimilative phase a plasmodium is free living and saprobic. These organisms exhibit phagotrophic nutrition and produce the following life cycle stages: 1. Three types of uninucleate cells, one of which is flagellate

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2. A multinucleate somatic phase as a plasmodium that moves and exhibits a reversible shuttle streaming of its protoplasm 3. A resistant stage consisting of a sclerotium 4. A reproductive phase that culminates in the production of stationary sporophores containing walled spores The phylum Myxomycota contains a single class Myxomycetes that is subdivided into three subclasses containing a total of six orders. These orders are Liceales, Echinosteliales, Trichiales, Physarales, Stemonitales, and Ceratiomyxales. Kingdom Chromista or Straminopila

This kingdom is divided into 3 phyla namely 1. Phylum Oomycota 2. Phylum Hyphochytridiomycota and 3. Phylum Labyrinthulomycota. Phylum Oomycota The characteristics that set the Oomycota organisms apart from true fungi and also define or delineate the phylum are as follows: 1. Asexual reproduction by a biflagellate zoospore with a longer tinsel flagellum directed forward and a shorter whiplash flagellum directed backward 2. Various features of ultra-structure of zoospores 3. The production of a diploid thallus and meiosis occurring at the time of gametogenesis. 4. Oogamous reproduction by gametangial contact results in the formation of a thick-walled sexual spore termed an oospore. 5. Cell walls made up of β-glucans but also containing the amino acid hydroxyproline as well as small amounts of cellulose. 6. Mitochondria with tubular cristae and 7. Various biochemical and molecular characteristics. The phylum Oomycota contains a single class Oomycetes which has six orders Leptomitales, Rhipidiales, Sclerosporales, Pythiales, Peronosporales and Saprolegniales (Dick, 1990a). However Alexopoulos et al. (1996) have recognized only 5 orders.

Key to the five orders of the class Oomycetes A. Spores always formed within the sporangium, monomorphic or dimorphic, rarely aplanetic: B,BB B. Eucarpic, hyphae regularly constricted with “cellulin” granules at constrictions: Leptomitales BB. Holocarpic or eucarpic, hyphae when present without constrictions: C, CC. C. Thallus holocarpic or eucarpic and mycelial, periplasm minimal, usually several oospores in an oogonium: Saprolegniales CC. Thallus monocentric, with rhizoids, periplasm persistant, usually a single oospore per oogonium; often in stagnant water: Rhipidiales A. Spores formed within the sporangium or, if not, then usually within an evanascent vesicle arising from the sporangium; monomorphic reniform: D, DD D. Holocarpic Lagenidiales DD. Eucarpic Peronosporales

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Aphanomyces euteiches (Saprolegniales) causes root rot of peas. Genus Pythium causes damping off seed decay, root rot and fruit rot, Phytophthora species cause blights, fruit rot, cankers and root rot of several economic plants. Genera belonging to Peronosporales such as Sclerospora, Peronospora, Bremia, Plasmopara, Pseudoperonospora, Peronosclerospora and Sclerophthora cause downy mildew of several crop plants. Phylum Hyphochytridiomycota These are fresh water or marine chytrid-like fungi whose motile cells are anteriorly uniflagellate. In species studied so far both chitin and cellulose have been shown in the cell wall of the thallus. Sexual reproduction has not been demonstrated so far. Fuller (1990) included them in the Phylum Hyphochytridiomycota, Kingdom Protoctista. Barr (1992) suggested the ancestry of the hyphochytrids was within heterokont algae, which have similar flagellar structures and a similar DNA sequence analysis which support this view. There is a single order Hyphochytriales containing the families Anisolpidiaceae, Rhizidiomycetaceae and Hyphochytriaceae which are separated on the basis of mature thallus morphology. Phylum Labyrinthulomycota The phylum has been the subject of many taxonomic debates. These have now been placed in the kingdom Straminopiles or Chromista. Porter (1990) recognized two families Labyrinthulaceae and Thraustochytriaceae. Several characters unite these two families, the most important character is the presence of an ectoplasmic network of branched, anastomosing wall less filaments produced by cells with a specialized organelle known as bothrosome or sagenogen. Other characters include the production of cell walls composed of Golgi-derived scales and in many species heterokont, biflagellate zoospores. The members belonging to both these families are associated with leaves of vascular plants, algae or organic debris. Most species are saprobes or weak parasites. Some evidence has been found that species of both the families are endobiotic in algae (Raghukumar et al. 1992). Kingdom Fungi Phylum Chytridiomycota The phylum Chytridiomycota contains the single class Chytridiomycetes. These are the only members of the kingdom Fungi that produce motile cells at some stage of the life history. The motile cells (both zoospores and gametes) of these organisms each possess a single, posterior flagellum. Other characteristics that these species of Chytridiomycota have in common but other fungi may also exhibit are 1) the coenocytic structure of the thallus, whether a globose or ovoid structure, an elongated simple hypha, or a well developed mycelium, and 2) the conversion of the zygote into a resting spore or a resting sporangium, or in one order, its growth into a diploid thallus. Cell walls of these fungi contain chitin and glucan. Nuclear divisions in the group are intranuclear and centric. Chytridiomycota are divided into following orders—Spizellomycetales, Neocallimasticales, Chytridiales, Blastocladiales, and Monoblepharidales.

Order Spizellomycetales Zoospores with several (sometimes one) lipid globules, sometimes amoeboid when in motion, rhizoids when present with blunt tips, less than 5 µm in diameter predominantly from soil. Some important genera belonging to this order are: Olpidium, Rozella and Spizellomyces. Two other interesting genera are Caulochytrium and Urophlyctis. Urophlyctis alfalfae is a plant pathogen causing crown wart disease of alfalfa, while Caulochytrium is a biotrophic parasite of higher fungi.

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Genus Olpidium A genus consisting of nearly 30 species of holocarpic, endobiotic plant parasites of algae, mosses, pollen and leaves and roots of higher plants. Development in the genus is exogenous monocentric. One best species, O. brassicae, is a vector for a number of plant viruses. This species attacks the roots of cabbage, the host for which it is named, but is capable of attacking a vide variety of monocots and dicots. The genus Rozella consists of 20 species virtually all of which are obligate endoparasites of aquatic fungi belonging to Chytridiomycota and Oomycota. Order Neocallimasticales The order once considered within Spizellomycetales consists of species of anaerobic chytrids that inhabit the rumen and also caecum of herbivore animals. They are placed in five genera including Neocallimastix. Both monocentric and polycentric thalli occur within the group. While some species have typical uniflagellate zoospores, others are exceptional within the chytrids because they are polyflagellate. Neocallimasticales is also unique among the fungi because its members are obligately anaerobic. Order Chytridiales In Chytridiales the zoospore is characterized by a constellation of characters. One or more mitochondria are included in the MLC (microbody-lipid globule complex) of the zoospore and the nucleus appears to occupy the space not taken by the MLC and the ribosomes. The nucleus is not connected to the kinetosome. The ribosomes do not occur dispersed throughout the cell, but rather are packaged by a double membrane in the central part of the cell. Rootlet microtubules typically extend from the side of the kinetosome into the cytoplasm. Most members of Chytridiales are water- or soil-inhabiting fungi, many of the former parasitic on algae and water moulds, many of the latter on vascular plants. A few parasitize animal eggs and protozoa while others are saprobic on decaying remains of dead plants. Some better known genera are Chytridium, Chytridiomyces, Polyphagus, Rhizophydium, Endochytrium, Synchytrium, Cladochytrium and Nowakowskiella. Synchytrium endobioticum causes the wart disease of potato. In the genus Synchytrium belonging to the family Synchytriaceae the thallus is holocarpic, formed exogenous to zoospore cyst, at maturity developing into a sorus, prosorus or resting spore. Order Blastocladiales Chiefly water and soil inhabiting fungi. Blastocladiales genera are characterized by the production of thick-walled resistant sporangia usually with pitted walls. Another feature linking the members of this group is the prominent nuclear cap present in the zoospore and planogametes. The order is now divided into four families, Blastocladiaceae, Coelomomycetaceae, Catenariaceae, and Physodermataceae (formerly placed in Chytridiales). Examples of the genera placed in Blastocladiaceae are Allomyces, Blastocladia, and Blastocladiella. Coelomomyces belonging to the family Coelomomycetaceae shows alternation of generations but is also heteroecious. Basically the life cycle consists of the diploid spore-producing phase which develops in the mosquito larvae while the haploid gamete-producing phase is produced in the copepod. In the family Catenariaceae the thallus is Eucarpic polycentric producing what has been described as a catenulate thallus consisting of branched or unbranched septate hyphae with rhizoids. The species of the Physoderma are obligate parasites of vascular plants producing thick-walled and thin-walled slipper shaped sporangia. Physoderma maydis causes brown leaf spot of maize or corn. Order Monoblepharidales Thallus unbranched, eucarpic with a basal or sub-basal, disc-like holdfast or differentiated into a well-developed hypha-like vegetative system, oogamous and saprobic. The order is divided into two families, Monoblepharidaceae and Gonapodyaceae. These are not of plant pathological importance.

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Phylum Zygomycota It is divided into two classes Zygomycetes and Trichomycetes. Saprobic or, if parasitic or predacious, having mycelium immersed in host tissue – Zygomycetes. Associated with arthropods and attached to the cuticle or digestive tract by a holdfast and not immersed in host tissue—Trichomycetes. The class Zygomycetes are divided into orders Mucorales, Entomophthorales, and Zoopogales. In the order Mucorales non-motile spores are produced in terminal sporangia. No zoospores are produced. The sexual spore is a zygospore. There are several families in the order Mucorales. The genus Rhizopus of the family Mucoraceae and the genus Choanephora and Blakeslea of the family Choanephoraceae are plant pathogens. The genus Rhizopus causes soft rots of fruits and vegetables, Choanephora species also cause soft rot of cucurbitaceous fruits and young flowers and of other plants including lady’s finger and others. Mucor species also cause bread mould and storage rots of fruits and vegetables. Phylum Ascomycota These fungi are characterized by the production of a sac-like structure containing ascospores, cleaved from within by free cell formation after karyogamy and meiosis. Eight ascospores are generally formed but this number may vary from one to over a thousand according to species. Mycelial ascomycetes are characterized further by compartmetalization of mycelium with distinctive walls, septa having simple pores. Group I Archiascomycetes A group of fungi difficult to characterize with morphological and biochemical characters. Members of the group lack ascogenous hyphae and ascocarps, and the asci have been homologized with sporangia. In some members of the group the asci are formed within thickwalled ascogenous cells that have been called cysts or chlamydospores. According to Alexopoulos et al., (1996) this is divided into three orders namely—Taphrinales, Schizosaccharomycetales (fission yeasts), and Protomycetales. According to some, Taphrinales contains two important genera, Taphrina and Protomyces. Some recognize the order Protomycetales to include the genus Protomyces separate from Taphrina. Order Taphrinales—Asci arise from binucleate ascogenous cells as thin-walled sacs. In the genus Protomyces, the ascospores are formed in a synascus. Taphrina species cause leaf curl of peaches and almond. The important species is T. deformans. T. pruni causes plum pockets and T. cerasi causes witches’ broom of cherries. T. maculans causes leaf spot of turmeric. Species of Taphrina are dimorphic with a saprobic, haploid, uninucleate yeast phase that probably undergoes plasmogamy to produce their infective mycelial phase in the presence of the host. Protomyces species cause galls. Protomyces macrosporous causes galls on coriander. Group II Saccharomycetes—Order Saccharomycetales (the Ascomycetous yeasts) Mostly unicelluar that produce by budding. As a group this order is characterized, not by the yeast phase, but by the absence of ascogenous hyphae and ascocarps. A number of ascomycetous or true yeasts also produce mycelium. Yeasts are important in fermentation, in baking, brewing, distilleries, and related industries. Some yeasts such as Candida albicans are human pathogens. Group III Filamentous Ascomycetes (Plectomycetes) These include Eurotiales and related species. Plectomycetes share the following characters: (1) asci typically thin-walled, globose to pyriform, evanescent, (2) asci scattered at various levels in the ascocarp and not forming a hymenium,

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(3) ascospores unicellular, (4) ascocarp typically a cleistothecium when one is present, (5) cleistothecial peridium of thin wefts to stromatic structures, (6) various types of anamorphs and conidial forms. Orders Ascosphaeriales Members of this order have been considered to be yeasts because they lack an ascocarp. Species of the genus Ascosphaera are associated with bees, most apparently are saprobic on the provisions of insects (pollen and nectar). Although a number of genera are saprobic, several members of Onygenaceae, notably species of Ajellomyces are important animal pathogens. Order Eurotiales In this order the members of the family Trichochomaceae are the most widely distributed of all fungi. The group is characaterized by a diversity of coverings over the asci. The teleomorph genera usually have phialidic anamorphs, including Aspergillus, Penicillium and Paecilomyces. Species of Aspergillus and Penicillium are important in the processing of foods, beverages. Some are important in the production of mycotoxins, yet others produce life-saving antibiotics. Species of Aspergillus and Penicillium play an important role in the post-harvest rotting of fruits and vegetables. Filamentous Ascomycetes (Pyrenomycetes; ascomycetes with perithecia) These are characterized by (1) perithecial or, occasionally, cleistothecial ascocarps that may be formed in a stroma, immersed in a subiculum or be unassociated with specialized stromatic structures, (2) ovoid-to-cylindrical unitunicate asci, usually formed from ascogenous hyphae and croziers in a hymenium or becoming secondarily scattered throughout the ascocarp, (3) persistent asci that have forcible discharge of ascospores through an ascal tip apparatus or evanescent asci with passive discharge (4) hamathecia that may consist of one or more types of sterile hyphae and pseudoparenchymatous tissue (5) one-to-several celled ascospores of various shapes and (6) diverse and often complex anamorphs and related conidial species which do not reproduce sexually. Pyrenomycetes are important as parasites and symbionts of arthropods, endophytes, mycotoxin producers, mammalian parasites, plant parasites, and saprobes. The orders discussed here are Hypocerales, Melanosporales, Microascales, Phyllachorales, Ophiostomales, Diaporthiales, Xylariales, Sordariales, and Meliolales.

Order Hypocreales (1) Pale-to-brightly coloured, fleshy stromata (2) perithecial ascocarps in most species (3) ovoid-to-cylindrical asci (4) ascospores spherical to needle-like, one-to-several celled, breaking into parts, spores in some species (5) ascospores forcibly discharged (6) conidia produced enteroblastically from phialidic conidiophores. Genus Hypocrea Cushion-shaped or effused stroma. Asci are narrowly cylindrical containing 8 two-celled ascorpores: Several types of anamorphs including Trichoderma and Gliocladium which are biocontrol agents. Family Nectriaceae

Genus Nectria Nectria produces its perithecia on the surface of a cushion-shaped stroma and the ascocarps are brightly coloured. N. galligena and related species cause serious canker diseases of a large number of hardwood trees including apple and pear.

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Genus Gibberella Giberella is characterized by deep blue or purple perithecia and fusoid one septate ascospores. Gibberella zeae with a Fusarium graminareum anamorph causes red ear rot of maize, while G. xylarioides destroys coffee plantations in Africa. G. fujikuroi with a F. moniliforme anamorph causes the foolish seedling disease of rice in Asia and is a source of gibberellic acid. It acts as a promoter of flowering, cell elongation, amylase production in seed germination and inducer of seedless fruit development. Family Clavicipitaceae Characterized by bright or darkly-pigmented, fleshy, stromata, long narrow cylindrical asci with thickened dome-like caps perforated by long cylindrical pores, and paraphyses formed on the lateral walls of the ascocarp but not occurring among the basally-tufted asci. Ascospores are thread-like and extend the length of the ascus. Important genera are Claviceps, and Cordyceps. Claviceps parasitic on grasses while Cordyceps is pathogenic on insects. Torrubiella is pathogenic on spiders, Epichloe is endophytic in grasses and Balansia is endophytic in grasses and sedges. Claviceps purpurea causes the ergot of rye (Secale cereale) while C. fusiformis causes the ergot of pearl millet. Order Melanosporales The important genus is Melanospora. The genus is characterized by the absence of stroma, production of short-to-long necked, pale coloured perithecia, hamathecia of pseudoparenchyma, absence of paraphyses but with periphyses, ovoid, evanescent asci, darkly pigmented, onecelled ascospores, fusoid lemon shaped with germ pores at each end. Phialophora and Gonatobotrys anamorphs parasitize the mycelium of many fungi, including the important plant pathogens. Order Microascales Lack stromata, perithecia in most species but some with cleistothecia. Asci are globoid or ovoid, disintegrating. Ascospores are one-celled. Ceratocystis and Ophiostoma are important genera of the family Ceratocystidaceae. Ceratocystis fagacearum causes the oak wilt in North America. C. fimbriala causes root rot of sweet potato, butt rot of pineapple is caused by C. paradoxa. The genus Ophiostoma primarily attacks gymnosperms. Order Phyllochorales The order is characterized by stromatic perithecial forms in most species, sometimes having a clypeus (a shield-shaped structure of differentiated stromatic tissue around the mouth of the perithecium) with oblong-to-cylindrical asci arranged basically or peripherally at the base of the perithecium, and ascus tips with pores encircled by narrow rings. Phyllachorales include the genera Phyllachora, Polystigma and Glomerella. Genus Phyllachora In Phyllachora the perithecia are immersed in the substratum. P. graminis causes leaf spot of grasses. Genus Glomerella Glomerella produce dark perithecia and there is the absence of stroma. Glomerella species are more encountered as anamorph in the genus Colletotrichum. G. cingulala causes anthracnose of many plants, bitter rot of apple is caused by G. cingulata; its anamorphic stage is Colletotichrum gloeosporioides. G. tucumanensis causes the red rot of sugarcane. Colletotrichum gloeosporioides causes mango anthracnose and that of papaya fruit rot. Order Ophiostomales The order is characterized by perithecial ascocarps, but with cleistothecia in one genus, globose-to-ovoid evanescent asci usually produced in a basal fascicle and released upward into the cavity or developing , from cells that line the periphery of the cavity and are released into the centre in the manner of Ceratocystis. Sporothrix and Leptographuim anamorphs are characteristics of some species.

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The genus Ophiostoma differs from Ceratocystis because it has rhamnose and cellulose in the cell walls, resistance to cycloheximide and nonphialidic anamorphs. Ophiostoma ulmi causes the Dutch elm disease. Order Diaporthales Perithecial ascocarps produced in a stroma of fungal and substrate tissues or directly from somatic hyphae on the substrate. Persistent clavate or cylindrical asci with pores. Genus Diaporthe Diaporthe is characterized by perithecia immersed in a stroma with long necks protruding. Egg plant fruit rot is caused by Diaporthe vexans. Diaporthe citri is the cause of citrus melanose. Species of Diaporthe have Phomopsis anamorph. Genus Gnomonia Perithecia of Gnomonia are buried in the substrate, apparently not in stromata. G.leptostyla is the cause of walnut anthracnose. Genus Gaeumannomyces The development pattern is similar to Diaporthe. It differs from other genera of the order by having multiseptate, filiform ascospores. It is a strong pathogen of plant roots, prominent appressoria referred to as hyphopodia and hyphomycetous Phialophora-like anamorphs. G. graminis causes the very destructive take all disease of wheat, rice and oats. Genus Magnaporthe M. grisea is best known by its anamorph Pyricularia grisea (formerly P. oryzae). It is the cause of rice blast. The ascospores are three-septate and the two middle cells are pigmented. A non-specific toxin pyricularin is involved in the disease. Genus Cryphonectria (formerly included in Endothia) C. parasitica causes the chestnut blight (Castanea dentata). Perithecia of C. parasitica are produced in a stroma, with long necks terminating at the surface in an ostiole. Order Xylariales It includes diverse group of pyrenomycetes with dark, leathery, woody, or carbonaceous perithecial or occasionally, cleistothecial ascocarps, sometimes embedded in stroma. Genus Xylaria Mostly saprobic or weak parasites on woody trees. The genus is characterized by the stromata, which are stipitate upright simple or branched; corky, leathery or woody in texture. Perithecia are produced over the entire portion of the stroma except the stipe. X. digitata is responsible for root rot of hard woods and tree cankers. Genus Rosellinia Rosellinia species especially R. necatrix causes root diseases of fruit trees and vines. The genus is characterized by a subiculum from which the anamorph and later stromata develop. The imperfect state of R. necatrix belongs to the form genus Dematophora necatrix characterized by the formation of synnemata. Order Sordariales The order is characterized by dark or pallid perithecia or cleistothecia with periphyses and membranous or leathery peridia; ascocarps sometimes are embedded in a subiculum. From an economic stand point it is not an important order but several species have proved of great value as experimental organisms. Neurospora is the Drosophila of the fungus world.

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Genus Chaetomium The genus has ascocarps with hyphal appendages on the ascocarp surface. In some species the hairs produced on the body of the ascocarp are different from those produced at the apex. Species of Chaetomium have strong cellulolytic ability. Order Meliolales It is a small homogenous group of fungi characterized by hyphopodiate mycelium produced on the surface of leaves and stems of host plants. Economically, members of the order are the least important plant parasitic fungi and none causes enough damage. Filamentous Ascomycetes with Apothecia (Discomycetes) Most of these fungi commonly called discomycetes are characterized by the production of ascocarps known as apothecia. Asci are cylindrical-to-ovoid, often interspersed with paraphyses. Ascospores are forcibly discharged. This is the major group of lichen-forming ascomycetes. Some of the important orders of the plant pathological importance are discussed briefly.

Order Rhytismatales Ascocarps are produced in stromata. The ascocarps are black externally and may be spherical, discoid or elongate in shape. Genus Rhytisma Most species of Rhytisma produce numerous apothecia in each stroma. R. acerinum causes the disease tar spot in maple leaves. Species of Lophodermium cause needlecast disease of conifers. Order Ostropales Includes several families of discomycetes with paragymnohymenial development. Graphidiaceae is one of the families that includes many lichen-forming fungi. Order Helotiales The order is characterized by either cup-or disc-shaped apothecia and asci that have only slightly thickened apices. The ascospores are several septate and variously shaped. Sclerotinia, Monilinia, Stromatinia, Pseudopeziza, and Diplocarpon are the important genera from plant pathological stand point. Genus Sclerotinia In Sclerotinia the sclerotia develop in leaf blades or rarely in twigs; apothecia are deep inoperculate to discoid. S. sclerotiorum is responsible for many rot and wilt diseases in vegetable crops. The species attacks 383 plant species in 225 plant genera placed in 64 families. The maximum number is in Compositae followed by Leguminosae and Cruciferae. Genus Monilinia In this genus the conidial stages is always a Monilia and spermatia are formed. Monilinia species cause brown rot disease of stone fruits. Filamentous Ascomycetes with Ascostroma (Loculoascomycetes) Loculoascomycetes are characterized morphologically by the production of asci within locules in a preformed stroma (ascostromata) that constitutes the ascocarp. Some are lichen-forming, and others are plant pathogens, endophytes and animal pathogens.

Order Myriangiales This is a small order of the small group Loculoascomycetes. Elsinoe is an important genus. In Elsinoe the ascostroma is simple, innate, infra- or subepidremal, partially erumpent at maturity. Small pulvinate-to-crustose, ascospores 3 septate with longitudinal septa in some cells.

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Elsinoe mangiferae causes scab of mango. Elsinoe fawcetti causes scab of sour oranges, rough lemon, and other citrus fruits. Elsinoe ampelina causes anthracnose of grapes. Order Dothidiales This order of Loculoascomycetes has 8 families with more than 70 genera and several thousand species. Important genera are Guignardia, Mycosphaerella, Didymella. In Guignardia the fungus develops perithecium-like structures in which clusters of 8-spored asci arising from the base of the locule are produced. Paraphyses are absent. Conidial stage is of Phoma type. Guignardia bidwellii causes the destructive black rot disease of grapes. Genus Mycosphaerella In Mycosphaerella the asci are 8-spored, psudothecia are small and immersed in the host tissue (usually on dead leaves). Ascospores are hyaline or pale green. The conidial forms are of several genera such as Ramularia, Cercospora, Phoma, Ascochyta, Septoria and Phyllosticta of Imperfect Fungi. Mycosphaerella ribis causes leaf spot of gooseberry and currant. M. fragariae – causes leaf spot of strawberry. M. berkeleyii has the conidial state in Cercosporidium personatum which causes tikka disease of groundnut. M. arachidicola has the conidial state in Cercospora arachidicola which also causes leaf spot of groundnut. M. musicola—has conidial state in Cercospora musae which causes leaf spot or Sigatoka disease of banana. M. pinodes—This species is the ascigerous stage of Ascochyta pinodes which causes blight and pod spot of pea. Genus Didymella In the genus Didymella the pseudothecia are separate, innate, immersed in the substratum and ascospores are hyaline. Order Capnodiales In this order Ascostromata or spherical pseudothecia may be grouped or separate and sometimes occur in a subiculum and produced in a loose mat of dark hyphae. Genus Capnodium Order Pleosporales variable.

being one of several fungi causing sooty mould on plants. In this order the asci are surrounded by psuedoparaphyses. Ascostromata are

Genus Cochliobolus The pseudothecia are often covered with conidiophores. They lack setae. Ascospores are more or less spirally coiled within the ascus. Anamorphs are in the genus Bipolaris and Curvularia cause leaf spots and root rots on grain crops and grasses. Genus Pyrenophora In this genus the pseudothecia are large, hairy (setose) and often bearing conidiophores on the surface. The ascospores are large, hyaline but commonly pale brownish, oblong with almost straight sides and broadly rounded ends, 3-,5,7-septate with a single longitudinal septum in 1 or all the cells. Anamorph is Drechslera species causing leaf spots on cereals and grasses.

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Genus Pleospora Anamorph of this genus is Stemphylium causing black mould rot of tomato. The genus differs from Pyrenophora mostly in shape and size and dark colour of the spores. Genus Setosphaera

(anamorph is Exserohilum) causing leaf spots on cereals and grasses.

Genus Leptosphaeria (anamorph is Phoma) In this genus the pseudothecial wall is composed of scleroplectenchyma of isodiametric thick-walled cells. The pseudothecial beak is short or lacking. The ascospores are fusoid to rarely scolecosporous, 3-to many septate and brownish-yellow-to-uniformly hyaline. Genus Venturia It has globose pseudothecia or asco-stromata with several locules. Pseudothecia are often setose at the apex. Conidial stage is found in the form genus Spilocaea. V. inaequalis causes apple scab while V. pyrina the pear scab. Other Filamentous Ascomycetes

Order Erysiphales The fungi belonging to this order are obligate biotrophs that cause a major group of plant diseases commonly known as powdery mildews. Asexual reproduction is by conidia formed singly or in a chain on simple, rarely-branched conidiophores. Sexual structures the ascocarps are completely closed like cleistothecia but produce their asci in a basal layer. The asci have been described as bitunicate. Genus Sphaerotheca Mycelium superficial, perithecium with one ascus only S. pannosa causes powdery mildew of roses and peach, S. fuligenea causes powdery mildew of cucurbits in India. Genus Podosphaera Mycelium superficial, Perithecium with one ascus and perithecial appendeges dichotomously branched. P. leucotricha causes powdery mildew of apple. Genus Erysiphe Mycelium superficial perithecium with several asci. Perithecial appendages simple (mycelioid). Erysiphe species causing powdery mildew of several herbaceous plants. E. graminis (on wheat), E. cichoracearum (on cucurbits), E. polygoni (on pea and Lathyrus etc.). Genus Microsphaera Mycelium superficial, perithecium with several asci, Perithecial appendages dichotomously branched. M. alphitoides causes powdery mildew of oak. Genus Uncinula Mycelium superficial, perithecium with several asci and perithecial appendages coiled at tip. U. necator causes powdery mildew of grapes while U. tectonae causes powdery mildew of Tectona grandis (teak tree). Genus Leveillula Mycelium partly internal, perithecial appendages simple and perithecium with several asci. L. taurica is a serious plant parasite of a number of plants. Genus Phyllactinia Mycelium partly internal, Perithecial appendages stiff with basal swelling and several asci in a perithecium. P. acaciae on leaves of Acacia arabica, P. ferruginea on Acacia catechu, P. corylea var. subspiralis on Dalbergia sissoo.

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Asexual Ascomycetes and Other Asexual Fungi (Deuteromycetes or Imperfect Fungi)

Mycelium well developed, septate, branched. Sexual reproduction and structures are rare or lacking or unknown. Asexual spores (conidia) formed on conidiospores existing singly, grouped in specialized structures such as sporodochia and synnemata, or produced in structures known as pycnidia and acervuli. Anamorph stage

Genus

Genus Genus

Genus

Genus

Genus

Mode of development of conidia Geotrichium G. candidum causing sour rot of fruits and vegetables. Aspergillus causing seed decays and bread mould. Penicillium causes blue mould rot of several fruits.

Paecilomyces biological control agent against insects and nematodes. Bipolaris causing leaf spots on grasses.

Drechslera causing leaf spots on grasses

D. oryzae brown leaf spot of rice. Genus Genus Genus

Exserohilum causing leaf spots on grasses. Curvularia weak parasites Botrytis B.cinerea gray mould rots on many plants including grapes. B. allici on onions

Thallic mode of conidiogenesis

Phialidic Conidium development blastic dry. Phialidic Blastic dry.

Phialidic Blastic (dry).

Certain or likely teleomorphic group Saccharomycetales

Eurotium There are as many as 11 different teleomorphs. Talaromyces is the teleomorph of the subgenus Biverticillium, Penicillium has no known teleomorphs Byssochlamys.

Porosporous conidia produced apically and laterally from determinate conidiophore, conidia germinate only by the two end cells. Porosporous conidia produced apically and laterally from determinate conidiophore. All the cells of a conidium are capable of germination. Porosporous conidia.

Cochliobolus

Poroconidium. Terminal cells of the conidiophore swell to produce sporogenous ampullae. Numerous conidia arise simultaneously on each ampulla.

Cochliobolus Botryotinia.

Pyrenophora.

Setosphaeria

(Contd.)

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Anamorph stage

Mode of development of conidia

Genus

Monilia causing brown rot stone fruits.

Genus

Oidium causing the powdery mildew diseases

Genus

Chalara causing oak wilt and as vegetable or fruit pathogen. Sporothrix, and Graphium cause Dutch elm disease. Trichoderma used as a biocontrol agent. Gliocladium used as biocontrol agent Verticillium causing wilt in many vascular plants. Fusarium causing vascular wilts, root rots etc. many species also infect a variety of vertebrates including fish and is an important pathogen in aquaculture. Colletotrichum causing anthracnoses of many plants and red rot of sugarcane.

Genus

Genus Genus Genus

Genus

Genus

Conidiophores in dense velvety tufts (sporodochium) conidia are formed in chains, which extend in length at their apices by budding of the terminal conidium. Oidia are produced by short hyphal branches, the oidiophores, which cut off oidia in succession, from tip of the oidiophore. Phialidic Enteroblastic

Certain or likely teleomorphic group Monilinia.

Erysiphe and other genera in Erysiphales.

Ceratocystis

Directly on the mycelium, Synnematous Phialidic blastic (sticky) Phialidic blastic (sticky) Phialidic blastic (sticky)

Ophiostoma

Two types of conidia macro and micro. Both types are produced on phialides.

Gibberella.

Phialidic conidia Dark setae in Acercvuli

Glomerella

Hypocrea Hypocrea. Hypocrea

(Contd.)

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Anamorph stage

Genus

Genus

Genus

Mode of development of conidia Cercospora cause Tikka disease of groundnut and Singatoka disease of banana. Septoria causing leaf spots on many crops S. tritici leaf spot of wheat. Phyllosticta

Genus

Alternaria causing leaf spots and blights A. solani causes early blight of potato. A. triticina leaf blight of wheat. A. brassicae leaf spots of crucifers . A. brassicicola causes leaf spot and pod spots of crucifers.

Genus

Rhizoctonia R. solani causing root and stem rots.

Genus

R. bataticola causing black scurf of potato. Rhizoctonia binucleate forms Sclerotium S. rolfsii causing root rots and blights of many plants. S.oryzae causes stem rot of paddy.

Sympodially extending conidiophore cicatrized (with conspicuous scars) Conidia (sympodulospores and filiform. Conidiogenous cells holoblastic, determinate or indeterminate

Conidiogenous cells enteroblastic, phialidic Conidia are poroconidia

In culture or in nature sclerotia formed

Certain or likely teleomorphic group Mycosphaerella

Mycosphaerella

Lewia

Basidiomycota Thanatephones

Ceratobasidium

Brown to black fairly large sclerotia with a pseudoparenchymatous rind.

Aethelium

Magnaporthe salvinii

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Phylum Basidiomycota It has a single class Basidiomycetes. These members are characterized by the production of basidiospores formed on the outside of the specialized microscopic spore producing structures called basidium. The class is differently classified by different authorities in the subject. For convenience sake we have followed here the old classification used by Alexopoulos and Mims (1979) in the third edition of the book “Introductory Mycology”.

Subclass Holobasidiomycetidae hymenium is exposed.

Basidia are single-celled. Basidiocarp present or, if not,

Order Aphyllophorales, also known as Polyporales all produce single-celled, club-shaped basidia in well-developed hymenia. The hymenia are borne on various ways on gymnocarpous sporophores. The hymenium is exposed before the spores are mature. Genus Schizophyllum In this genus the hymenial layer consists of thick lamellae that are split longitudinally with both edges folded back. Basidiocarps are soft textured. Not much of plant pathological importance. Genus Poria

Sporophore a thick crust-like layer without pileus and resupinate.

Genus Fomes It produces hoofed perennial trimitic sporophore containing stratified tubes. Fomes species attack heart wood of various trees. Genus Heterobasidion This differs from Fomes in the absence of clamp connections. H. annosum is a serious problem in freshly cut stumps and causes root rot of trees. It also causes heart rot of trees. Genus – Lenzites – It produces radially elongated pores with gill-like structures, corky-to-leathery in texture and trimitic in construction. Genus – Trametes – The fructifications are leathery-to-corky, effused, pileate and dimtic in construction. Pore tubes are of different lengths. Genus – Irpex – Sporophores are effused reflexed with dimitic hyphal system. It is a lignicolous species. Genus – Ganoderma – This genus contains lignicolous species that produce spores with an inner brown layer. It produces hard shelt-like fruiting bodies. It causes root and basal stem rot of various trees including Dalbergia sissooo, Acacia catechu and other trees. Genus – Polyporus – The fruit bodies in this genus are not so hard as that of Fomes. In addition they are annual sporophores. The fruit bodies may be stipitate, sessile or effused. Most of the species are wood rotters. Order – Agaricales – These produce fleshy, sometimes tough umbrella-like sporophores that bear their basidia on the surface of gills or plates (lamellae). In Boletus, the basidia are borne not on gills but instead line the inside of tubes or, more rarely shallow pits. Genus – Boletus – Most of the species are Mycorrhizal. Genus – Amanita – Well known white spored genus. Members are characterized by free gills, and the presence of an annulus and vulva. Most of the species are poisonous. Genus – Marasmius – The genus consists of small, rather tough mushrooms with thin stalks. Grows generally on grasses and leaves. M. ordeades cause fairy rings on turf grasses.

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Genus – Armillaria – A. mellea is the honey mushroom. It produces rhizomorphs and is responsible for root rot of forest trees. Genus – Psilocybe – It produces attached gills, and dark spores. Psilocybe produces conis-tocampanulate caps with attached gills. P. mexicana is hallucinogenic. Genus Agaricus White to brown sporocarp, free gills and an annulus but no vulva. The genus contains edible mushrooms. Holobasidiomycetidae The third group in this subclass contains orders Exobasidiales, Tulasnellales, Dacrymycetales, and Brachybasidiales. Order Exobasidiales Basidiocarp is lacking, basidia are produced directly from dikaryotic hyphae between the epidermal cells of the host, push through the cuticle and form a layer on the surface of the host. Exobasidium vexans is responsible for the blister blight of tea in India and Sri Lanka. The other orders are not of plant pathological importance. Gasteromycetidae These include puff balls, earth stars, stink horns and birds nest fungi. These produce holobasidia, the development of the basidiocarp is typically angiocarpic. Most are saprobic in soil, dead wood of dung, although a few are mycorrhizal. All produce well developed septate hyphae, that may or may not possess clamp connection. Dolipore septa are common in the group. The orders are Hymenogastrales, Sclerodermatales, Tulostomatales, Lycoperdales, Phallales, and Nidulariales. Phragmobasidiomycetidae This subclass consists of fungi whose metabasidium is typically divided in to four cells by transverse or longitudinal septa. The subclass consists of the orders Tremellales, Auriculariales, and Septobasidiales. The Tremellales and Auriculariales are saprobic forms and the Septobasidiales are parasittic or parasitic-symbiotic on scale insects. Subclass Teliomycetidae (rusts, smuts and basidiomycetous yeasts) The rusts and smuts are characterized by the production of a thick-walled, binucleate resting spore termed the teliospore which upon germination, gives rise to a promycelium into which the diploid nucleus moves. Following meiosis, haploid basidiospores are formed either directly on the surface of the promycelium or on the tips of small out growths from the mycelium. Order Uredinales Basidia arising from thick-walled probasidium. Basidia becoming septate bearing 2-4 (mostly four) basidiospores, one at each septum and one terminal. Biotrophs. Several types of spores being produced. Autoecious or heterocious. Genus Puccinia The teliospores of Puccinia are stalked and two-celled. P. graminis tritici causes black rust of wheat, P. recondita (syn. P. triticina, P. rubigovera) causes leaf rust or brown rust of wheat, P. striiformis (syn. P. glumarun) causes yellow rust of wheat and barley. P. arachidis causes rust on the leaves of groundnut. Genus Uromyces

The teliospores are one-celled and dark and stalked.

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Uromyces fabae causes rust on Vicia fabae and Lathyrus spp., U. pisi on pea, U. ciceris – arietini on gram (chickpea). Genus Hemileia The teliospores in this rust are one-celled and pedicellate, round and turnip shaped. The urediniospores are reniform. H. vastartix causes coffee rust disease. Genus Gymnosporangium The teliospores in this rust are produced in long tongue-like structures with very long pedicels. G. juniperi – virginianae causes cedar rust. Genus Phragmidium Teliospores in this rust are multicellular with pedicels covered with a gelatinous structure. Phragmidium causes rust on rosaceous plants. Genus Melampsora The teliospores in this rust are formed in a crust or column below the surface of the host. M. lini causes the rust of linseed (or flax). Genus Coleosporium The teliospores are laterally united laterally, forming a crust-like structure on the host. No external promycelium is formed. The aecia form on pine needles with prominent white lip that puts them in the form genus Peridermium. Order Ustilaginales The order includes the smut fungi and the basidiomycetous yeasts. The teliospores in Ustilaginales are formed from intercalary cells of the binucleate mycelium. Basidiospores are sickle-shaped, elliptical, or hyphae-like, sessile and not discharged violently. Number of basidiospores is indefinite (generally) and clamp connections are more common in the mycelium. The dikaryotic mycelium is obligately parasitic in the host plants. Genus Ustilago The sori contain 1-celled teliospores, dusty at maturity and are covered by the membrane of host origin. U. segetum var. tritici (U. tritici) causes loose smut of wheat. U. zeae causes smut of corn or maize. Genus Sphacelotheca In Sphacelotheca the sori are similar to Ustilage but are covered by a peridium of fungal cells and has a central columella of host tissue. S. sorghi causes grain smut of jowar, S. cruenta the loose smut of jowar. Genus Sorosporium In Sorosporium the sori consist of spore balls entirely of fertile cells, are evanescent or tending to fragment into single spores. S. reliana causes head smut of jowar. Genus Tolyposporium In Tolyposporium the sori consist of permanent spore balls not tending to fragment. T. penicillariae causes smut of pearl millet (bajra). Genus Tilletia In the genus Tilletia the teliospores are large in size which on germination form a nonseptate promycelium bearing 8 filiform sporidia (basidiospores). The sporidia form characteristic H-shaped pieces as a result of fusion. T. caries and T. foetida cause hill bunts or stinking smut of wheat.

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Genus Neovossia The genus differs from Tilettia in the method of spore germination. A large number of sporidia are formed terminally when the spore germinates and which do not fuse in situ. N. indica is responsible for the Karnal bunt of wheat. Genus Graphiola Sori in this genus are formed under the epidermis of the palm host. There is a cuplike dark outer peridium, which surrounds the central sporogenous portion. G. phoenicis causes spots on palm.

REFERENCES Alexopoulos, C.J. and C.W. Mims, (1979), Introductory Mycology, Third Edition John Wiley and Sons, New York, pp. 632. Alexopoulos, C.J. and C.W. Mims, and M. Blackwell, (1996), Introductory Mycology, John Wiley and Sons Inc., New York, pp. 869. Barr, D.J.S. (1992), “Evolutions and Kingdoms of Organisms from the Perspective of a Mycologist”, Mycologia, 84: 1–11. Burnett, J.H. (1976), Fundamentals of Mycology, Second Edition, Edward Arnold, London. pp. 546 Dick, M.W. (1990), “Oomycota”, in: Hand book of Protoctista, L.Margulis et al. (Eds.), Jones and Bartlett, Boston, MA. pp. 661–685. Fuller, M.S. (1990), “Phylum Hyphochytridiomycota”, in: Hand book of Protoctista, L. Margulis et al., (Eds.), Jones and Bartlett, Boston, MA, pp.380–387. Porter, D. (1990), “Phylum Labyrinthulomycota”, in: “Handbook of Protoctista , L. Margulis et al. (Eds.), Jones and Bartlett, Boston, MA. pp. 380–387. Raghukumar, C. S. Nagarkar and S. Raghukumar (1992),“Association of Thraustochytrids and Fungi with Living Marine Algae”, Mycol. Res., 96: 542–546. Raper, J.R. (1954), “Life cycles, sexuality and sexual mechanisms in the fungi”, in: Sex in Microorganisms, D.H. Wenrich (Eds.), Ann. Association for advancement of Science Washington, D.C. pp. 41–81. Whittaker, R.H. (1969), “New Concepts of Kingdoms of organisms”, Science, 163: 150–160.

QUESTIONS 1. Fungi and fungal-like organisms are now placed in 3 kingdoms. What are these three Kingdoms? How are Oomycota different from other true fungi? 2. What are the criteria for classifying fungi and fungal like organisms? 3. Give an outline classification of fungi and fungal like organisms. 4. What is centrum? Discuss its importance in the classification of Ascomycota. 5. What are anamorphs and teleomorphs of a fungus? 6. Give and outline classification of Chytridiomycota based on zoospore ultrastructure.

13 Rots, Damping Offs, Downy Mildews and White Rusts

DAMPING OFF FUNGAL PATHOGENS, DOWNY MILDEWS AND WHITE RUSTS

Damping off fungi, white rusts and downy mildews all belong to the order Peronosporales. Many fungi in this group are pathogens of higher plants and may cause diseases of economic importance, such as late blight of potato, downy mildew of grapevine and white rust of crucifers. Some of them, particularly the members of the Peronosporaceae and Albuginaceae appear to be biotrophs (obligate parasites) and do not survive saprophytically in soil or decaying vegetable matter. Peronosporales are coenocytic mycelial fungi, which are parasitic (pathogens) in plants or saprobic in water or soil. The cell walls are mainly made up of a glucan-cellulose complex in addition to proteins containing hydroxyproline and lipids. The parasites (pathogens) form haustoria. Asexual reproduction occurs by zoosporangia or conidia, which vary greatly in shape. The sporangia possess one or few pores with or without a papilla and produce biflagellate zoospores or a plasma, either in a vesicle or naked, or a germ tube. Sexual reproduction is oogamous. KEY TO THE FAMILIES OF THE ORDER PERONOSPORALES

Obligate parasites of plants with unbranched, club-shaped sporangiophores. Each bearing a basipetal chain of deciduous sporangia in dense subepidermal clusters on the host, white or creamish sori; oogonial periplasm, persistent and conspicuous haustoria are knob-like. Albuginaceae. (Albugo)-A. candida grows worldwide on crucifers, A. tragopogonis is common on Compositae (Asteraceae); A. bliti on Amaranthaceae and A. ipomoeae panduranae on Convolvulaceae. Strict parasites of plants, some are biotrophs with branched tree-like sporangiophores or conidiophores of determinate growth, emerging singly or in tufts usually via stomata, producing sporangia or conidia singly at the branch tips, periplasm persistent and conspicuous, haustoria varied usually branched-Peronosporaceae.

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Non-obligate parasites or saprophytes, sporangiophores or condiophores usually undifferentiated from the mycelium, branched, indeterminate, if determinate then the sporangiophore similar to the vegetative mycelium. Periplasm a thin layer or absent, haustoria absent or branched—Pythiaceae. Key to the Important Genera of Peronosporaceae

Sporangiophores determinate, hypha like short, unbranched or sympodially branched, sporangia citriform or obpyriform, not maturing synchronously, germinating by zoospores; antheridia always paragynous; oogonial wall thick and confluent with that of oospore; oospore germinate by germ tube or a sporophore teminating in a sporangium—Sclerophthora. Sporangiophore determinate, macronemous, unbranched, apex swollen with short sterigmata bearing papillate sporangia, germinating by zoospores, oospore aplerotic—Basidiophora. Sporangiophore 2-3 times dichotomously branched in the upper part, sporangia usually non-papillate, mature synchronously germinating by zoospores or germ tube, oospore, aplerotic, oospore germinate by a germ tube—Sclerospora. S. graminicola on sorghums, millets, maize and grasses, S. sacchari and S. spontanae on sugarcane, S. maydis, S. philippinensis and S. sorghi on maize and Sorghum. Sporangiophores determinate, narrow dichotomously branched at acute angles and taper to gracefully curved pointed tips on which sporangia are borne. Sporangia always germinate by germ tube— Peronospora. P. parasitica on crucifers, P. destrustor on leeks and other alliums. P. tabacina on tobacco, P. viciae on peas, and so on, P. farinosa on spinach. Sporangiophores determinate, branched, branching of the sporangiophores at right angles, irregularly spaced, tips of the branches blunt, sporangia germinating by zoospores or germ tubes—Plasmopara, P.viticola on grapes. Sporangiophores determinate narrow, branched upper third of the sporangiophore is 3-5 times branched, either dichotomously or intermediate between dichotomous and monocotomous branching habits. The tips are subacute, sporangia germinating by zoospores—Pseudoperonospora, P. cubensis on cucurbits. Sporangiophores determinate, dichotomously branched at acute angles. The tips of the branches are expanded into saucer-shaped structures—Bremia. B. lactucae on lettuce. Key to the Important Genera of Pythiaceae

Sporangiophores micronemous (undifferentiated), sporangia ovoid or obyriform or filamentous, protoplasm emerging almost or completely undifferentiated into a spherical vesicle usually at the tip of a short discharge tube. Zoospores formed in a vesicle, antheridia, always paragynous—Pythium. P. ultimum and P. irregulare (damping-off and root rots), P. aphanidermatum (common in warmer areas as root rot pathogen), P. debaryanum is rare. Sporangiophores macronemous, or sympodially branched, sporangia pyriform or globose. Sporangia do not mature synchronously, zoospores differentiated inside the sporangia. Antheridia mostly amphigynous (in some paragynous), oogonia smooth or occasionally ornamented, oospore aplerotic, oospore germination direct by a germ tube—Phytophthora.

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P. infestans (late blight of potato and tomato), P. palmivora (cocoa black pod and diseases of rubber and other tropical crops); P. megasperma, P. cactorum, P. cinnamomi and P. nicotianae and its variety parasitica (on a large number of hosts such as tobacco, castor and Piper betle). DAMPING OFF DISEASE OF SEEDLINGS

Damping off of seedlings is very common all over the world. It occurs in agricultural and forest soils, in tropical and temperate climates and in almost every greenhouse or nursery. Damping off can be of two types, that is, pre-emergence damping off in which the seed and radicle rot before the seedlings emerge from the soil and the post-emergence damping off in which the newly-emerged seedlings are killed at ground level after they emerge from the soil, causing them to collapse or topple over. This is a common symptom of post-emergence damping off. This disease is most noticeable in nursery beds, greenhouse flats and row crops because symptoms develop suddenly, killing large numbers of seedlings in the vicinity. Among the pathogens in this class are species of Pythium, Phytophthora, Corticium and Fusarium; the former two will form the subject of our present discussion. Most species of Pythium mainly affect the juvenile or succulent tissues. This restricts their parasitism to seedlings, the feeder roots or root tips of older plants, and to watery fruits or stem tissues. They also cause fruit rots of crops, such as beans, squash and watermelons. At a later stage, when the cells of stems and main roots have developed secondary thickenings, infection is restricted to feeder roots. This causes seedlings to become stunted and chlorotic. Peach decline, which kills thousands of trees each year, has been shown to be a feeder root problem caused by a combination of factors, including Pythium spp. Pectolytic and cellulolytic enzymes produced by Pythium and Phytophthora spp. play an important role in tissue disintegration and maceration. Mathews (1931) did much work to clarify the taxonomic position of the known Pythium spp. during his time. Middleton (1943) compiled an extensive monograph of the genus Pythium. Waterhouse (1967, 1968) compiled the original descriptions and illustration of all the species and published a key to the genus in 1967. Literature up to 1961 has been compiled by Rangaswamy (1961). Vander Plaats – Niterink (1981) has given a key to the species of Pythium. Dick (1990) gave a key to the world species of Pythium while Misra and Hall (1996) gave the characteristics, distribution and host range of forty one species of the genus Pythium in India. Tucker (1931) published a monograph of the genus Phytophthora. Waterhouse (1956, 1963) compiled the original descriptions and illustrations of all described species and published a key to the genus in 1963. There is an interesting article by Hickman (1958) on “Phytophthora as a plant destroyer”. There are two recent books on “Phytophthora diseases. Phytophthora diseases worldwide” by Erwin and Ribeiro (1996), and “Phytophthora diseases in India” by Mehrotra and Aggarwal (2001). In contrast to the species of Phytophthora, Pythium spp. have a high degree of competitive saprophytic ability. Pythium debaryanum Hesse and several other species of Pythium such as P. aphanidermatum (Eds.) Fitz; Pythium ultimum Trow and P. arrhenomanes Drechsler are pathogens. Symptoms

As has already been stated, damping off of vegetable seedlings occurs in two stages, that is, the preemergence and the post-emergence phase. In the pre-emergence phase the young seedlings are killed before they reach the soil surface. The young radicle and plumule are killed and there is complete rotting

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of the seedlings. The post-emergence phase is characterized by infection of the young, juvenile tissues of the collar at the ground level. The infected tissues become soft and water soaked. The seedlings topple over or collapse. Such symptoms are very common in seed beds in nurseries. Causal Organisms

The mycelium of P. debaryanum is branched, hyaline and has coenocytic hyphae, the main hyphae being up to 5 mm in diameter. The ends of the blunt terminal hyphae penetrate the cell walls of the hypocotyl and ramify within and between the tissues of the cortical parenchyma; sporangia and oospores are formed in the parenchymatous tissues of the host. Sporangia are terminal or intercalary and are 1526 mm in size. They are spherical, oval, or barrel-shaped depending upon their position. Just before germination, a prominent beak is formed on the sporangium, followed by a thin-walled vesicle into which the sporangial contents are emptied. Zoospores are differentiated in the vesicle. The zoospores are reniform, with two lateral flagella. They measure upto 8 mm in diameter. Sexual reproduction is oogamous, oogonia spherical and terminal on the short side branches with a smooth and thin wall, 15 to 28 mm antheridia monoclinous or diclinous, one to six per oogonium; oospores smooth, aplerotic 12 to 20 mm. Among other common species of Pythium are P. aphanidermatum and P. ultimum. These are characterized below: P. aphanidermatum—Sporangia inflated, lobulate branched or unbranched; zoospores 7 by 12 mm; oogonia spherical, terminal, 22 to 27 mm; antheridia monoclinous or diclinous, intercalary or terminal, one or two per oogonium, dome shaped, 9 to 11 by 10 to 14 mm; oospores aplerotic, single, 17 to 19 mm. Associated with soft rot of storage organs and damping off of many host species. It is responsible for cottony leak of cucurbit fruits in storage, cottony blight of turf grasses, root and stalk rot of maize, stem or foot rot of papaya, rhizome rot of ginger, rhizome rot and root rot, of turmeric. Other hosts are sugarbeet, tobacco, sugarcane, pineapple, bean and cotton. P. ultimum – Sporangia mostly terminal and spherical, with a diameter varying from 28 mm and more to 12 mm and less, but occasionally intercalary and barrel–shaped, with the dimensions varying from 27.8 mm ¥ 22.9 mm to17 mm ¥ 14 mm, antheridia monoclinous, arising just below the oogonium, usually one per oogonium; ooospores aplerotic, single, spherical, 14 to 18 mm . It is a common causal organism of damping off, root rot and soft rot of many species. Disease Cycle and Predisposing Factors

Pythium spp. are natural inhabitants of the soil where they are probably low grade parasites (pathogens) on fibrous roots. As a plant parasite, the pathogen can infect succulent organs of plants but tissues that have matured and developed secondary thickenings become resistant to its attack. In soil, Pythium spp. are susceptible to antagonism by a large number of fungi (Trichoderma) and bacteria (Bacillus and Pseudomonas) and some amoebae. This weakness of the pathogen is compensated for by its ability to form oospores. The oospores serve as the overwintering or oversummering organs. They are not vigorous competitors and their saprophytic activities are greatly restricted. Soil moisture is important in the saprophytic growth of Pythium spp. and survival by resistant structures is more important than saprophytic persistence.

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Royle and Hickman (1964) showed that zoospores in water suspension are attracted to the region of elongation behind the tips of pea roots, to wounds in the epidermis and to the exposed stele at cut ends of roots where they encyst and germinate. Factors influencing infection include inoculum density, soil moisture, soil temperature, pH, cation composition, light intensity, and the presence of members of other microorganisms. Soil temperature and moisture are the most important factors. Generally, a high soil moisture is necessary for disease development. The disease cycle of a typical Pythium species causing damping off and seed decay is given in Fig. 13.1. Germ tube Infection Seedling Soil line

Encysted zoospore Seedling

Seed Zoospores

Germ tube

Seed

Germ tube

Vesicle Encysted zoospore Zoosporangium Germ Oospore tube

Intracellular mycelium Zoospores Vesicle

Germination

Sporangium

Mycelium

Oospore

Sporangio phores

Oogonium Antheridium

Sporangia

Overwintering Oospore Fertilization of oogonium

Fig. 13.1

Dying

seedling

Disease cycle of damping- off and seed decay caused by Pythium sp. (From G.N. Agrios, Plant Pathology, Academic Press, New York and London, 1969.)

Temperature has as much effect on disease development as has soil moisture. Generally, P. irregulare, P. spinosum and P. ultimum are more damaging at lower temperatures, while P. myriotylum, P. aphanidermatum, P. arrhenomanes and related species are damaging at higher temperatures. But the disease is most severe in poor soils, ill-aerated and ill-drained soils. Pythium spp. are most destructive when soil temperatures are between 24°C and 30°C.

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Disease Management

Most Pythium spp. produce oospores and chlamydospores and persist for many years. Direct control of Pythium on field scale is difficult and expensive. On a small scale, Pythium spp. can be eliminated from the soil for greenhouse use by heating. The common method is by steam treatment or pasteurization. The latter method is preferred as it does not change the microflora drastically. Fumigation with chloropicrin or methyl bromide is a standard practice in many nurseries and horticultural operations but these chemicals are now banned. Very often the most effective measure against damping off of fungi involves the use of seed protectants to keep away the pathogens in the pre-emergence phase and to adopt sanitary precautions in the nursery to check post-emergence damping off. The most commonly used seed-protectant fungicides now are Thiram, Captan, Zineb and some systemic fungicides such as Metalaxyl. Drenching the soil with suitable fungicides, such as Cheshunt compound, 1% Bordeaux mixture, 0.1% Thiram, 0.3% Blitox – 50 or 0.2% Esso Fungicide 406 has been found useful in eliminating soil-borne infections. Dexon and other soil fungicides, applied at low rates, have been successfully used to retard the rate of reinfestation of fumigated soils by Pythium. The use of healthy seeds in crops (ginger and turmeric) which are vegetatively propagated by rhizomes, and so on form an important control measure in such diseases. Crop rotation is another method of reducing the population of soil pathogens, but because the more common and destructive Pythium spp. have a wide host range, this practice is not of much help. Seed treatment with spores of Trichoderma harzianum and Penicillium oxalicum and cells of Pseudononas cepacia, P. fluorescens have been found very effective biological control agents. A combination of seed and soil treatment is best for management of damping off. Some cultural practices recommended are: thin sowing of seeds, light sandy soil for nurseries, use of well-decomposed manure, light but frequent irrigation, and sterilization of soil by burning a 30 cm thick stack of farm trash on the nursery beds. Some new methods such as osmopriming of seeds (seed priming with inorganic salts) or with a fine silicate clay (solid matrix priming) before planting have been found promising. Some of the other fungicides used these days are Thiram (3g per kg of seed), Captan (3g per kg of seed) and some systemic fungicides such as Metalaxyl (2g per kg of seed). FRUIT ROT OF CUCURBITS

This is a very common disease of cucurbits in India. The disease occurs on bottlegourd (Lagenaria vulgaris), spongegourd (Luffa cylindrica and L. acutangula), snakegourd (Trichosanthes anguina), cucumber (Cucumis sativus), bitter gourd (Memordica charantia) and other cucurbitaceous fruits. Cottony leak disease is very widespread in cucurbit fruits. Sometimes 50% or more of muskmelons or watermelons undergo rotting after rains due to this disease (Singh and Chouhan, 1977). Symptoms

A watery soft rot develops, especially in fruits touching the soil surface. The disease is common in the field during and after the rains. It also spreads among fruits during storage and transit. As a result of

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infection the diseased fruits have a luxuriant woolly mycelial growth. The decaying fruits also emit a bad smell. Causal Organism

The causal organism are mainly Pythium aphanidermatum and P. butleri but other fungal pathogens, such as Fusarium, Rhizoctonia and Phytophthora spp., may also be involved. The pathogen P. aphanidermatum has been described earlier. P. butleri and P. aphanidermatum are responsible for cottony leak disease (or fruit rot) of cucurbits in north Indian conditions. P. butleri can be distinguished from P. aphanidermatum in more copious robust mycelium, larger and more swollen and much branched sporangia, larger zoospores, oogonia and oospores, and larger (usually diclinous) antheridia. The susceptibility of six cucurbits, chilli, tobacco and papaya to P. butleri has been reported. P. butleri, a papaya isolate causes fruit rot of many cucurbits on inoculation. Damping off of ‘toria’ seedlings (Brassica campestris var. toria) also occurs. P. butleri grows best between 30—35°C. This observation is in conformity with the study by Desai and Pathak (1969) regarding the effect of temperature on the growth of P. butleri in vitro. Desai and Pathak (1969) also reported that 30°C was the optimum for fruit rot of cucurbits due to P. butleri. Disease Cycle and Predisposing Factors

The pathogens P. aphanidermatum and P. butleri are soil-inhabiting ones and survive as saprophytes on dead organic matter, causing the disease when a suitable host is available. A slight injury or wound on the surface of fruits or vegetables facilitates infection. High soil moisture and temperature favour the development of the disease. Disease Management

Efforts should be made to keep the fruits away from the soil surface. Soil disinfection, as recommended for damping off, is not practicable due to the prohibitive costs involved. STEM OR FOOT ROT OF PAPAYA (CARICA PAPAYA )

It is a severe disease found in many parts of India, Sri Lanka, Cuba, Hawaii and South Africa. The disease often appears during the rainy season. The severity of the disease depends directly on the temperature and rainfall. Symptoms

It is characterized by the appearance of spongy, water soaked patches or areas on the bark, at the collar region or immediately at the soil line. The patches enlarge rapidly and girdle the stem, causing the tissues to rot. The tissues become black and the entire tree topples down under a slight wind pressure and

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dies. If the bark is opened, the internal tissues appear dry and give a honeycomb appearance. The rotting spreads to the roots and they are destroyed. The typical stem rot symptoms are common in 2-3 year-old plants but younger plants have also been seen dying due to early infection. A damping-off of papaya seedlings in the nurseries is also common. Seedlings grown in such nurseries carry the disease and when transplanted, develop symptoms. Causal Organism

The morphology of organism P. aphanidermatum has been described earlier. Disease Cycle and Predisposing Factors

The pathogen is a soil inhabitant and produces abundant oospores in the soil. It is capable of growing and surviving on residues left in the soil. According to Trujillo and Hine (1965), the optimum temperature for disease development is 36°C. Water-logging increases chances of disease development. Disease Management

Stem rot of papaya can be avoided if plants are grown in well-drained soil. Affected plants should be carefully pulled out and destroyed (by burning). Replanting should not be done in a pit where the disease has once appeared. At the time of cultivation care should be taken so that no injury is caused to the basal portion of the stem. The disease can be kept in check by drenching the soil with Bordeaux mixture with a sticker (6:6:50) or 2% Captan which considerably reduces the incidence of stem rot. According to Rawal and Muniyappa (1988) soil drenching with Calixin (0.1%), Topsin-M (0.1%), and Daconil (0.2%) at bimonthly intervals gave very good stand of the crop. The damping off phase of the disease has been reported to be effectively controlled by seed treatments with Thiram, or Difolatan (0.25%) along with soil treatment as described earlier with suitable fungicides. The affected areas on the plant should be removed and fungicidal paste should be applied. RHIZOME ROT OF GINGER (ZINGIBER OFFICINALE)

Rhizome rot or soft rot or ginger (Zingiber officinale) occurs in India wherever this crop is grown. The disease is quite severe in south India. Pellicularia spp. and Fusarium spp. are also known to cause rhizome rot of ginger. The disease is not confined only to the crop in the field but also causes 80-90% loss of corms in storage. In some low-lying infested fields, there may be total loss but generally losses in the field vary from 8–15%. Symptoms

The first visible symptoms are seen at the basal portion of the plant which become watery and soft. The leaves become slightly pale in colour, their tips turn yellow, and the yellowing spreads down the leaves. The rhizomes start rotting and are converted into a pulpy mass giving out a putrefying smell.

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If plants are raised from infected rhizomes the young shoots show damping-off and if infection occurs after the emergence of shoots, the pseudostem withers and dies which results in a poor crop. Causal Organisms

At least 8 species of Pythium are known to attack ginger in different parts of world. According to Rajan and Agnihotri (1989) in India, P. aphanidermatum, P. butleri, P. myriotylum, P. graminicola, P. deiense, P. pleroticum, and P. vexans have been reported on ginger. Pythium myriotylum, P. aphanidermatum, Pythium monospermum, and Pythium gracile have been isolated from the diseased plants. The pathogenicity of P. myriotylum and P. aphanidermatum has been proved so far. Pythium myriotylum Drechsler—Hyphae straight or sinous, not much branched except at the terminations, upto 8.5 mm in diameter, forming numerous clavate or knob-like appressoria, often in branching clusters. Sporangia are terminal or intercalary, consisting of long simple or branched portions of unswollen hypha or often with lateral, swollen, lobulate or digitate branches up to 17 mm in diameter. Oogonia abundant, terminal or intercalary, 15-44 mm, smooth wall. Antheridia up to 10, usually 3-6 per oogonium, usually diclinous, stalks slender, often once or twice branched, investing with a single reserve globule, wall up to 2 mm thick, smooth, pale golden in colour. P. aphanidermatum —This been described earlier. Disease Cycle and Predisposing Factors

It is a soil-and seed-borne disease and it is transported over long distances on ginger rhizomes. The incidence of this disease is very high in virgin soil containing abundant decomposing matter. Disease Management

The first requisite for checking this disease is to choose healthy seed pieces. Soft rot of ginger has proved amenable to control in India by the preplanting treatment of rhizomes and the soil with copper fungicides. Dohroo and Sharma (1983) had recommended a half-hour dip of seed rhizomes in 0.25% Antracol or 0.3% Fycop or 0.3 % Blitox-50 to control rhizome rot. Since the pathogen is present deep in the tissue of the host (corm or rhizome), surface disinfection of seed is not effective. Spraying of the soil with Bordeaux mixture (6:6:50) at the rate of 7 litres per square foot once before sowing and then after germination gives partial control of the disease. Ramachandran et al. (1989) found best control of the disease by seed and soil treatment with Metalaxyl (Ridomil 5G or Apron 35 WS). LATE BLIGHT OF POTATO (SOLANUM TUBEROSUM)

The great famine in Ireland and elsewhere in Europe during the 1840’s can be attributed mainly to the failure of the potato crop. Potato is a native of the North Andes (South America) and the late blight of potato was initially an endemic disease. However, due to good communication links, it spread to the USA and Europe in 1830-1840. The pathogen was well established in Ireland, England and Europe by

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1842. By 1845, the epiphytotics of late blight became widespread throughout Europe where it devastated the potato fields and brought famine. In India, the disease was first introduced into the Nilgiri hills between 1870 and 1880. Very soon it spread to the Darjeeling district in the Himalayas after the introduction of the English potatoes. Losses due to the ravages of this disease have been recorded in different parts of our country by several workers (Bhattacharya, 1990). These have been estimated at 20-25% in Punjab, 40-50% in Haryana, 15-50% in U.P., 5-10% in Bihar and West Bengal. The overall losses range from 20-25% amounting to 4 million tonnes of potatoes worth Rs. 4 crores. The disease is endemic and more severe in the east than northern plains. According to Bhattacharya (1990) with the release and large-scale cultivation of Kufri Jyoti in early seventies, losses came down considerably. Singh and Bhattacharya (1998) have reviewed the work on late blight of potato recently. Symptoms

The first symptoms of late blight in the foilage of potato and tomato are hydrotic areas with indefinite margins at the tips or on the margins of the leaflets [Fig. 13.2a ]. These lesions then become necrotic and turn brown to almost black. Very often a chloranemic border develops around the necrotic areas. If moist weather prevails, the entire leaf may be killed within a couple of days. If dry weather intervenes, following the appearance of lesions, the infection advances slowly and the affected areas curl and shrivel. However, under moist conditions, they remain limp and soon decay, producing a characteristic

(a)

Fig. 13.2 (a)

(b)

Late blight symptoms due to Phytophthora infestans (b) sporangiophores and sporangia on leaf.

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offensive odour. If the weather remains dry after the development of spots on the leaflets, the diseased areas seem hard and easily break away from the leaf blade. At this stage symptoms are very similar to those due to Alternaria solani, which is responsible for early blight. The wet rot in warm humid weather advances rapidly and affects all the leaves and the stem but it does not go as far down as the tubers. Tubers are infected separately. Slightly sunken hydrotic areas develop in the superficial tissues of the potato tubers. These areas become necrotic, and if conditions remain favourable, the infection spreads inwards so that the entire tuber turns brown and decays before harvest. In drier soils the progress of the disease is slower and a rot may result. During wet and humid conditions the sporangiophores and sporangia can be seen as a downy growth, particularly on the abaxial surface of the leaflets. The fungus penetrates the host mostly through stomata. The morpho- anatomical characters of the host are reported to be related to resistance (Kaur et al. 1990). Causal Organism and Disease Cycle

The causal organism of late blight of potato is Phytophthora infestans (Mont.) de Bary. The causal organism of late blight of potato is carried through tubers used as seed, volunteer plants that develop from diseased tubers left in the field of sprouts produced from infected potatoes in cell piles or dumps. Such infected shoots form foci within the crop from which the disease spreads. Sporangiophores and sporangia formed on the diseased shoots are blown to healthy areas, where they germinate either by the formation of germ tubes or zoospores. Zoospore production is favoured by temperatures between 9 and 15°C. After swimming for a time the zoospores encyst and then form germ tubes which usually penetrate the epidermal walls of the potato leaf or occasionally enter the stomata. An appressorium is formed at the tip of the germ tube and penetration of the cell wall is probably both by mechanical as well as enzymic action. Penetration occurs within two to two and half-hours. Optimum temperature for the growth of the fungus is 16-18°C. Sporulation occurs at temperatures of 9-26°C but the optimum temperature for their production is 21°C. The optimum temperature for germination of sporangia by zoospores is 12°C and by germ tube, 21°C. The minimum temperature for sporangial germination is as low as 2-3°C, while the maximum temperature may go up to 24°–30°C. It has been noted that a relative humidity of 100% is necessary for abundant production of sporangia, none being formed at relative humidities of less than 90%. P. infestans is a heterothatllic fungus. The oospores of P. infestans appear to be very rare in the eastern hemisphere, the USA and Canada, and therefore, may not play a significant role in the survival of the fungus. However, in Mexico, oospores have been discovered by Gallegly and Galindo (1958). It is quite possible that in this part oospores play a significant role in the survival and recurrence of the disease from year to year. The disease cycle of late blight pathogen in our country is given in Fig. 13.3. It is not possible for the fungus to survive in the form of mycelium. The living mycelium in the tuber is killed by exposure to 40°C for four hours or to 30°C for 65 hours. Moisture and temperature also affect the viability of the sporangia in the soil. The optimum soil moisture is between 15–20% saturation. The viability and infectivity of sporangia lasts for nine to ten weeks at low temperature and optimum moisture levels. Kaung (1965) also studied the viability of zoospores in soil and found them to be viable only for two to three weeks.

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Zoosporangium

Sporangium Germination

Zoosporangium

Oospore

Zoosporangiophore on infected seedling

Ongonium

Zoosporanium

Antheridium

Oogonium

mycelium from tuber infects seedling

Zoosporangiophore on infected tuber (in spring) Sporangiophore on leaf

Zoospores Zoospores infect leaf

Antheridium Infected leaf Infected tuber

Zoospores infect tuber

Sexual reproduction extremely rare in nature Infected plant

Fig. 13.3

Disease cycle of late blight of potato caused by Phytophthora infestans. (From G.N. Agrios, Plant Pathology, Academic Press. New York and London. 1969).

In India, the main potato-growing season is followed by hot summer months in the plains, and therefore, the fungus is eliminated from the soil. Potato tubers which are stored at ordinary temperatures also become free from disease. In the Indian plains, the late blight is rare because the two cardinal factors—high humidity and low temperature—must be simultaneously present before infection can develop. Such conditions are encountered only in the northern hills of India and therefore, late blight epidemics are confined to the hills. However, late blight epidemics have now been regularly reported in the northern plains from places such as Shahabad (Ambala District), Kurukshetra (Haryana), Meerut, and Kanpur in Uttar Pradesh and Patna (Bihar). It is believed that the primary source of infection for late blight in these areas is the seed potatoes stored in cold storages. The environment plays a very significant part in the occurrence of a late blight epidemic. Van Everdingen (1933) was one of the first to define the weather conditions necessary for an outbreak of blight in a potato crop. His work was developed by Beaumont (1947) who examined critically the meteorological data for the periods preceding blight outbreaks in southwestern England. He found that if for two days the temperature remained above 10°C and relative humidity was 75% or above the blight

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could be expected approximately ten days afterwards. These periods are called “Beaumont periods” and are the basis of a blight forecasting service in England and Wales. Physiological Specialization and Origin of New Races

Schick (1932) and Muller (1933) discovered physiological specialization and occurrence of races of Phytophthora infestans on different cultivars of potato from crosses of S. demissum and S. tuberosum. In the hybridization work, resistance genes from S. dimissum were tranferred to the commercial cultivar. On the basis of this, Black et al. (1953) determined four major genes for resistance (R1, R2, R3 and R4). They suggested an international system for classifying races and genes for resistance. According to this system, a particular race receives the resistant genes of the plant it is able to infect. The four R genes produce 16 possible combinations and 15 of the total 16 races were identified experimentally. In India now several races are found. Later, six major genes for blight resistance in potato were identified (Black, 1960). On this basis probably 64 races of P. infestans can be identified. Another type of resistance, the so-called minor gene resistance or field resistance, was found in numerous cultivars of Solanum tuberosum. This type of resistance is relatively weaker and is not race specific. It is a recessive character and probably depends on the interaction of several genes. Resistance of the tops is not necessarily correlated with that of the tubers. Several workers from different parts of India have reported a number of physiological forms of this fungus. Up to 1965, races O and 1 were predominant in north-western hills, and O, and 4 in the eastern hills. Gradually, more complex races have appeared since 1966 in the eastern hills and by 1971 in the Simla hills. By the end of 1980, 8 to 9 complex races became prevalent particularly in the hills (Bhattacharya, 1990, Singh and Bhattacharya, 1998). It is believed that the race flora has reached more than 80 or 100. The races from different parts of India contain 2-10 genes in different combinations. It is believed that the pathogen produces new and complex races through mutation and somatic recombination particularly on resistant genotypes carrying major genes. The rate of development of new races is higher in areas such as the eastern and western hills having longer blight favourable periods as compared to the southern and northern hills. The identification of some high spectrum races of P. infestans in the Khasi hills has been done by Khanna et al. (1982). Sokhi et al. (1993) reported 11 pathogenic races of P. infestans on potato and 3 pathotypes on tomato in the plains of Punjab. The origin of physiological races is not properly understood. Sexual reproduction is a rare phenomenon in P. infestans. Mutation could be responsible for new races. Another possibility is that the mycelium of P.infestans is heterokaryotic, carrying nuclei of more than one race. It has been suggested that there is some evidence that the pathogenicity of a strain of the parasite can be increased by continued passage through resistant hosts. Epidemiology and Forecasting

Infected tubers are the main source of the initial inoculum. In the hills, temperatures are favourable for the survival of the pathogen in tubers in soil and in country stores up to the next planting season (Bhattacharya, 1990). In the plains the pathogen does not survive in infected potato stalks buried in soil for more than 5 days. However, it was able to survive for 150 days at Simla and for 40 days at

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Ootacamund in potato stalks left in the field. According to Singh and Bhattacharya (1990), the pathogen can survive in tomato seeds. In our country, 10–20% of potato true seeds collected from fruits or berries have been found to carry the pathogen. However, the role of potato stalks and true potato seeds as the effective primary source of infection in the hills is still to be confirmed (Singh and Bhattacharya, 1990). In the plains, the initial source of the disease is the tubers placed in cold stores or the seed tubers which have been found to carry the late blight infection. Moisture and temperature are the main factors limiting the development of blight and these conditions determine the time of the appearance of the disease. The forecasting is based on weather data collected in meteorological observations. The main factors under consideration are: rainfall or humidity, temperature, cloudiness and dew deposition. Disease Management

Certified and disease-free tubers should be used for cultivation. Seed tubers should be obtained from uninfected areas. Harvesting of a diseased crop should be delayed until the plants are fully mature. The danger of infection of tubers by sporangia falling on to them from foliage at lifting time can be minimized by ensuring that all foliage is destroyed beforehand. This is achieved by spraying the foliage two to three weeks before harvesting with sprays, such as copper sulphate, tar acid compounds or sodium chlorate. High ridging at the time of earthing helps to reduce chances of tuber infection. The spread of the disease is favoured by cloudy, warm weather with occasional rain. Since the time of the appearance of the disease in the hills and plains is more or less known, spraying should be done with suitable fungicides. In advanced countries, there is a regular disease forecasting service where accurate forecasts of the disease can be made on the basis of the temperature and humidity. In the plains, Paharia (1961) tried different fungicides for the control of late blight in Patna, and Dithane Z-78 gave good results. In the Nilgiri hills, Bordeaux mixture provided good protection to the crop and helped to increase yields. In the eastern Himalayas experiments were conducted in Darjeeling with a few fungicides and Dithane Z-78 was found to give good protection to the crop. In the western Himalayas, at Kufri in the Simla hills, Vasudeva and Azad (1962) tried different fungicides and found Burgundy mixture to be effective. Dutt (1962) tried 16 different fungicides in these hills during 1958-1960 and found Bordeaux mixture to be the best. Dutt (1970) demonstrated that Bordeaux mixture was found to give better results than Burgundy mixture or any other proprietary product or fungicide. According to Nagaich (1977), in the northern hills, a protective spray of Dithane M-45 at 2kg/ha is essential in the last week of June before the disease appears. This should be followed by a spray of Bordeaux mixture, 5:5:50, at intervals of ten days. In the eastern hills, the crops should be sprayed with Bordeaux mixture. Other fungicides, such as Brestan (1kg/ha) can also be sprayed. In the Nilgiri hills, spraying may be done with Brestan or Difolatan. For spraying in the hilly regions some sticker like Triton HE or Sandont (0.1%) should be added to the fungicide. In the plains. any fungicide, for example, Dithane M-45(2kg/ha), Brestan (1kg/ha), and Difolatan 80 WP(2.5 kg/ha) can be sprayed. The discovery of Metalaxyl, a systemic fungicide gave a new dimension to late blight control strategies. According to Bhattacharya et al. (1983) and Khanna and Sharma (1981), Metalaxyl (1kg/ha) and Propineb (1.5 kg/ha) were found superior over other fungicides in inhibiting the infection completely and increasing the yield about 3 times over unsprayed controls.

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Oxadixyl, a new systemic fungicide has also been found effective against the disease. At Kufri, Shillong, and Darjeeling the formulation of Oxadixyl (Oxadixyl + Mancozeb; Oxadixyl + Copper oxychloride) were very effective. This treatment did not reduce tuber infection. According to Bhattacharya et al. (1987) eradicative properties of this chemical were also observed. According to Singh et al. (1988), the pathogen was found to be least adaptable to Mancozeb and Oxadixyl + Copper oxychloride but readily adaptable to Metalaxyl + Ziram and Oxadixyl + Mancozeb. The pathogens in the tubers can be readily killed if these are dipped in water at a temperature of 45°C for 4-5 hours or at 40°C for 24 hours (Butler 1903, CPRI 1987). According to Singh and Bhattacharya (1988) dipping the tubers suspected to be carrying infection up to 5% in a 1500-2000 ppm solution of Oxadixyl or Metalaxyl for 30 minutes is very effective in removing infection from the tubers but this holds true only when the tubers have overcome the dormancy. Thind et al. (1989) have emphasized the importance of the time of application of fungicides in the control of late blight of potato. A detached-leaf technique was developed by Thind et al. (1989) to study the efficiency of fungicides after artificial inoculation. Their work further showed that the contact fungicides provided better control when applied before infection, therefore these should be used as a prophylactic measure only. Systemic fungicides exhibited a good control potential to check established infection also. However, to avoid build up of resistant strains of the pathogen as reported in some countries, it is advisable to use these fungicides rationally and in combination or alternation with other fungicides. Metalaxyl-resistant isolates were first reported by Arora (1991 a,b). Navneet et al. (1995) have studied the effect of two effective fungicides, Dithane M-45 and Ridomil, on the phylloplane mycoflora of potato to know the succession of microbes on treated and untreated leaves of potato plants. Singh et al. (1998) have examined Metalaxyl residue in potato tubers. Accumulation of Matalaxyl within potato tubers after two sprays on foliage was within prescribed limits. However, the best control measure lies in cultivating resistant varieties. Hybridization work has been done with Solanum tuberosum (South American variety), S. neoantipoviezi, and others showing a high and promising degree of resistance. Solanum demissum, a Mexican species, shows complete resistance with commercial susceptible varieties. There has been considerable progress in obtaining resistant varieties for India at the Central Potato Breeding Station in Simla. Kufri Kundan and Kufri Kumar were the first set of varieties, possessing resistance of late blight, released for commercial cultivation. Later on hybrids were released for commercial cultivation as Kufri Jeevan, Kufri Neela, Kufri Muthu, Kufri Jyoti, Kufri Khasigaro, Kufri Naveen, Kufri Badshah and Kufri Sherpa. Cultural Methods of Control

The following are some cultural methods, which have been found useful in controlling the disease. 1. Tubers from infected fields should be discarded for seed purposes or very carefully inspected at planting time to remove all those suspected to be carrying or harbouring infection. 2. The tubers should be harvested after proper skin curing (mature tubers). 3. In an infected field harvesting should be delayed until the plants are mature. The plants should be allowed to dry completely before harvesting. Alternatively the infected haulms be cut and buried or burnt so that the tubers do not get in touch with infected portions of the potato plants. 4. Infected tubers and aerial portions of the plants should be collected and buried deep in the soil. 5. All sanitary precautions must be enforced to keep the crop free from weeds. Under hill conditions ground keepers or volunteer potato plants harbour the infection and therefore, it is essential to remove these as soon as they appear.

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6. Tubers should be planted on high ridges. High ridges will cover the tubers thoroughly with soil and thus prevent growing out of the fungus from mother tuber and also infection in daughter tubers. 7. The tubers should be stored either in cold storage at low temperature or in a cool, dry and well aerated store. The best storage temperature is 38-40°F. 8. In the plains, the disease normally appears with the onset of winter rains. Therefore irrigation should be restricted during this period. 9. Planting of alternate beds of susceptible and resistant varieties will reduce spread of the disease. 10. In order to reduce humidity, field-spacing and reduced nitrogen are recommended in endemic areas. Biocontrol

Arora (1999) has reported the antagonism of a number of phylloplane mycroflora of potato such as Trichoderma viride, Penicillium viridecatum and Chaetomium brasilense while Roy et al. (1991) reported reported Myrothecium verrucaria and Penicillium aurantiogriseum. Jindal et al. (1988) have reported Epicoccum purpurascens, Stachybotrys atra and Trichoderma koningii. Evaluation of the fungal antagonists against potato late blight disease has been done in greenhouse (Roy et al.1994) while mass culture of antagonists and their application on a large scale in field conditions have been done by Arora (1994) with some success. Khanna (1992) and Puri (1995) have done field experiments in small plots against some fungal antagonists of P. infestans. BLIGHT OF COLOCASIA (COLOCASIA ESCULENTA)

Taro (C. esculenta) is an important tropical tuber crop for millions of people in developing countries. Leaf blight and corm rot are the important diseases of taro causing yield losses of 25–50 per cent. Besides, the pathogen also causes serious post-harvest decay of corms. Comprehensive reviews have been published by Ghosh et al. (1989), Thankappan (1985), and Aggarwal et al. (1990). The disease was first reported in India by Sydow and Butler (1907). Butler and Kulkarni (1907) were the first to make detailed investigations on the disease and causal organism, P. colocasiae Raciborski. In India, two Colocasia types, C. esculenta var. esculenta and C. esculenta var. antiquorum, are generally cultivated and both are equally susceptible to Phytophthora blight. The pathogen affects at crucial stages of crop growth. In C. esculenta var. esculenta, the crop is in the stage of tuber (corm) development whereas in C. esculenta var. antiquorum, it is in the initial stages of crop growth at the time of infection. Infection at these stages in both taro leads to heavy yield losses. Misra (1993) reported 33-64 per cent yield losses in the farmers field in Orissa whereas in the experimental farm 50.39 per cent and 26.26 per cent yield loss was recorded in susceptible and tolerant cultivars, respectively due to leaf blight. The local variety (Jankhari) in Orissa seems to have some tolerance to the disease. Symptoms

The disease first shows itself ordinarily on the leaf, the earliest attack occurring in August or September, in the form of small, dark, roundish specks, which increase in area rather quickly by centrifugal growth

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[Fig. 13.4]. The spots may remain circular or assume an oval or irregular appearance. Several such spots may appear on the leaf and the whole leaf lamina may get infected. Clear, yellow liquid drops ooze from the surface of the spots in the initial stages; later the central portions become dry and drop off, and thus forming holes in the leaf. The periphery of the spots has a clear zonation pattern of different shades of brown, green, and yellow.

(a)

(b)

Fig. 13.4

(a) Symptoms on Colocasia due to Phytophthora colocasiae (b) sporangiophores and sporangia.

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Towards the periphery these zones reveal on careful observation, a delicate white haze which is caused by the sporangial stage of the pathogen. As the spots increase in size or diameter, sporangia continue to be produced in a zonate fashion at progressively increasing distances from the centre. In severe cases of infection when humidity is high and temperatures are congenial for the development of blight symptoms, the petioles of the leaves get infected. The tissues in the infected region often become so softened as to be unable to bear the weight of the leaves, which fall or even break off completely from the plants. Extension may continue down to the sheathing base of the petiole and probably into the short stem. Inflorescence is also attacked. In severe attacks all the leaves are completely destroyed and the underground corms are infected and rot completely. Causal Organism

P. colocasiae Raciborski is the pathogen responsible for the leaf blight and corm rot of Colocasia esculenta which was first described by Raciborski in the year 1900 as the causal agent of the leaf spot of taro in Java (Indonesia). P. colocasiae is primarily a foliar pathogen and there is little information on its origin, but there are indications that it is Asiatic. According to the latest revised key to the species of Phytophthora, P. colocasiae belongs to group IV of Stamps et al. (1990). The parasitic mycelium grows within the tissues, the hyphae being coenocytic, unseptate and profusely branched. In the leaf they grow between the cells, except in the epidermis. Butler (1918) has given a detailed account of the histopathological account of the pathogen inside the tissues. Longish, unbranched, slender haustoria are sent into the cells. Hyphae have not been seen to enter the fibro-vascular bundles in the leaf or petiole. In the corms on the other hand, not only are hyphae found in storage cells, the mycelium being within the cells rather than between them, but the bundles are commonly penetrated. The effect of infection leads to the gradual disappearance of the green colouring matter and the collection of the cell contents into a shapeless and grumous mass. Gradually, all the starch is destroyed and the cells turn brown in colour. From the internal hyphae, branches of sporangiophores grow out to the surface which ultimately produce sporangia. The sporangia are found on both the surfaces of the leaves, petioles or influorescence. These are produced in a successive manner as the spots continue to grow. According to Butler (1918) similar sporangia can be induced to form on the corms in water or in moist air, for some months after growth elsewhere has ceased. These incipiently infected corms can further rot in storage and spread the pathogen. Sporangiophores are slender (2-4 mm), narrowing at the tip and unbranched. On the leaf they are short, sometimes scarcely longer than the sporangium. In culture, branching of the sporangiophores is irregular to sympodial with swellings at the point of origin of the branches as well as the sporangiophore. These branches bear single, elongated, ellipsoidal, ovoid or pear shaped sporangia which measure 38– 60 ¥ 18-26 mm; sporangia are semi-papillate (