Seed-borne Plant Diseases

K. Subramanya Sastry

Seed-borne Plant Virus Diseases

123 K. Subramanya Sastry Emeritus Professor Department of Virology S.V. University Tirupathi, AP India

ISBN 978-81-322-0812-9 ISBN 978-81-322-0813-6 (eBook) DOI 10.1007/978-81-322-0813-6 Springer New Delhi Heidelberg New York Dordrecht London

Library of Congress Control Number: 2012945630

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Springer is part of Springer Science+Business Media (www.springer.com) About the Author

Prof. K. Subramanya Sastry, Ph.D., is emeritus professor in the department of virology at S.V. University, Tirupathi Ð 517502, A.P., India. (India). He obtained his M.Sc. (Botany) and Ph.D. (Botany) with plant virology special- ization in 1966 and 1973, respectively, from Sri Venkateswara University, Tirupati (India). He joined Indian Council of Agricultural Research (ICAR) as a scientist (Plant Virology) during the year 1971 and retired in 1999. He has served at Indian Institute of Horticultural Research, Hessaraghatta, Bangalore 560 089, Karnataka, and also at Directorate of Oil Seeds Research, Hyderabad 500 080, Andhra Pradesh. Prof. Sastry’s research has been primarily on epidemiology and management of virus and virus-like diseases of Horticultural and Oil Seed crops. He has done pioneer research on Begomoviruses of and okra. He has research experience in molecular and biotechnological approaches for characterization and control of viral diseases of horticultural crop plants. Further, he has also published over 120 research papers both in national and international journals. He has also published “Compendium of the Plant Virus Research in India (1903–2008)” having 8,652 plant virus references that is considered as one of the rich sources of information for Indian plant virus research.

v

Foreword

I am delighted to write the foreword for a book on seed-borne , since many economically important viral diseases are spread in nature through seeds. Increasing our knowledge on all aspects of seed-borne viruses is the first step towards developing a strategy for their control. This book represents a comprehensive up-to-date treatise for seed-borne viruses, including detec- tion methods, ecology, epidemiology and control. Attention is also placed on the importance of integrated management to reduce losses caused by seed- borne viruses. I congratulate Dr. K. S. Sastry for his sincere effort and many years of hard work to assemble existing information on seed-borne viruses and make them available in a well organized manner to a wide audience: research scientists, graduate students, extension workers, progressive farmers as well as individuals who are interested in agricultural production at large. I am confident that this book will serve as an important reference for seed-borne viruses which affect agricultural crops, globally. Legume crops are the main source of for the majority of people in developing countries. Around 50% of the viruses which infect legumes are seed-borne, and some of them could lead to a complete crop failure. Improving our knowledge on these viruses can be well translated to improved legume crops production, worldwide. It is hoped that this book will serve as an important resource for all agricultural workers dedicated to improved and stabilized crop production through adoption of environment-friendly practices, including the use of virus-free seeds.

Khaled M. Makkouk Advisor for Agriculture and Environment National Council for scientific Research (CNRS) P.O. Box 11-8281 Riad El-Solh 1107 2260, Beirut, LEBANONCNRS, Beirut, Lebanon

vii

Preface

Seed, a highly ordered plant structure, is the basic input in crop production. It possesses the qualities necessary for division, morphogenesis and regeneration of species. The study of seed itself is as good as the study of life. Seed is one of the vital inputs in the development of agriculture in any country. To increase agricultural production, viability of quality seed is one of the prerequisites. The seed is also one of the most important source for the perpetuation of fungi, bacteria, , insects, viruses, etc. and acts as an efficient carrier for their spread to new areas through introduction and/or seed trade which is a global enterprise. Among these plant pathogens, viruses are unique in nature and behaviour. As is the case with any seed- transmitted plant pathogens, virus transmission through seeds of higher plants also result from complex interactions between the genetic systems of the host, pathogen and the environment. There is increasing awareness of seed- transmitted viruses with particular reference to their mode of transmission, survival and management. Till date, more than 231 viruses have been reported to be seed transmitted in different food, fiber, weed and ornamental crops. Virus-free seed has assumed multifold significance in quarantine and seed certification for ensuring initial crop health. Circumstantial evidence shows that several viruses have spread to different geographical regions during the process of liberalized seed exchange of crop plants in recent years. Seeds are instrumental in an effective worldwide spread of a range of diseases through international exchange of seeds. The techniques of identification and management of seed-transmitted viruses are completely different from those of other pathogens like fungi, bacteria and phytoplasmas. The information on reliable techniques for detection of seed-transmitted viruses and their management is of immense use in the present international seed trade. The coverage of seed-transmitted plant viruses is limited to few chapters in books on seed pathology. Considerable information in respect of new seed- transmitted viruses, their detection and identification techniques, transmission and management has been generated in recent years. This publication is an endeavor to compile up-to-date literature available on seed-transmitted viruses in a comprehensive form. It is hoped that this book will have an important role to play in the context of the government agencies on new seed policy for liberal import of the seeds of coarse cereals, oilseeds and pulses. The knowledge of seed-transmitted virus diseases on the isolation and identification of viruses in fresh seed lots and their management not only restricts the entry of virus diseases but also will help to prevent the spread

ix x Preface of unrecorded virus diseases in the country. Within the scope of this book, elaborate attempts have been made to present a comprehensive account on identification, mode of transmission, ecology, epidemiology and manage- ment of seed-transmitted virus and viroid diseases covered in ten chapters. An up-to-date list of all seed-transmitted viruses and viroids of different host plants is presented in the form of a table for ready reference. The information given on latest molecular techniques for virus detection and management included in this volume will be of immense practical value to researchers and field workers. This work has immensely benefited from critical comments and con- structive suggestions made by Prof. M. V. Nayudu, Dr. S. E. Albrechtsen, Dr. P. Sreenivasulu, Dr. G. P. Rao, Dr. R. K. Khetarpal, Dr. D. V. R. Saigopal, Dr. V. C. Chalam and also assistance offered by our scholarly friends and colleagues. I am highly thankful to all the persons, organizations and various publishers for their prompt help in providing information, photographs and consents for reproduction. I also wish to express my sincere gratitude to Mr. C. Nagaraja for secretarial work. I thank my wife Mrs. B. N. K. Kumari for her continuous support during the preparation of this book. I dedicate this book to the memory of my parents late K. Panduranga Sastry and Smt. K. Subadramma who have sacrificed everything to give me the best education possible and for their eternal blessings. I hope this book will be of value and interest to many teachers, students, seed biologists, seed technologists, seed companies and researchers at quaran- tine stations as a comprehensive, accurate and easily readable reference book on seed-transmitted plant virus and viroid diseases. I shall deem it an honour and reward if readers find this book useful to them. I welcome suggestions and comments for the improvement of this book in future editions.

K. Subramanya Sastry Contents

1 Introduction ...... 1 1.1 Introduction ...... 1 1.1.1 Seed (Sexual Propagule) ...... 2 1.2 Seed Transmission of Viruses ...... 2 1.2.1 History of Seed-Transmitted Plant Virus Research 4 1.3 Seed Transmission of Partitiviridae ...... 5 1.4 Seed Transmission of Viroids ...... 5 1.4.1 Extent of Seed Transmission ...... 6 1.5 Viruses Erroneously Listed as Seed Transmitted ...... 7 1.5.1 Seed-Transmitted Plant Virus Names That Appeared Only Once in the Literature ...... 8 1.5.2 Establishing Certain Erotic Positive Seed- Transmitted Viruses to Be Non-seed Transmissible 9 References ...... 32 2 Identification and Taxonomic Groups ...... 55 2.1 Identification ...... 56 2.2 Classification of Viruses ...... 56 2.3 Variability in Certain Seed-Transmitted Viruses ...... 62 References ...... 65 3 Economic Significance of Seed-Transmitted Plant Virus Diseases ...... 67 3.1 Introduction ...... 67 3.2 Assessment of Crop Losses ...... 67 3.3 Viruses and Seed Viability ...... 69 3.4 Factors Affecting Yield Losses ...... 69 References ...... 70 4 Virus Transmission ...... 75 4.1 Vector Transmission ...... 75 4.1.1 ...... 76 4.1.2 ...... 77 4.1.3 Thrips ...... 77 4.1.4 Whiteflies ...... 78 4.1.5 Mites ...... 78 4.1.6 Nematodes ...... 78 4.1.7 Bumblebees ...... 79

xi xii Contents

4.1.8 Fungi ...... 79 4.1.9 ...... 79 4.2 Nonvector Transmission ...... 80 4.2.1 Mechanical Spread ...... 80 4.2.2 Wind ...... 80 4.2.3 Water ...... 80 4.2.4 Obligate Symbiosis ...... 80 4.3 Conclusions ...... 81 References ...... 81 5 Mechanism of Seed Transmission ...... 85 5.1 Embryology and Development of Seed Structures ...... 85 5.2 Distribution of Virus in the Seed ...... 87 5.3 Virus Longevity in Seeds ...... 88 5.4 Genetics of Seed Transmission ...... 88 5.5 Factors Influencing Rate of Seed Transmission ...... 88 5.5.1 Number of Infection Sources ...... 90 5.5.2 Virus Strain/Isolate ...... 90 5.5.3 Mixed Infections...... 90 5.5.4 Host Species ...... 90 5.5.5 Stage of Infection ...... 92 5.5.6 Environmental Factors ...... 92 5.6 Reasons for Failure of Seed Transmission ...... 93 5.6.1 Inability to Infect Embryos ...... 93 5.6.2 Inability of Virus Survival in the Embryos ...... 94 5.7 Conclusions ...... 95 References ...... 95 6 Detection of Plant Viruses in Seeds ...... 101 6.1 Introduction ...... 101 6.1.1 Seed Health Testing ...... 102 6.1.2 Low Seed Transmission/ Symptomless Carriers . . . 103 6.2 Biological Methods ...... 105 6.2.1 Visual Examination ...... 105 6.2.2 Grow-Out Test ...... 107 6.2.3 Indicator Hosts ...... 108 6.2.4 Biological Properties ...... 109 6.3 Physical Methods ...... 109 6.3.1 Inclusion Bodies ...... 109 6.3.2 Electron Microscopy ...... 111 6.4 Serological Techniques ...... 111 6.4.1 Monoclonal and Polyclonal Antibodies ...... 112 6.4.2 Immunodiffusion Tests ...... 114 6.4.3 Labelled Antibody Techniques ...... 116 6.4.4 Dot-Immunobinding Assay (DIBA or DIA) ...... 126 6.4.5 Disperse Dye Immunoassay (DIA) ...... 128 6.4.6 Rapid Immunofilter Paper Assay (RIPA) ...... 128 6.4.7 Immunosorbent Electron Microscopy (ISEM) .... 129 Contents xiii

6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis . 131 6.5.1 Introduction ...... 131 6.5.2 Molecular Hybridisation ...... 132 6.5.3 Double-Stranded RNA (dsRNA) Analysis ...... 133 6.5.4 Gel Electrophoresis ...... 134 6.5.5 Nucleic Acid-Specific Hybridisation ...... 135 6.5.6 Array Technologies ...... 137 6.5.7 Polymerase Chain Reaction (PCR)-Based Detection ...... 138 6.5.8 Real-Time PCR ...... 143 6.6 Molecular Markers ...... 144 6.7 Conclusions ...... 145 References ...... 145 7 Ecology and Epidemiology of Seed-Transmitted Viruses ..... 165 7.1 Introduction ...... 166 7.1.1 Primary Inoculum Source ...... 166 7.2 Host Plant ...... 167 7.3 Vectors ...... 170 7.3.1 Factors Influencing Vector Movement ...... 170 7.4 Pollen Transmission ...... 172 7.4.1 Epidemiological Role of Pollen-Transmitted Viruses ...... 173 7.5 Viruses ...... 177 7.6 Conclusion ...... 178 References ...... 179 8 Methods of Combating Seed-Transmitted Virus Diseases ..... 185 8.1 Introduction ...... 185 8.2 Avoidance of Virus Inoculum from Infected Seeds ...... 186 8.2.1 Removal of Infected Seeds ...... 186 8.2.2 Chemical Seed Disinfection ...... 186 8.2.3 Seed Disinfection by Heat ...... 187 8.2.4 Storage Effect ...... 189 8.2.5 Irradiation Effect ...... 189 8.3 Reducing the Rate of Virus Spread Through Vector Management ...... 189 8.3.1 Avoiding of Continuous Cropping ...... 189 8.3.2 Elimination of Weed, Volunteer and Wild Hosts . . 190 8.3.3 Roguing ...... 190 8.3.4 Crop Rotation ...... 191 8.3.5 Planting Dates ...... 191 8.3.6 Plant Density ...... 191 8.3.7 Barrier and Cover Crops ...... 192 8.4 Integrated Cultural Practices for Seed-Transmitted Virus Disease Management ...... 192 8.5 Crops Hygiene ...... 193 8.5.1 Raising Transplants ...... 194 xiv Contents

8.6 Control of the Vectors ...... 195 8.6.1 Insecticides ...... 195 8.6.2 Mineral Oils ...... 196 8.6.3 Repelling Surfaces ...... 196 8.7 Virus Avoidance ...... 197 8.7.1 Exclusion ...... 197 8.8 Resistance ...... 199 8.8.1 Host Resistance ...... 201 8.8.2 Sources of Resistance ...... 201 8.8.3 Conventional Breeding of Natural Resistance Genes ...... 203 8.8.4 Cultivars with Low Seed Transmission ...... 205 8.8.5 Vector-Resistant Cultivars ...... 206 8.9 Immunisation ...... 207 8.10 Approved Seed Certification Standards ...... 209 8.11 Stages of Seed Multiplication ...... 210 8.12 Inoculum Threshold ...... 210 8.13 International Seed Testing Association (ISTA) ...... 213 8.13.1 Objectives of ISTA ...... 214 8.13.2 ISTA Certificates ...... 214 8.13.3 Conditions for Issuance of ISTA Certificates ..... 214 8.13.4 Accredited Laboratory ...... 215 8.14 Seed Certification Against Plant Virus Diseases ...... 215 8.14.1 The Quality Control by ELISA ...... 216 8.14.2 Certification Schemes Against Crops ...... 217 8.15 Conclusion ...... 220 8.16 Quality Control of Bulk Seed Lots ...... 220 8.16.1 Determination of Seed Transmission Rate ...... 220 8.16.2 Infection Status of a Bulk Seed Lot with Respect to a Tolerance Limit ...... 222 8.16.3 Infection Status of a Bulk Seed Lot with Respect to a Virus Not Known in the Importing Country . . . 222 8.17 World Trade Organization (WTO) Regime and Its Implications ...... 222 8.17.1 Examples of Introduced Plant Viruses Through Seed Exchange ...... 223 8.18 The Role of Plant Biosecurity in Preventing and Controlling Emerging Plant Virus Disease Epidemics . . 223 8.19 Pest Risk Analysis (PRA) ...... 225 8.19.1 Pest and Pathogen Risk Analysis ...... 225 8.19.2 Pest Risk Analysis for Viral Diseases of Tropical Fruits ...... 225 8.20 Biosafety ...... 227 8.20.1 Biosafety Regulations ...... 227 8.20.2 History of Biosafety Protocol and Regulations .... 227 8.20.3 Biosafety Regulations of Asia-Pacific Countries . . 228 8.21 Risks Associated with Genetically Modified Crops ...... 229 Contents xv

8.22 Quarantines ...... 229 8.22.1 Plant Quarantine ...... 230 8.22.2 Plant Quarantine Measures ...... 230 8.22.3 Functions of Plant Quarantine ...... 231 8.22.4 Pathways of Spread of Pests and Pathogens ...... 232 8.22.5 Quarantine Status of Plant Importations ...... 233 8.22.6 Types of Materials Received ...... 233 8.23 Role of FAO/IBPGR in Germplasm Exchange ...... 234 8.23.1 Conceptual Guidelines for Exchange of Legume Germplasm, Breeding Lines and Commercial Seed Lots as Follows ...... 234 8.23.2 The Technical Guidelines for Exchange of Germplasm and Breeding Lines ...... 235 8.23.3 Movement of Germplasm ...... 235 8.24 Steps in Technical Recommendations for Seed Germplasm Exchange in the Country of Origin or Destination ...... 236 8.25 Part of the Planting Material to Be Tested and Post-Entry Quarantine ...... 237 8.26 Exclusion of Exotic Plant Viruses Through Quarantine .... 238 8.26.1 International Scenario ...... 238 8.26.2 National Scenario ...... 239 8.27 Challenges in Diagnosis of Pests in Quarantine ...... 241 8.28 Quarantine for Germplasm and Breeding Material...... 241 8.29 Role of IPGRI and NBPGR in Germplasm Maintenance and Exchange ...... 243 8.29.1 Objectives of NBPGR ...... 243 8.29.2 Methods of Testing at Quarantine Stations ...... 244 8.30 Important Cases of Introduction ...... 246 8.31 Important Diseases Restricted to Some Countries ...... 248 8.32 Effective Methods of Plant Importations ...... 249 8.32.1 Phytosanitary Certificates ...... 249 8.32.2 Closed Quarantines ...... 249 8.32.3 Quantity of Plant Materials ...... 250 8.32.4 Open Quarantine ...... 250 8.32.5 Examination of Exportable Crops During Active Growth ...... 250 8.32.6 The Intermediate Quarantine ...... 251 8.32.7 Aseptic Plantlet Culture ...... 251 8.32.8 Embryo Culture ...... 252 8.32.9 Use of Shoot Tip Grafting or Micrografting ...... 253 8.33 General Principles for the Overall Effectiveness of Quarantines ...... 253 8.34 Quarantine Facilities ...... 254 8.35 Need for Networking for the Developing Countries ...... 254 8.36 Perspectives ...... 255 8.37 Conclusion ...... 256 8.38 Biotechnology and Virus-Derived Resistance ...... 256 8.38.1 Capsid Protein-Mediated Resistance ...... 257 xvi Contents

8.39 Molecular Approaches ...... 257 8.39.1 Molecular Interactions of Seed-Transmitted Viruses ...... 257 8.39.2 Transgenic Approach ...... 258 8.40 Conclusion ...... 266 References ...... 266 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction) ...... 285 9.1 Different Vegetative Propagative Plant Materials ...... 286 9.2 Role of Vegetatively Propagated Plant Materials in Virus Spread ...... 286 9.3 Different Virus, Phytoplasma and Viroid Diseases ...... 286 9.4 Virus Transmission ...... 286 9.5 Yield Losses...... 288 9.6 Virus Diagnosis ...... 289 9.7 Vegetatively Transmitted Plant Virus and Virus-Like Disease Management by Certification Schemes ...... 291 9.7.1 Success Stories of Production of Virus-Free Plant Propagules ...... 291 9.7.2 Certification Schemes ...... 295 9.8 IPGRI’S Role in Controlling Virus Diseases in Fruit Germplasm ...... 296 9.9 Conclusion ...... 296 References ...... 296 10 Future Strategies and Conclusions ...... 307 10.1 Introduction ...... 308 10.2 Plant Virus Management by Integrated Approach ...... 310 10.3 Challenges for the Future ...... 313 References ...... 315

Index ...... 317 List of Standard Acronyms of Plant Virus and Viroids

Virus and viroid names Acronym Abaca mosaic virus AbMV African cassava mosaic virus ACMV Alfalfa cryptic virus ACV Alfalfa mosaic virus AMV Andean latent tymovirus APLV Apple chlorotic spot virus ACLSV Apple mosaic virus ApMV Apple scar skin viroid ASSVd Apple stem grooving virus ASGV Arabis mosaic virus ArMV Arracacha virus A AVA Artichoke Italian latent virus AILV Artichoke latent virus ALV Artichoke yellow ringspot virus AYRSV virus I AV 1 Asparagus virus II AV 2 Avocado sunblotch viroid ASBV Bamboo mosaic virus BaMV Banana bract mosaic BBMV Banana bunchy top virus BBTV Banana streak badnavirus BSV Barley stripe mosaic virus BSMV Barley yellow dwarf virus BYDV Bean common mosaic virus BCMV Bean common mosaic necrosis virus BCMNV Bean pod mottle virus BPMV Bean southern mosaic virus BSMV Bean yellow mosaic virus BYMV Beet 1 alpha cryptovirus BCV-1 Beet 2 alpha cryptovirus BCV-2 Beet 3 alpha cryptovirus BCV-3 Beet mild yellowing virus BMYV Blackeye mosaic virus BICMV Blackgram mild mottle virus BMMV Blackgram mottle virus BgMV Bramble yellow mosaic virus BrmYMV Broad bean mottle virus BBMV Broad bean stain virus BBSV Broad bean true mosaic BBTMV Broad bean wilt virus BBWV Brome mosaic virus BMV Carnation necrotic fleck virus CNFV Carrot red leaf virus CtRLV (continued)

xvii xviii List of Standard Acronyms of Plant Virus and Viroids

(continued) Virus name Acronym Cassava brown streak virus CBSV Cassava common mosaic virus CsCMV Cauliflower mosaic virus CaMV Cherry leaf roll virus CLRV Cherry necrotic rusty mottle virus CNRMV Cherry rasp leaf virus CRLV Chicory yellow mottle virus CYMV Chrysanthemum stunt viroid CSV Citrus exocortis viroid CEVd Citrus mosaic virus CiMV Citrus psorosis virus CPsV Citrus ringspot virus CRSV Citrus tristera virus CTV Citrus variegation virus CVV Citrus yellow mosaic virus CYMV Clover yellow mosaic virus ClYMV Clover yellow vein virus CYVV Cocao necrosis virus CoNV Cocao swollen shoot virus CSSV Cocao yellow mosaic virus CYMV Coconut cadang cadang viroid CCCV Coffee ringspot virus CoRSV Coleus blumei viroid CbVd Cow parsnip mosaic virus CpAMV Cowpea -borne mosaic virus CABMV Cowpea banding mosaic virus CpBMV Cowpea chlorotic mottle virus CCMV Cowpea chlorotic spot virus CpCSV Cowpea green vein-banding virus CGVBV Cowpea mild mottle virus CMMV Cowpea Moroccan aphid-borne mosaic CABMV Cowpea mosaic virus CPMV Cowpea mottle virus CPMoV Cowpea severe mosaic virus CpSMV Crimson clover latent virus CCLV cryptic virus CuCV Cucumber pale fruit viroid CPFVd Cucumber green mottle mosaic virus CGMMV Cucumber leaf spot virus CLSV Cucumber mosaic virus CMV Cymbidium ringspot virus CyRSV Dahlia mosaic virus DMV Dapple apple viroid DAV Dasheen mosaic virus DsMV Desmodium mosaic virus DesMV Dioscorea bacilliform virus DBV Echtes Ackerbohnen mosaik viruses EAMV Eggplant mosaic virus EMV Elm mosaic virus EIMV Elm mottle EMoV Euonymus mosaic EuoMV Fescue cryptic virus FCV Fig latent virus 1 FLV-1 Garland chrysanthemum temperate GCTV Garlic common latent virus GCLV Grapevine Bulgarian latent virus GBLV (continued) List of Standard Acronyms of Plant Virus and Viroids xix

(continued) Virus name Acronym Grapevine fanleaf virus GFLV Grapevine yellow speckle viroid GYSV Groundnut bud necrosis virus GBNV Guar symptomless virus GSLV Hibiscus latent ringspot virus HLRSV High plains virus HPV Hop stunt viroid HsVd Hop trefoil cryptic 1 HTCV1 Hop trefoil cryptic 2 HTCV2 Hop trefoil cryptic 3 HTCV3 Hosta virus x HSVX Humulus japonicas virus HJV Hydrangea mosaic virus HdMV Indian cassava mosaic virus ICMV Indian citrus ringspot virus ICRSV Indian peanut clump virus IPCV Iris mild mosaic virus IMMV Iris yellow spot virus IYSV Kalanchoe top-spotting KTSV Leek yellow stripe virus LYSV mosaic virus LMV Lilac ring mottle virus LRMV Lucerne Australian latent virus LALV Lucerne (Australian) symptomless LASV Lucerne transient streak virus LTSV Lychnis ringspot virus LRSV chlorotic mottle virus MCMV Maize dwarf mosaic virus MDMV Maize mosaic virus MMV Maize streak virus MSV Melon necrotic spot carmovirus MNSV Melon rugose mosaic virus MRMV Mibuna temperate virus MTV Mulberry ringspot virus MRSV Muskmelon mosaic virus MuMV Muskmelon necrotic spot virus MNSV Nicotiana velutina mosaic virus NVMV Oat mosaic virus OMV Olive latent virus OLV yellow dwarf virus OYDV Panicum mosaic virus PMV PRSV Paprika mild mottle tobamovirus PaMMV Parsley latent virus PLV Passionfruit woodiness virus PWV Peach rosette mosaic virus PRMV early browning virus PEBV Pea enation mosaic virus PEMV Pea mild mosaic virus PMiMV Pea mosaic virus PMV Peanut clump virus PCV Peanut mottle virus PeMoV Pea streak virus PeSV Peanut bud necrosis virus PBNV Peanut stripe virus PStV Peanut stunt virus PSV (continued) xx List of Standard Acronyms of Plant Virus and Viroids

(continued) Virus name Acronym Pea seed-borne mosaic virus PSbMV Pelargonium zonate spot virus PZSV Pepino mosaic virus PepMV Pepper mild mosaic virus PMMV Pepper mild mottle virus PMMoV Piper yellow mottle virus PYMoV Plum pox virus PPV Potato leaf roll virus PLRV Potato spindle tuber viroid PSTVd Potato virus M PVM Potato virus S PVS Potato virus T PVT Potato virus U PVU Potato virus X PVX Potato virus Y PVY Prune dwarf virus PDV Prunus necrotic ringspot virus PNRSV Radish yellow edge virus RYEV Raspberry bushy dwarf virus RBDV Raspberry ringspot virus RRV Red clover cryptic virus RCCV Red clover mottle virus RCMV Red clover vein mosaic virus RCVMV Red pepper crypticÐ1 RPCV1 Red pepper crypticÐ2 RPCV2 Rhubarb temperate virus RTV Rice yellow mottle virus RYMV Rubus Chinese seed-borne RCSV Rye grass cryptic virus RGCV Santosai temperate virus STV Satsuma dwarf virus SDV Soil-borne wheat mosaic virus SBWMV Southern bean mosaic virus SBMV Sowbane mosaic virus SoMV mild mosaic virus SMMV SMV Spinach latent virus SpLV Spinach temperate virus SpTV Squash mosaic virus SqMV Strawberry latent ringspot virus SLRSV Subterranean clover mottle virus SCMoV Sugarcane mosaic virus SCMV Sunflower mosaic potyvirus SuMV Sunflower rugose mosaic virus SRMV Sunn-hemp mosaic virus SHMV Sweet potato feathery mottle virus SPFMV Sweet potato ringspot virus SPRSV Tobacco mosaic virus TMV Tobacco necrosis virus TNV Tobacco rattle virus TRV Tobacco ringspot virus TRSV Tobacco streak virus TSV Tomato apical stunt viroid TASVd Tomato aspermy virus TAV (continued) List of Standard Acronyms of Plant Virus and Viroids xxi

(continued) Virus name Acronym Tomato black ring virus TBRV Tomato bushy stunt virus TBSV Tomato chlorotic dwarf viroid TCDV Tomato mosaic virus ToMV Tomato ringspot virus ToRSV Tomato spotted wilt virus TSWV Tomato streak virus TSV Turnip mosaic virus TuMV Turnip yellow mosaic virus TYMV Urdbean leaf crinkle virus ULCV Vicia cryptic virus VCV Watermelon mosaic virus WMV Wheat soil borne mosaic virus WSBMV Wheat streak mosaic virus WSMV White clover cryptic virus WCCV White clover mosaic virus WClMV Zucchini yellow mosaic virus ZYMV

Introduction 1

Abstract Good quality seeds must be genetically and physically pure, healthy vigorous and high in germination percentage. The term ‘seed transmission’ refers to the passage of pathogen from seeds to seedlings and plants. Besides fungal and bacterial pathogens, the viruses are also established to be seed transmitted in a number of crops. The rate of seed transmission depends on the host, virus, environment, vectors and their interactions. The spread of seed-transmitted virus diseases will be rapid and irreversible if initial inoculum is high and vector flight activity is great. Seed-transmitted plant viruses are also small, submicroscopic infec- tious particles composed of a protein coat and a nucleic acid. They are prevalent in vegetable, fruit, cereal and ornamental crops and are great concern to successful crop production. The up-to-date list of seed- transmitted plant virus and viroid diseases is presented in the form of tabular form in this chapter. There are nearly 231 plant virus and viroid diseases which are reported to be seed transmitted from different parts of the world. Seed transmission of nearly 68 viruses is more common in leguminous species than in any other species of crop plants. Seed transmission of plant viruses plays a pivotal role in the spread and survival of a number of important plant viral and viroid diseases. Viroid diseases are effectively transmitted vertically through pollen and ovule to the seed and seedlings. Attempts are also made to list out the virus diseases, which are erroneously listed as seed-transmitted viruses in the earlier days, which in the future are not to be considered as seed- transmitted viruses.

of the growing population of the world constitutes 1.1 Introduction the first priority in the agricultural productionÐ The agricultural scenario as a basis for socio- demand equation. There are 800 million under- economic and physical development of a country nourished people in the world, and with the is undergoing rapid changes all over the world. present rate of population growth, this number Satisfying the basic hunger and nutritional needs is expected to reach 1,000 million and result into

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 1, 1 © Springer India 2013 2 1Introduction starvation deaths due to food shortage. At present seed that starts the cyclic process all over again. nearly 1.3 billion population live on less than $1 When man learnt cultivation of plants, he also a day. According to the FAO, food prices have realised that he had to save part of the seed pro- increased by over 75% since 2000 and alarming duced for sowing in the subsequent year. About increase in prices has been witnessed especially 90% of food crops grown are propagated through since 2006. In 2008, internationally the prices seed (Maude 1996). Since seed is the carrier of of rice increased by 74% and wheat by more the genetic potential for higher crop production, than double of the previous year. As a result of improved varieties of seed have been produced this sharp increase in food prices, widespread by modern selection and breeding techniques to protests and clashes were reported in several help in increasing the yield per unit area and parts of Latin America, Africa and South Asia. in turn to boost agricultural production leading According to FAO, about 14% of the 6.5 billion to green revolution. Asexually or vegetatively world population is affected by hunger. Twenty- clonally propagated crops in which no true seeds nine countries including India have ‘alarming’ are involved are also covered in this book. or ‘extremely alarming’ levels of hunger. The situation is likely to be aggravated by 2050, as the global population is expected to increase by 40%. 1.2 Seed Transmission of Viruses This desperate overpopulation situation cannot be reduced. On the other hand, there should Seeds, from the time of their inception at flower- be rapid agricultural and industrial development, ing of the parent plants till their germination and and out of the two, intensive agriculture will development into seedlings, are prone to micro- play major role. Expansion of cultivated land can bial attacks. They are known to be the most effi- increase agriculture production to some extent, cient vehicles of transport for a number of plant but high-yielding varieties and hybrids along with pathogens comprising fungi, bacteria and viruses advanced agro-production technologies will play and cause catastrophic yield and economic losses. amajorrole. Worldwide, plant pathogens cause about 12% of The tremendous increase in population in re- crop losses, weeds about 13% and insect pests cent years has given an impetus to man’s urgent about 15%. The value of crop losses due to pests quest for rapid means of increasing agricultural is estimated to be more than $1 1012 per year. production. This will mean multiple cropping and Probably developing countries suffer the most increasing yields per unit land. Among the major from crop attacks by plant pathogens. It is re- factors which influence agricultural productivity, ported that plant pathogens cause several billions seed has a place of prime importance. of dollars in crop losses each year (http://apsnet. org). In intensive agricultural systems, constant coordinated efforts are required to control the 1.1.1 Seed (Sexual Propagule) diseases to ensure higher food production and provide raw material to agro-based industries. Seed is a productive propagule to perpetuate the Among the plant diseases, virus diseases are species that germinates to produce a new plant. prevalent in cultivated plants in highly intensive It is a fertilised mature ovule that possesses an agricultural systems as well as under more tra- embryonic plant, stored material (sometimes ab- ditional farming conditions. The most important sent) and a protective coat or coats. Most species effects of seed transmission of plant viruses are as are adapted to environments that allow the seed follows: (1) direct and/or indirect injury as even to desiccate and survive adverse environmental a low incidence of infected seeds sown results in conditions in which the species normally cannot numerous randomly scattered foci of inoculum, grow. When the environmental conditions are facilitating early secondary spread in the crop congenial for plant growth, the seeds germinate, through insect vectors; (2) survival of viral in- differentiate, grow and set flower, followed by oculum from one crop season to the next; and 1.2 Seed Transmission of Viruses 3

Fig. 1.1 Symptomatology of some seed-borne virus diseases

(3) several viruses and viroid diseases have been which may be enveloped in a few cases by a lipid and undoubtedly still are disseminated worldwide envelope as in Tomato spotted wilt virus (TSWV). through exchange of seeds having undetected The genome may be a single- or double-stranded infection (Fig. 1.1). or a segmented form enclosed in a single coat Viruses are defined as ultramicroscopic, fil- protein or occur in two or three particles. They terable and pathogenic entities which multiply may be rigid rod-shaped or flexuous filamentous in living cells, using host components and in- particles or icosahedral particles. duce diseases in higher animals, plants, bacteria, Seed transmission plays a pivotal role in the phytoplasmas, spiroplasmas, insects, fungi and spread and survival of a number of important algal organisms (Agrios 2005; Khan and Dijkstra plant viral and viroid diseases. Infected seed is 2002; Nayudu 2008). They have ss or ds RNA probably the most important source of viruses or DNA not both, enclosed in a protein coat and subviral pathogens in commercial plantings. 4 1Introduction

In fields, the seedlings raised from the randomly seed transmission of Cucumber mosaic virus dispersed infected seeds serve as initial sources (CMV) in wild cucumber (Echinocystis lobata) of virus inoculum or foci of infection from which was reported by Doolittle and Gilbert (1919), secondary spread occurs within and outside the Lettuce mosaic virus in lettuce by Newhall field by suitable vectors. Certain viruses like (1923)andSoybean mosaic virus in soybean Lettuce mosaic (LMV) and Bean common mosaic by Kendrick and Gardner (1924). Later the (BCMV) have the least number of alternate hosts, BCMV transmission through seed was reported their principal crop hosts do not overwinter and by several workers (Burkholder and Muller seed transmission is one of the chief ways of virus 1926;Merkel1929; Pierce and Hungerford survival from one season to another. Besides 1929). Regarding listing of seed-transmitted being a source of inoculum, the seed also helps plant viruses as early as 1951, eight seed- in perpetuation of the virus over long periods. For transmitted viruses were reported by Smith example, BCMV in French bean seed and Prunus (1951), and 6 years later around 20 seed- necrotic ring spot virus in Prunus pensylvanica transmitted viruses were reported in 40 species seed persists for about 38 and 6 years, respec- of various plants by Crowley (1957a, b). Thirty- tively (Walters 1962a; Fulton 1964). Many coun- six seed-transmitted viruses in 63 plant species tries import plant germplasm to diversify the ge- were recorded by Fulton (1964), later Bennett netic base of crop to improve yields and raise the (1969) reported 47 and Phatak (1974) listed 85. levels of disease resistance and other economic Agarwal and Sinclair (1988) listed 156 viruses and agronomic characteristics. But due to indis- to be transmitted through seed of several plant criminate international exchange of germplasm, species. Neergaard’s (1977) and Agarwal and areas hitherto free of certain pathogens now have Sinclair’s (1987) voluminous compilations and new virus and virus-like diseases. There are num- other reviews by Crowley (1957a), Fulton (1964), ber of established reports from many countries Baker and Smith (1966), Kunze (1968), Bennett that some new virus and virus strains are be- (1969), Baker (1972), Shepherd (1972), Phatak ing introduced along with the germplasm/food (1974, 1980), Bos (1977), Richardson (1979, grains when imported for research/consumption 1981, 1983), Mandahar (1981, 1985), Quiot purposes, and the examples are fruit tree, peanut, et al. (1982), Mishra et al. (1984), Stace-Smith pea and bean viruses (Chalam and Khetarpal and Hamilton (1988), Bos et al. (1988), Frison 2008). et al. (1990), Rishi and Nain (1992), Sastry et al. (1992), Mink (1993), Johansen et al. (1994), Gaur et al. (1996), Maude (1996), Maule and Wang (1996), Sutic et al. (1999), Hull (2002), Power 1.2.1 History of Seed-Transmitted and Flecker (2003) and Albrechtsen (2006) Plant Virus Research provided a great deal of information on seed- transmitted viruses in terms of identification, The first seed transmission of Tobacco mosaic ecology, epidemiology and management aspects. virus through tomato seed was suspected by During 1988, Stace-Smith and Hamilton have Westerdijk in 1910 and later by Allard (1914). As stated that about 18% of the described plant early as 1915, Soybean mosaic virus was reported viruses are seed transmitted in one or more hosts. in the annual report of Connecticut Agricultural Later Mink (1993) has listed 108 viruses (exclud- Experiment Station (Clinton 1916). Subsequently ing Partitiviridae) and 7 viroid diseases to be seed seed transmission of virus disease of Lima bean transmitted in one or more hosts. Hull (2002) mosaic was studied by Mc Clintock (1917). reported that one seventh of the known plant Further, instances of transmission of Bean viruses are transmitted through the seed. Dur- common mosaic virus (BCMV) through bean ing 2006, Albrechtsen from Danish seed health seed were reported (Stewart and Reddick 1917; centre for developing countries, Denmark, has Reddick and Stewart 1918, 1919). Similarly, listed seed-transmitted 113 conventional viruses, 1.4 Seed Transmission of Viroids 5

31 cryptoviruses and 12 viroids. During 2003, Carrot temperate virus 1, 2, 3 and 4; Cucumber Power and Flecker have reported 131 viruses to cryptic virus; Fescue cryptic virus; Hop trefoil be carried through seed. Since then, the number cryptic 1, 2 and 3; Red clover cryptic virus 2; of seed-transmitted viruses has phenomenally in- Spinach temperate cryptic virus; Vicia cryptic creased year after year, and presently more than virus; White clover cryptic virus 1, 2 and 3; and 231 viruses are reported to be transmitted through Ryegrass cryptic virus (Boccardo et al. 1983, seed in different cultivated and weed hosts. An 1987; Milne and Natsuaki 1995; Albrechtsen updated list of seed-transmitted virus diseases 2006). is furnished in Table 1.2. Descriptions and lists of VIDE database edited by Brunt et al. (1990, 1996) and CMI/AAB descriptions and also the 1.4 Seed Transmission of Viroids virus diseases of plants on CD by Barnett and Sherwood (2009) will provide more information Viroids are independently replicating circular on seed-transmitted plant viruses. However, an RNAs capable of causing diseases in infected attempt is made in this book by collecting the plants. They consist of naked RNA which information on seed-transmitted viruses from Re- does not code for any protein nor is protein view of Plant Pathology and CAB abstracts and associated with it and replicate independent of other information sources including VIDE/ICTV, any associated plant viruses. The two families of and the list is prepared and presented in the viroids are the Pospiviroidae and Avsunviroidae form of Table 1.2. Approximately, out of the with five and two genera, respectively. Viroid total 1,500 plant viruses reported on different diseases are effectively transmitted through seed crops and weed plants, nearly 231 virus and and pollen (Mink 1993;Diener1999;Hadidi viroid diseases are found to be seed transmit- et al. 2003; Flores et al. 2005; Hammond and ted. In Table 1.2, some synonyms or strains of Owens 2006). For the first time, Diener (1971) seed-transmitted viruses are also listed as the reported that potato spindle tuber disease was author has included all the publications on seed- induced by small, unencapsidated molecules transmitted plant viruses without having any dis- of autonomously replicating circular RNA, and crimination. Future studies by taking many char- the term viroid has been adopted. Some of the acters including molecular detection techniques properties of viroids that differentiates them from may reveal authentic data on seed-transmitted the viruses are as follows: (1) the pathogen exists viruses. in vivo as an unencapsidated RNA, that is, no virion-like particles are detectable in infected tissue; (2) the infectious RNA is of low molecular 1.3 Seed Transmission weight; (3) despite its small size, the infectious of Partitiviridae RNA is replicated autonomously in susceptible cells, that is, no helper virus is required; and (4) Partitiviridae (cryptoviruses) are unique in nature the infectious RNA consists of one molecule and do not induce any type of symptoms in in- only. Viroid species are clustered into the fected plants. Cryptoviruses are transmitted only families Pospiviroidae and Avsunviroidae, whose through seed and pollen and consist of a double- members replicate (and accumulate) in the stranded RNA genome encapsidated in protein, nucleus and chloroplast, respectively. Out of the without lipid envelope (Fraser 1989). The virions 28 viroid diseases known, 10 are reported to be are small and isometric and no vectors were iden- seed transmitted. For more details about viroid tified. Cryptoviruses as per ICTV report classified diseases, refer to Hadidi et al. (2003). in the family Partitiviridae (Boccardo et al. 1983, Viroid diseases are effectively transmitted 1987; Milne and Natsuaki 1995). The examples vertically through pollen and ovule to the seed are Alfalfa cryptic virus 1 and 2; Beet cryptic and seedling. Some of the viroid diseases have virus 1, 2 and 3; Carnation cryptic virus 1 and 2; now been shown to be transmitted through seed, 6 1Introduction namely, Apple scar skin viroid, Apple dapple and Holdeman 1965) and as high as 80Ð100% viroid, Chrysanthemum stunt viroid, Coleus with Pea seed-borne mosaic (PSbMV), Soybean viroid, Grapevine viroid, Hop stunt viroid and stunt, Cucumber mosaic (CMV), BCMV and Potato spindle tuber viroid. High degree of seed Red clover vein mosaic viruses (RCVMV) in transmission is reported from viroid diseases certain legume cultivars (Sander 1959;Medina of Potato spindle tuber, Cucumber pale fruit, and Grogan 1961; Stevenson and Hagedorn 1973; Avocado sunblotch and Chrysanthemum stunt. Brunt and Kenten 1973; Iizuka 1973; Takahashi et al. 1980; Truol et al. 1987; Morales and Castano 1987;Khetarpal1989). 1.4.1 Extent of Seed Transmission 1.4.1.2 Viroids 1.4.1.1 Viruses High degree of seed transmission is noticed The extent of seed transmission depends on in certain viroid diseases, namely, Australian particular hostÐvirus combination. Variable grapevine viroid; Avocado sunblotch viroid; estimates for seed transmission in a majority Chrysanthemum stunt viroid; Citrus exocortis of cases have been due to the use of different viroid; Coconut cadang-cadang viroid; Coleus cultivars of the same host species or different blumei viroid 1,2 and 3; Grapevine yellow speckle strains of the same virus. Complete absence viroid 1 and 2; Hop stunt viroid; Potato spindle of seed transmission is virtually impossible to tuber viroid; Apple scar skin viroid; Apple demonstrate with certainty in some virusÐhost dapple viroid;andTomato apical stunt viroid combinations. Lack of seed transmission in (Mink 1993; Pacumbaba et al. 1994;Hadidi a small sample does not preclude its possible et al. 2003; Albrechtsen 2006; Antignus et al. transmission. Hence, negative results can at best 2007), and various aspects of seed-transmitted be tentative or inconclusive. Seed transmission of viroid diseases were discussed in the relevant viruses predominates in some plant families and chapters. is rare in others. Seed transmission of nearly 68 viruses is more common in leguminous 1.4.1.3 Suspected Seed Transmission species than in any other species of crop plants of Phytoplasma Diseases (Table 1.2). Some viruses with nonlegume During Khan et al. (2002), have reported the principal hosts are also seed transmitted in transmission of phytoplasma in both seed and legumes. seedling progeny of alfalfa plants affected by In Rosaceae family, there is an affinity witches’ broom disease, indicating the seed between a group of hosts and seed-transmitted transmission in certain plant hostÐphytoplasma viruses. In Prunus avium, P. cerasus, P. pathosystem. Necas et al. (2008) have reported mahaleb, P. pensylvanica and P. persica,the the preliminary information of possibility of seed transmission of Prunus necrotic ring spot European stone fruit yellows phytoplasma (PNRSV) was most common (Millikan 1959; (ESFY) transmission through apricot seeds. Wagnon et al. 1960; Megahed and Moore 1967). Seeds from ESFY-infected trees showed very low As a group, greater seed transmission of viruses viability (21.6%) and practically no germination is seen with which may show up to activity. Earlier Cordova et al. (2003)have 100% (Athow and Bancroft 1959;Murantand observed the presence of phytoplasma DNA Lister 1967; Allen et al. 1970;Hansenetal. in coconut embryos by PCR studies, raising 1974). Tobraviruses in general have a low rate the possibility of seed transmission of lethal of transmission (1Ð6%) except Dutch isolate yellowing disease of coconut palms. These of Pea early browning virus, PEBV (Fiedorow research reports warrant further research on the 1983). Among the aphid-borne seed-transmitted role of seed transmission in the epidemiology of viruses, it is as low as 0.1Ð0.4% in corn infected phytoplasma diseases, and the observations cited with Sugarcane mosaic virus (SCMV) (Shepherd here requires further study. 1.5 Viruses Erroneously Listed as Seed Transmitted 7

Bove et al. (1988) stated in their review ar- for number of times over periods of up to 3 years. ticle that the occurrence of stubborn symptoms No symptoms typical of huanglongbing, such as on citrus seedling trees or plants was taken as blotchy leaf mottle, chlorotic shoots or dieback evidence for natural spread of the disease because of branches, were observed in these seedlings, Spiroplasma citri has never been found to be and none of these 723 seedlings tested positive transmitted vertically through seeds, even though for the presence of ‘Ca. L. asiaticus’, even after the seed coats from infected citrus trees can carry repeated testing by sensitive quantitative PCR S. citri. The aetiological agent, Spiroplasma,in assays. Some sour orange seedlings did have citrus stubborn and corn stunt diseases is sus- quite pronounced and atypical growth, including pected to be seed transmitted, but confirmation is stunting and mild to severe leaf malformation. warranted. These atypical growth habits were limited to seedlings that arose from zygotic embryos as 1.4.1.4 Lack of Evidence for determined by expressed sequence tag simple Transmission of Citrus sequence repeat analyses. Thus, no evidence of Huanglongbing transmission of ‘Ca. L. asiaticus’ through citrus Lot of controversy existed regarding the aeti- seed was obtained by Hartung et al. (2010), and ology of citrus greening disease. As early as an earlier report of transmission of the pathogen Ghosh et al. (1971) have reported the associ- through seed was not confirmed. ation of phytoplasma with citrus greening. In the subsequent researches based on electron mi- croscopy (Garnier and Bove 1983) and PCR stud- 1.5 Viruses Erroneously Listed as ies (Jagoueix et al. 1994), the presence of bac- Seed Transmitted terium Candidatus Liberibacter asiaticus (Ca.L. asiaticus) was observed and reported to be the Nearly 13 review articles with comprehensive aetiological agent of Citrus greening which was lists of seed-transmitted plant virus and viroid later named as huanglongbing (HLB). Transmis- diseases were published since 1951, and each sion of ‘Ca. L. asiaticus’ through infected citrus reviewer had added a substantial number of seed has been reported (Tirtawidjaja 1981) based names to the lists of their predecessors. The book on the rapid appearance of HLB-like symptoms entitled Testing methods for seed-transmitted in seedlings when apparently healthy seed har- viruses: Principles and Protocols by Albrechtsen vested from symptomatic fruit was sown. ‘Ca. is the latest and has covered various aspects L. asiaticus’ was also readily detected throughout of seed-transmitted viruses. Perusal of the HLB-affected fruit by quantitative qPCR (Tate- review articles of Mandahar (1981), Agarwal neni et al. 2008;Lietal.2009) and in the seed and Sinclair (1987), Mink (1993), Niazi et al. coat but not the embryos of limited numbers of 2007 and others reveal that at least 14 virus seed collected from HLB-affected fruit (Tateneni or virus disease names are erroneously listed et al. 2008). as seed transmitted and has been furnished in Citrus germplasm is often exchanged as seed, Table 1.1.Mink(1993) has furnished details of and thus, a pathway for the potential introduction the 14 erroneously listed seed-transmitted viruses of huanglongbing (HLB) into previously HLB- in his review article. free regions would exist, if the pathogen is Of the 14 virus disease names considered seed transmitted. Hence, intensive researches by Mink (1993), four have been perpetuated were carried out by Hartung et al. (2010)to in the literature as seed transmitted on the provide evidence as to whether or not ‘Ca. basis of erroneous reports or erroneously cited L. asiaticus’ can be transmitted through seed. reports: ACLSV, Barley mottle mosaic,CaMV They have grown various citrus species from and TuMV. These should be eliminated from the seed collected from symptomatic ‘Ca.L. future lists of seed-transmitted viruses. Four other asiaticus’, and the seedlings were tested by PCR viruses should also be removed from lists of seed- 8 1Introduction

Table 1.1 Virus or disease names erroneously listed as seed transmitted in earlier reviews Virus listed as Disease name and virus Seed transmitted Seed borne Comment Abutilon mosaic Yes Yes Geminivirus Apple chlorotic leaf spot Yes Yes Misidentification Barley mottle mosaic Yes No No such name listed Barley yellow dwarf Yes No Luteovirus Bean yellow dwarf No Yes Luteovirus Beet western yellows No No Luteovirus Carrot motley dwarf No Yes Not seed transmitted Carrot red leaf Yes Yes Luteovirus Cauliflower mosaic Yes No Not seed transmitted Cherry necrotic rusty mottle Yes Yes Not seed transmitted Citrus psorosis Yes Yes Misidentification Potato leaf roll No Yes Luteovirus Sugarcane mosaic Yes Yes Not seed transmitted Turnip mosaic Yes Yes Not seed transmitted Source: Mink (1993) transmitted viruses because of nomenclatural seed-transmitted viruses in previous reviews that problems: Abutilon mosaic virus, Beet curly occurred only once, or very few times, in the top virus, Citrus psorosis virus and Sugarcane literature. Plant virus literature searches have mosaic virus. Six of the names in Table 1.1 failed to provide much additional information are either luteoviruses or associated with a regarding most of the causal agents. Usually, luteovirus. In each case (except bean yellow transmission through seed was demonstrated dwarf), reports claim transmission through by the occurrence of symptomatic seedlings. In seed based on the occurrence of symptomatic some cases, the causal agents were demonstrated seedlings. These reports have not been taken to be viruses, which were at least partially seriously, either by many reviewers or the ICTV. characterised in the original reports although Credibility of any future reports of luteovirus their relationships to other viruses were not seed transmission will be enhanced by a clearly established (Mink 1993). Forty-one of the demonstration that virus can be transmitted from 46 names were reported prior to 1980 and some seedlings (whether symptomatic or not) using of these names may represent newly recognised aphid vectors. The occurrence of symptomatic seed-transmitted viruses that have received little but apparently noninfected seedlings for several attention since the initial report. However, most luteoviruses, CNRMV and the causal agent of were more likely applied to diseases that were Abutilon variegation represents a phenomenon eventually found to be caused by commonly that deserves additional study. recognised viruses, and the disease names were well documented in the review article of Mink (1993). Although many agents are indubitably 1.5.1 Seed-Transmitted Plant Virus seed transmitted, Mink (1993) has listed their Names That Appeared Only names in a separate table in his article to call Once in the Literature attention to their plight. Specialists working with one or more of these agents may be able to Excellent exercise has been done by Mink provide up-to-date information regarding their (1993) in his review article while listing the identity. seed-transmitted plant viruses. Forty-six disease Mink (1993) and also other reviewers of seed- and virus names were found among the lists of transmitted viruses have complied the available 1.5 Viruses Erroneously Listed as Seed Transmitted 9 literature in the form of tables. Primarily Mink TSV isolate, and comparison has to be made with (1993) has observed many names of seed Ð trans- seed-transmissible strain of TSV from different mitted viruses that appeared on earlier lists are countries as Walter et al. (1995) observed extra synonyms of other viruses. Because of these minor RNAs in non-seed-transmissible strain synonyms, a list of 50 such names that have often (MelF) of TSV. So, differentiation has to be made been cited as distinct seed-transmitted viruses has between the seed-transmissible strains of TSV been discussed thoroughly. In some cases, several with respect to their host range, basic chemical synonyms of a single virus have been used. The properties and genetic diversity. five synonyms for Prunus necrotic ring spot virus Similarly, seed transmission of Tomato spotted are an extreme example: cherry necrotic ring wilt virus in tomato and Senecio cruentus were spot, peach latent, peach necrotic leaf spot, peach reported by Jones (1944). Reports exist regarding ring spot and stone fruit ring spot. These disease seed transmission of Groundnut bud necrosis names convey no unique strain identity (Mink virus (GBNV) in peanut; however, the recent 1991). Other synonym names identified by Mink studies carried out at NBPGR and ICRISAT, Hy- (1993) are also not strain specific, and to include derabad, and elsewhere have proved that GBNV multiple synonyms on lists of seed-transmitted is not seed transmitted in peanut and other hosts. viruses creates redundancy. Rice yellow mottle virus (RYMV), genus Sobemovirus, causes severe yield losses ranging from 25 to 100% in most countries of Africa. 1.5.2 Establishing Certain Erotic Studies carried out by Konate et al. (2001) Positive Seed-Transmitted and Allarangaye et al. (2006) have established Viruses to Be Non-seed the evidence of non-transmission of RYMV Transmissible through seeds of rice and wild rice species. RYMV was infectious in freshly harvested seed In recent years, ilar- and tospoviruses which extracts, whatever the plant species or the virus cause enormous yield losses in majority of isolate. However, most infectivity was lost in economically important crops of both positive dried seeds, possibly due to virus inactivation and negative seed transmission in different crops following dehydration of the seeds. Therefore are reported. Positive seed transmission reports virus dissemination or epidemics of rice yellow of (Tobacco streak virus) are reported mottle do not originate from infected seeds. in soybean by Ghanekar and Schwenk (1974), Similarly, Beet curly top virus transmitted by Kaiser et al. (1982), Truol et al. (1987)andJain Cicadellidae vector Circulifer tenellus was seed et al. 2006; in tomato by Sdoodee and Teakle transmitted in to the extent of (1988); in beans by Kaiser et al. (1991); and in 11Ð25% as reported by Abdel-Salam and Amin Parthenium hysterophorus by Sharman et al. (1990) requires confirmation. (2009). Extensive seed transmission studies Although plant virus and viroid diseases infect by Prasada Rao et al. (2003, 2009), Reddy number of hosts belonging to different families, et al. (2007) and Vemana and Jain (2010)have either in natural or controlled conditions, seed established the negative seed transmission of transmission is noticed only in certain virusÐhost TSV in groundnut and sunflower. It was further combinations (Table 1.2). The mere presence of established by Vemana and Jain (2010)thatTSV virus in/on the seed does not always lead to is not true seed transmitted in V. mungo, V. seedling infection. Some viruses may be confined radiata, G. max.andDolichos lablab.Sofar only on seed coat or in cotyledons or in embryos. TSV seed transmission was not proved from Some viruses are present before seed matura- any crop or weed hosts in India. Hence, it is tion and drying, whereas certain viruses will get concluded that non-seed-transmissible strain eliminated with seed maturation and drying. The of TSV may be existing in India. Moreover, reasons for positive or negative seed transmission complete genome sequence is lacking for Indian are discussed in Chap. 5. 10 1Introduction

Table 1.2 Per cent transmission of virus and viroid diseases through seeds in different hosts Virus/viroid Host Per cent References Alfalfa mosaic (Syn. Berseem mosaic) Amaranthus albus 1.9Ð15.5 Kaiser and Hannan (1983) Capsicum annuum 1Ð5 Sutic (1959) Cicer arietinum Ð Jones and Coutts (1996) Lathyrus cicera 2 Latham and Jones (2001a) Lathyrus sativus 0.9Ð4 Latham and Jones (2001a) Lens culinaris Ð Jones and Coutts (1996); Latham et al. (2004) Lupinus angustifolius 0.8 Jones et al. (2008) Medicago lupulina Ð Paliwal (1982) Medicago polymorpha 80Ð100 Jones and Nicholas (1992) Medicago sativa 6 Belli (1962) M. sativa 55 Zschau and Janke (1962) M. sativa 1Ð4 Frosheiser (1964); Jones (2004a, b) M. sativa 0.2Ð6 Frosheiser (1970) M. sativa 0.6Ð17 Beczner and Manninger (1975); Tosic and Pesic (1975); Hemmati and McLean (1977) M. sativa 4 Ekbote and Mali (1978) M. sativa 10.6 Pesic and Hiruki (1986) M. sativa 0.3Ð74 Jones and Pathipanawat (1989); Pathipanawat et al. (1995, 1997); Jones (2004a, b) M. sativa 3.5Ð6 Avegelis and Katis (1989a, b) Medicago scutellata Ð Paliwal (1982) Nicandra physalodes 23 Gallo and Ciampor (1977) Petunia violacea 30.3 Phatak (1974) 0.7Ð4.9 Kaiser and Hannan (1983) Trifolium alexandrinum 60Ð70 Mishra et al. (1980); Fugro and Mishra (1996) Trifolium michelianum 0.05 Latham and Jones (2001b) Trigonella balansae 7 Latham and Jones (2001b) Vicia sativa 0.04Ð0.7 Latham and Jones (2001b); Latham et al. (2004) Alfalfa cryptic Medicago sativa High Boccardo et al. (1983); Natsuaki et al. (1986) Alfalfa temperate M. sativa High Natsuaki et al. (1984, 1986) Apple chlorotic leaf spot Rubus spp. 30Ð40 Cadman (1965); Converse (1967) Apple dapple viroid Prunus americana Ð Hadidi et al. (1991) Apple mosaic Prunus amygdalus ÐBarba(1986) Vigna unguiculata 2 Gottlieb and Berbee (1973) Apple scar skin viroid Prunus americana Ð Hadidi et al. (1991) Apricot gummosis Prunus americana Ð Fridlund (1966) P. avium 15 Fridlund (1966) P. domestica Ð Traylor et al. (1963) P. serrulata Ð Fridlund (1966) Arabis mosaic Beta vulgaris 13 Lister and Murant (1967) Capsella bursa-pastoris 6Ð33 Lister and Murant 1967 80 Lister and Murant 1967 Fragaria x ananassa 6.9 Lister and Murant (1967) Glycine max 6.3 Lister (1960) Humulus spp.10Kriz(1959) Lactuca sativa 60Ð100 Walkey (1967) Lamium amplexicaule 1.2Ð25 Lister and Murant (1967) Lycopersicon esculentum 1.8 Lister and Murant (1967) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Myosotis arvensis 19Ð95 Lister and Murant (1967) 35 Tomlinson and Walkey (1967) Petunia hybrida 20 Lister (1960) P. violacea 20Ð37 Phatak (1974) Plantago major 5.4Ð28 Lister and Murant (1967) Poa annua 4 Taylor and Thomas (1968) Polygonum persicaria 21Ð100 Lister and Murant (1967) Rheum rhaponticum 10Ð24 Tomlinson and Walkey (1967) Rosa rugosa Ð Thomas (1981) Senecio vulgaris 2.2 Lister and Murant (1967) Stellaria media 57 Lister and Murant (1967) Arracacha virus A Nicotiana clevelandii Ð Jones and Kenten (1980) Arracacha virus B Solanum tuberosum 4Ð12 Jones (1982); Jones and Kenten (1981) C. 4Ð12 Kenten and Jones (1979); Jones and Kenten (1981); Jones (1982) Artichoke Italian latent Cynara scolymus ÐVide(1996); Bottalico et al. (2002); Albrechtsen (2006) Artichoke latent Cynara scolymus 5Ð10 Bottalico et al. (2002) Artichoke yellow ring spot Cynara scolymus High Jones (1979); Kyriakopoulou et al. (1985) 9Ð33 Avegelis et al. (1992) Asparagus bean mosaic Vigna sesquipedalis 35 Snyder (1942) Asparagus latent (Syn. Asparagus officinalis 65 Paludan (1964) Asparagus virus II) A. officinalis 0.5 Uyeda and Mink (1981); Fujisawa et al. (1983); Falloon et al. (1986) A. officinalis 3Ð27 Bertaccini et al. (1990) Petunia hybrida Ð Fujisawa et al. (1983) Ð Fujisawa et al. (1983) Asparagus virus I A. officinalis 18Ð53 Bertaccini et al. (1990) Australian Lucerne latent Medicago sativa Ð Jones et al. (1979) Avocado sunblotch Persea americana 76 Wallace and Drake (1953), (1962) P. americana 86Ð100 Thomas and Mohamed (1979) Avocado viruses 1,2,3P. americana High Jordan et al. (1983) Banana streak Musa spp. Ð Daniells et al. (1995) Banana viruses Musa acuminate High Gold (1972) M. balbisiana High Gold (1972) Barley mottle mosaic Hordeum vulgare 2Ð45 Dhanraj and Raychaudhuri (1969) Barley stripe mosaic (Syn. Aegilops spp. Ð Nitzany and Gerechter (1962) Barley false stripe) Agropyron elongatum Ð Inouye (1962) Avena fatua 22 Chiko (1975) A. sativa 0Ð9.5 Mckinney and Greely (1965) Avena spp. Ð Nitzany and Gerechter (1962) Bromus inermis 8 Inouye (1962) Bromus spp. Ð Nitzany and Gerechter (1962) Commelina communis 4 Inouye (1962) Hordeum depressum 3 Inouye (1962) H. glaucum 2 Inouye (1962) H. glaucum 58 McKinney (1951a, b) H. glaucum Up to 90 McKinney (1953) H. glaucum 50Ð100 Gold et al. (1954) H. glaucum 4Ð64 Eslick and Afanasiev (1955) H. glaucum 38Ð86 Inouye (1962) H. glaucum 3Ð53 Mckinney and Greely (1965) H. vulgare 55Ð75 Phatak and Summannwar (1967) H. vulgare 38 Catherall (1972) (continued) 12 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References H. vulgare 5Ð60 Phatak (1974) H. vulgare 38Ð45 Slack et al. (1975) H. vulgare 61Ð70 Carroll and Mayhew (1976a, b) H. vulgare 52 Lange et al. (1983) H. vulgare 45 Makkouk et al. (1992) Lolium spp. 3Ð8 Inouye (1962) Poa exilis Ð Nitzany and Gerechter (1962) Triticum aestivum 71 Hagborg (1954) T. aestivum 6.7Ð80 McNeal and Afanasiev (1955) T. aestivum 70 Lange et al. (1983) Bean common mosaic Cyamopsis tetragonoloba 94 Gillaspie et al. (1998a, b) (Syn. Bean western mosaic, Lupinus luteus 16 Frencel and Pospieszny (1979) Syn. Azuki bean mosaic) Macroptilium lathyroides 5Ð33 Kaiser and Mossahebi (1974); Provvidenti and Braverman (1976) Phaseolus aborigineus 6.7 Klein et al. (1988) Phaseolus acutifolius var. latifolius 7Ð34 Lockhart and Fischer (1974) P. acutifolius var. latifolius 7Ð20 Provvidenti and Cobb (1975) P. angustifolius 1.0 Klein et al. (1988) P. mungo ÐNene(1972); Agarwal et al. (1977, 1979) P. vulgaris 2Ð3 Scotland and Burke (1961) P. vulgaris 50 Reddick and Stewart (1919) P. vulgaris 43 Archibald (1921) P. vulgaris 10Ð25 Kendrick and Gardner (1924) P. vulgaris 50 Burkholder and Muller (1926) P. vulgaris 21Ð51 Merkel (1929) P. vulgaris 10Ð30 Fazardo (1930) P. vulgaris 20Ð60 Harrison (1935) P. vulgaris 2Ð66 Smith and Hewitt (1938) P. vulgaris 10Ð86 Medina and Grogan (1961) P. vulgaris 2Ð3 Skotland and Burke (1961) P. vulgaris 5Ð33 Ordosgoitty (1972) P. vulgaris 7Ð20 Phatak (1974) P. vulgaris 20Ð80 Muniyappa (1976); Drijfhout and Bos (1977) P. vulgaris Ð Uyemoto and Grogan (1977) P. vulgaris 12Ð66 Capoor et al. (1986) P. vulgaris 1.3 Tsuchizaki et al. (1986a, b) P. vulgaris 93 Edwardson and Christie (1986) P. vulgaris 39.7Ð54.4 Morales and Castano (1987) Phaseolus vulgaris 1Ð18 Puttaraju et al. (1999) P. vulgaris 12.4 Njau and Lyimo (2000) P. vulgaris 39 Nalini et al. (2004, 2006a, b); Suteri (2007) Vigna angularis Ð Tsuchizaki et al. (1970a, b); Hampton et al. (1978) Vigna mungo 2Ð10 Agarwal et al. (1979) Vigna mungo 20Ð48 Provvidenti (1986) Vigna mungo 67 Dinesh et al. (2007) Vigna radiata 25 Kaiser et al. (1968) Vigna radiata 4.1Ð7.2 Tsuchizaki et al. (1986a, b) Vigna radiata 8Ð32 Kaiser and Mossahebi (1974) Vigna radiata 1Ð4.9 Choi et al. (2006) Vigna radiata 78 Dinesh et al. (2007) Vigna sesquipedalis 37 Snyder (1942) V. sinensis 25Ð40 Sachchidananda et al. (1973) Vigna unguiculata 0.8Ð12.4 Hao et al. (2003) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Bean common mosaic necrosis Phaseolus vulgaris 36.6 Njau and Lyimo (2000) Bean pod mottle Glycine max 0.1 Lin and Hill (1983) Bean southern mosaic (Syn. Phaseolus vulgaris 1Ð30 Crowley (1959); Smith (1972); Southern bean mosaic) Jayasinghe (1982); Morales and Castano (1985) Vigna sinensis 1Ð40 Shepherd and Fulton (1962); Givord (1981); O’Hair et al. (1981) V. unguiculata 1Ð3 Shepherd and Fulton (1962); Lamptey and Hamilton (1974) Bean yellow mosaic Lens culinaris Ð Bayaa et al. (1998) Lens culinaris ÐKumarietal.(1994); Kumari and Makkouk (1995) Lens esculenta Ð Erdiller and Akbas (1996) Lupinus albus Ð Blaszczak (1963, 1965) L. luteus 5Ð6.2 Mastenbroek (1942); Corbett (1958); Zschau (1962) L. luteus 7Ð21 Porembskaya (1964) Melilotus alba 3Ð5 Phatak (1974) Phaseolus vulgaris 7Crowley(1957a, b) Phaseolus radiata Ð Benigno and Favali-Hedayat (1977) P. vulgaris ÐHino(1962) Pisum sativum 10Ð30 Inouye (1967) Trifolium pratense 12Ð15 Hampton (1967) Vicia faba Low Quantz (1954a, b); Bos (1970) V. faba 0.1Ð2.4 Kaiser (1972a, b, 1973); Evans (1973) V. faba 0.1Ð0.2 Fiedorow (1980) V. faba 1.8 Eppler and Kheder (1988) V. faba 2.6 Aftab et al. (1989) V. faba 9.8 El-Dougdoug et al. (1999) V. radiata Ð Benigno and Favali-Hedayat (1977) Vigna sinensis 1Ð37 Snyder (1942) Beet 1 alpha crypto (Syn. Beet temperate) Beta vulgaris 100 Natsuaki et al. (1983a, b, 1986) Beet 2 alpha crypto Beta vulgaris 100 Kassanis et al. (1977, 1978); White and Woods (1978); Accotto and Boccardo (1986); Natsuaki et al. (1986); Osmond et al. (1988) Beet 3 alpha crypto Beta vulgaris Low VIDE (1996) Beet mild yellowing Beta vulgaris Ð Fritzsche et al. (1986) Beet 41 yellows Beta vulgaris 47 Clinch et al. (1948) Blackgram mild mottle Vigna mungo 4.9Ð14.9 Krishnareddy (1989) Blackgram mottle Vigna radiata 8Phatak(1974, 1983); Scott and Phatak (1979) Vigna radiata 1Ð1.5 Saleh et al. (1986) Vigna mungo 5.3Ð16.70 Krishnareddy (1989) Vigna mungo 1.3Ð15.9 Varma et al. (1992) Vigna mungo 5Ð10 Dinesh Chand et al. (2004) Blackeye cowpea mosaic Vigna sinensis 30.9 Anderson (1957); Zettler and Evans (1972) V. sinensis 1.8 Lin et al. (1981a, b); Tsuchizaki et al. (1984) Vigna mungo 14 Provvidenti (1986) Vigna sinensis 1.2Ð30.9 Edwardson and Christie (1986) (continued) 14 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References Vigna sinensis 2.3Ð38.4 Sumana and Keshava Murthy (1992) Vigna unguiculata 6Ð41.6 Pio-Ribeiro et al. (1978); Mali and Kulthe (1980) Mali et al. (1987, 1988, 1989) Bashir and Hampton (1996a, b); Puttaraju et al. (2000), (2004a, b); Shilpashree (2006) Black raspberry latent Rubus occidentalis 10 Lister and Converse (1972) Chenopodium quinoa 12 Uyemoto et al. (1977) Vaccinium corymbosum 29 Ramsdell and Stace-Smith (1983) Vitis labrusca 5 Uyemoto et al. (1977) Bramble yellow mosaic Rubus rigidus Ð Engelbrecht (1963); Vide (1996) Brinjal ring mosaic S. melongena 6.5 Sharma (1969) Brinjal severe mosaic S. melongena 10.1 Sharma (1969) Broad bean mild mosaic Vicia faba Ð Devergne and Cousin (1966) Broad bean true mosaic Vicia faba 0Ð28 Brunt (1970); Neergaard (1977); Mali et al. (2003) Broad bean mottle Cicer spp. Ð Erdiller and Akbas (1996) Phaseolus vulgaris 7Phatak(1974) Vicia faba 1.37 Makkouk et al. (1988) Broad bean stain Lens culinaris 2.1 AlÐMabrouk and Mansour (1998) Lens esculenta Ð Erdiller and Akbas (1996); Bayaa et al. (1998) Pisum sativum Ð Kowalska and Beczner (1980) Vicia faba 1 Lloyd et al. (1965) V. faba 1 Varma and Gibbs (1967) V. faba 10 Gibbs and Smith (1970) V. faba 5 Moghal and Francki (1974) V. faba 7.3 Cockbain et al. (1976) V. faba 4Ð16 Vorra-urai and Cockbain (1977) V. faba 3.2 Vorra-urai and Cockbain (1977) V. faba (minor) 0.06Ð2.7 Jones (1978) V. faba 6.7 Eppler and Kheder (1988) V. faba 16.0 El-Dougdoug et al. (1999) V. faba 3Ð20 Mali et al. (2003) V. faba 6.7Ð15.4 El-Kewey et al. (2007) Vicia palastina Ð Makkouk et al. (1987) Broad bean true mosaic Vicia faba 28 Mali et al. (2003) Broad bean wilt Vicia faba 0.6 Putz and Kuszala (1973); Makkouk et al. (1990) Brome mosaic Triticum aestivum > 50 Von Wechmar et al. (1984) Cacao necrosis Phaseolus vulgaris 1Ð24 Kenten (1972) Phaseolus lunatus ÐKenten(1977); Vide (1996) Glycine max ÐKenten(1977); Vide (1996) Cacao swollen shoot 34Ð54 Quainoo et al. (2008) Carlavirus Arachis hypogaea Low Sivaprasad et al. (1990) Carrot motley leaf ÐScott(1972) Carrot red leaf Daucus carota 25 Watson and Serjeant (1962) Carrot temperate 1 Daucus carota Ð Natsuaki et al. (1983a, b) Carrot temperate 2 Daucus carota Ð Natsuaki et al. (1983a, b); VIDE (1996) Carrot temperate 3 Daucus carota Ð Natsuaki et al. (1983a, b); VIDE (1996) Carrot temperate 4 Daucus carota Ð Natsuaki et al. (1983a, b); VIDE (1996) Cassia yellow spot (poty) Cassia occidentalis Ð Souto (1990); VIDE (1996) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Cauliflower mosaic Capsella bursa-pastoris Ð Tomilson and Walker (1973) Raphanus raphanistrum Ð Tomilson and Walker (1973) latent Apium graveolens 34 Bos (1973); Bos et al. (1978) Amaranthus caudatus Ð Bos et al. (1978) Amaranthus hybridus 0.3 Luisoni and Lisa (1969); Bos et al. (1978) Chenopodium quinoa 89 Luisoni and Lisa (1969); Bos et al. (1978) Trigonella foenumgraecum 0.5 Luisoni and Lisa (1969) Cherry leaf roll Arabidopsis thaliana Ð Artemis Rumbou et al. (2009) Betula pendula 4Ð22 Cooper (1976); Schimanski et al. (1980) Chenopodium amaranticolor 100 Lister and Murant (1967) Glycine max 100 Lister and Murant (1967) Juglans regia 4Ð32 Quacquarelli and Savino (1977); Mircetich et al. (1980) Juglans regia 52.3 Kolber et al. (1982) J. regia 6 Cooper and Edwards (1980) Nicotiana clevelandii Ð Hansen and Stace-Smith (1971) Nicotiana megalosiphon Ð Hansen and Stace-Smith (1971) 1 Schmelzer (1965, 1966) Olive spp. 41Ð90 Saponari et al. (2002) Phaseolus vulgaris 12Ð48 Lister and Murant (1967) Prunus serotina 0.5Ð0.8 Schimanski et al. (1976) Rheum rhaponticum 72 Tomlinson and Walkey (1967) Sambucus racemosa 13Ð44 Schimanski and Schmelzer (1972) Solanum acaule Ð Crosslin et al. (2010) Ulmus americana 1Ð48 Bretz (1950); Callahan (1957) Viola tricolor 1.2Ð6.1 Lister and Murant (1967) Cherry necrotic rusty mottle Prunus avium ÐNyland(1962) P. cerasus ÐNyland(1962) Cherry rasp leaf Chenopodium amaranticolor 4Ð25 Hansen et al. (1974) Prunus spp. Ð Hansen et al. (1974) Taraxacum officinale 30 Hansen et al. (1974) Cherry ring spot Prunus avium 5Ð56 Cochran (1946); Cation (1949, 1952) Chicory yellow mottle Cichorium intybus 3Vovlas(1973) Chrysanthemum stunt viroid Chrysanthemum morifolium 0Ð75.7 Monsion et al. (1973); Chung and Pak (2008) Lycopersicon esculentum Ð Kryczynski et al. (1988) Cineraria mosaic Senecio cruentus Ð Jones (1944) Citrus exocortis viroid Impatiens walleriana 66 Singh and Baranwal (2008); Singh et al. (2009) Verbena x hybrida 28 Singh and Baranwal (2008); Singh et al. (2009) Citrus leaf blotch Citrus spp. Ð Guerri et al. (2004) Citrus mosaic Citrus sinensis Ð Murthy and Subbaiah (1981) Citrus psorosis Citrus sinensis x Poncirus trifoliata 19 Childs and Johnson (1966) Citrus spp. Trace Wallace (1957) Trifoliate orange 1Ð10 Campiglia et al. (1976) Citrus tatter leaf Chenopodium quinoa Ð Inouye et al. (1979) Citrus yellow mosaic Citrus spp. 1.2 Reddy et al. (1996) Citrus veinal chlorosis Citrus spp. 70 Sawant et al. (1983) Citrus xyloporosis Citrus aurantifolia 66 Childs (1956) (continued) 16 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References Clover (red) vein mosaic Trifolium pratense Ð Matsulevich (1957) Vicia faba 100 Sander (1959) Clover (red) mosaic Trifolium pratense 12Ð18 Hampton (1967) Vicia faba Ð Sander (1959) Clover (white) mosaic Trifolium pratense 1Ð12 Hampton (1963); Hampton and Hanson (1968) Clover yellow mosaic T. pratense 7.6 Hampton (1963) Cocksfoot alphacryptovirus Ð Ð VIDE (1996) Coconut cadang-cadang viroid Cocos nucifera Ð Pacumbaba et al. (1994) Coffee ring spot Coffea excelsa 8.1 Reyes (1961) Coleus blumei viroid 1 Coleus scutellarioides 16Ð68 Singh et al. (1991a) Coleus blumei viroid 2 Coleus scutellarioides 71.4 VIDE (1996) Coleus blumei viroid 3 Coleus scutellarioides ÐVIDE(1996) Cowpea aphid-borne mosaic Arachis hypogaea 0.15 Gillaspie et al. (2001) Arachis hypogaea Ð PioÐRibeiro et al. (2000a, b) Phaseolus angularis Ð Tsuchizaki et al. (1970a, b) Vigna sesquipedalis Ð Chang and Kno (1983) V. sinensis 23 Capoor and Varma (1956) V. sinensis 0.3Ð1.6 Lovisolo and Conti (1966) V. sinensis 5Ð16 Chenulu et al. (1968) V. sinensis 35 Phatak (1974); Ramachandran and Sunmanwar (1982) V. unguiculata 27 Snyder (1942) V. unguiculata 0Ð30 Kaiser et al. (1968); Phatak (1974) V. unguiculata 0Ð2 Bock (1973) V. unguiculata 3Ð19 Phatak (1974) V. unguiculata 1.1Ð39.8 Kaiser and Mossahebi (1975); Ndiaye et al. (1993) V. unguiculata up to 20.9 Ladipo (1977) V. unguiculata 6.3Ð18.3 Ata et al. (1982); Bashir and Hampton (1996a, b) V. unguiculata 4.7 Chang and Kno (1983) V. unguiculata 5.0Ð20 Mali et al. (1987, 1988, 1989) V. unguiculata 5.9 PioÐRibeiro et al. (2000a, b) V. unguiculata 3.8Ð5.4 El-Kewey et al. (2007) V. unguiculata 0.67Ð13.49 Udayashankar et al. (2009) Cowpea green vein banding V. sinensis ÐVIDE(1996) Cowpea chlorotic spot virus V. sinensis 3Ð18 Sharma and Varma (1975a, b, 1986) Cowpea mild mottle Glycine max 90 Brunt and Kenten (1973) G. max 0.9 Iwaki et al. (1982) G. max 0.5 Thouvenel et al. (1982) Phaseolus vulgaris 6 Brunt and Kenten (1973) V. unguiculata 90 Brunt and Kenten (1973) Cowpea mosaic Vigna catjang 17 Capoor and Varma (1956) V. sinensis 17.5 Diwakar and Mali (1977) V. sinensis 23 Capoor and Varma (1956) V. sinensis 0Ð55 Anderson (1957); Haque and Chenulu (1972) V. sinensis Ð Khatri and Chohan (1972) V. sinensis 1Ð5 Gilmer et al. (1974) Vigna unguiculata 75Ð84 Mahalakshmi et al. (2008) Cowpea mottle Phaseolus vulgaris Ð Shoyinka et al. (1978) V. unguiculata 3Ð10.3 Shoyinka et al. (1978) V. unguiculata 0.4 Allen et al. (1982) Voandzeia subterranea 2BirdandCorbett(1988); Robertson (1966) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Cowpea Moroccan aphid-borne Vigna unguiculata ÐVIDE(1996) mosaic Cowpea severe mosaic Vigna sesquipedalis 8Dale(1949) Vigna sinensis 3.3Ð5.8 Haque and Persad (1975) Vigna unguiculata 10 Shepherd (1964); VIDE (1996) Cowpea severe mottle Vigna sinensis 0.7 Dos (1987) Crimson clover latent Trifolium incarnatum 97 Kenten et al. (1980a, b) Cucumber green mottle mosaic Citrullus vulgaris 5 Komuro et al. (1971) Citrullus vulgaris Ð Lee et al. (1990) Cucumis sativus 44 Yakovleva (1965) C. sativus 4.2 Kawai et al. (1985); Rama Murthy et al. (2008) Lagenaria siceraria Ð Komuro et al. (1971); Sharma and Chohan (1973) Cucumber leaf spot carmovirus Cucumis melo 10Ð40 VIDE (1996) Cucumber mosaic (Syn. Arachis hypogaea 1.3 Xu and Barnett (1984) Cowpea banding mosaic, Syn. Benincasa hispida 1 Sharma and Chohan (1973) Cowpea ring spot, Syn. Soybean Capsicum annuum 1 Glaeser (1976) stunt) Capsicum frutescens 53Ð83 Akhtar Ali and Kobayashi (2010) Cerastium holosteoides 2 Tomlinson and Carter (1970a, b) Cicer arietinum Ð Jones and Coutts (1996); Makkouk et al. (2001) Cucumis melo 2.1 Kendrick (1934) C. melo 16 Mahoney (1935) C. melo 11.37Ð23.07 Sandhu and Kang (2007) C. sativus 1.4 Doolittle (1920) Cucurbita moschata 0.7 Sharma and Chohan (1973) C. pepo 0.07 Reddy and Nariani (1963); Sharma and Chohan (1973) Echinocystis lobata 9.1 Doolittle and Gilbert (1919) E. lobata 55 Doolittle and Walker (1925) E. lobata 15 Lindberg et al. (1956) G. max 50 Koshimizu and Iizuka (1963) G. max 95 Iizuka (1973) G. max ÐShenetal.(1984) G. max > 70 Honda et al. (1988) Lamium purpureum 4 Tomlinson and Carter (1970a, b) Lens culinaris 7.0Ð64.2 Jones and Coutts (1996); Fletcher et al. (1997); Makkouk et al. (2001, 2003); Jones (2004a, b) Lupinus angustifolius 12Ð18 Alberts et al. (1985); Jones (1988) Lupinus luteus ÐTroll(1957) L. luteus 14 Porembskaya (1964) Lycopersicon esculentum 0.2 Van Koot (1949) Medicago sativa 0.1Ð0.3 Jones (2004a, b) Phaseolus vulgaris 41 Bos and Maat (1974) P. vulgaris 30 Marchoux et al. (1977) P. vulgaris 0Ð49 Davis and Hampton (1986) P. vulgaris 30Ð100 Bhattiprolu (1991) Pisum sativum 1 Latham and Jones (2001a) Solanum melongena Ð Sriram and Doraiswamy (2001) Spergula arvensis 2 Tomlinson and Carter (1970a, b) Stellaria media 1Ð30 Hani et al. (1970); Hani (1971) S. media 5Ð8 Tomlinson and Carter (1970a, b) (continued) 18 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References S. media 3Ð40 Tomlinson and Carter (1970a, b) S. media 1Ð4 Tomilson and Walker (1973) Spinacia oleracea 15 Yang et al. (1997) Trifolium subterraneum 8.8 Jones and Mc Kirdy (1990); Jones (1991a, b) Vicia faba Ð Latham and Jones (2001a) Vigna radiata 8Ð32 Kaiser et al. (1968); Kaiser and Mossahebi (1974) V. radiata 5Phatak(1974) V. radiata 0.61 Purivirojkul et al. (1978) V. radiata 11 Iwaki (1978) V. sesquipedalis 4Ð28 Anderson (1957) V. sinensis 30 Meiners et al. (1977) V. sinensis 10 Iwaki (1978) V. sinensis 15Ð31 Sharma and Varma (1975a, b, 1984); Prakash and Joshi (1980) V. unguiculata 4Ð28 Anderson (1957) Vigna unguiculata 5 Iizuka (1973) V. unguiculata 10Ð30 Phatak (1974) V. unguiculata 15Ð20 Phatak et al. (1976) V. unguiculata 26 Fischer and Lockhart (1976) V. unguiculata 3Ð10 Pio-Ribeiro et al. (1978) V. unguiculata 4Ð18 Mali et al. (1987) V. unguiculata 1.2Ð2 Dos (1987); Bashir and Hampton (1996a, b) V. unguiculata 1.5Ð37 Gillaspie et al. (1998a, b) V. unguiculata 1.3Ð25.8 Mali et al. (1989) V. unguiculata 1.5Ð37 Gillaspie et al. (1998a, b) V. unguiculata 10Ð30 Abdullahi et al. (2001) Cucumis sativus cryptic Cucumis sativus Ð Jelkmann et al. (1988) Cucumber cryptic Cucumis sativus Ð Boccardo et al. (1983, 1987) Cycas necrotic stunt Chenopodium amaranticolor 30 VIDE (1996) C. serotinum 80 VIDE (1996) Dahlia mosaic Dahlia pinnata Ð Pahalawatta et al. (2007) Desmodium mosaic Desmodium canum 8 Edwardson et al. (1970) Desmodium trifolium mottle Desmodium trifolium Ð Suteri and Joshi (1978) Dodder latent mosaic Cuscuta californica 2.4 Bennett (1944) C. campestris 4.9 Bennett (1944) Dulcamara mottle Solanum dulcamara 23 Gibbs et al. (1966) Solanum tuberosum < 1 Jones and Fribourg (1977) Echtes Ackerbohnen mosaic (Syn. Vicia faba 1Ð3 Quantz (1953) Broad bean true mosaic) V. faba 15 Gibbs and Smith (1970); Gibbs and Paul (1970) V. faba 0.8 Cockbain et al. (1976) V. faba 5 Vorra-urai and Cockbain (1977) V. faba 4.8 Eppler and Kheder (1988) Eggplant mosaic (Syn. Andean Andigena clone 1 Jones and Fribourg (1977) potato latent) Nicotiana clevelandii Ð Jones (1982) Petunia hybrida Ð Gibbs et al. (1966) Solanum tuberosum <1 Jones and Fribourg (1977) Elm mosaic Ulmus americana 1Ð3.5 Bretz (1950) Ulmus americana 48 Callahan (1957) Elm mottle Ulmus glabra Ð Jones and Mayo (1973) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Eucharis mottle nepovirus Eucharis candida ÐVIDE(1996) Euonymus mosaic Euonymus europaeus Ð Bojannsky and Koslarova (1968) Fescue cryptic Festuca pratensis Ð Boccardo et al. (1983) Fig latent virus 1 Ficus carica Ð Castellano et al. (2009) Foxtail mosaic potexvirus Setaria italica ÐVIDE(1996) Fragaria chiloensis Fragaria chiloensis 30Ð50 VIDE (1996) French bean mosaic Phaseolus vulgaris 12Ð66 Capoor et al. (1986) Garland chrysanthemum temperate Chrysanthemum coronarium Ð Natsuaki et al. (1983a, b) Grape decline Vitis vinifera Ð Dias and Cation (1976) Grapevine Bulgarian latent Vitis vinifera 4.54 Uyemoto et al. (1977) Chenopodium quinoa 12.50 Uyemoto et al. (1977) Grapevine fanleaf C. amaranticolor 1.3 Dias (1963) C. quinoa 3 Bruckbauer and Rudel (1961); Cory and Hewitt (1968) Glycine max 59 Cory and Hewitt (1968) Vitis vinifera 0Ð66 Cory and Hewitt (1968) Grapevine yellow mosaic Chenopodium amaranticolor 0.7 Dias (1963) Grapevine yellow speckle Vitis vinifera Ð Wan chow wah and Symons (1999) Guar symptomless Cyamopsis tetragonoloba 12Ð28 Hansen and Leseman (1978); Behncken (1983) Hibiscus latent ring spot Hibiscus cannabinus 26 Rubies-Autonell (1997) C. amaranticolor 11 Rubies-Autonell (1997) C. quinoa 1 Rubies-Autonell (1997) Hemp streak Cannabis sativa ÐBrierly(1944) Hippeastrum mosaic Hippeastrum hybridum Ð Brants and Vanden Heuvel (1965) High plains virus Zea mays Low Forster et al. (2001) Hop chlorosis Humulus lupulus 27 Salmon and Ware (1935) Hop stunt viroid (Syn. Cucumber Lycopersicon esculentum Ð Kryczynski et al. (1988) pale fruit, Syn. Grapevine viroid) Vitis vinifera Ð Wah and Symons (1999); Mink (1993) Hop trefoil cryptic1 Medicago lupulina High Boccardo et al. (1983) Hop trefoil cryptic 2 Medicago lupulina High Boccardo et al. (1983, 1987) Hop trefoil cryptic 3 Medicago lupulina High Boccardo et al. (1983, 1987) Hosta virus x Hosta longipes 7.5 Ryu et al. (2006) Humulus japonicus Humulus japonicus ÐVIDE(1996) Hydrangea mosaic Chenopodium quinoa 82.3 Thomas et al. (1984) Kalanchoe top-spotting Kalanchoe blossfeldiana Ð Hearon and Locke (1984); Brunt (1992) Leek yellow stripe Allium cepa Ð Vishnichenko et al. (1990) Lettuce mosaic Chenopodium quinoa 0Ð1 Phatak (1974) Lactuca sativa 3.1 Newhall (1923) L. sativa 10 Ogilvie et al. (1935) L. sativa 2Ð8 Ainsworth and Ogilvie (1939) L. sativa 6Ð15 Kramer et al. (1945) L. sativa 1Ð8 Grogan and Bardin (1950); Grogan et al. (1952) L. sativa 3Ð10 Couch (1955) L. sativa 11 Herold (1956) L. sativa 13 Rohloff (1962) L. sativa 5 Ryder (1964) L. scariola 0.2Ð6.2 van Hoof (1959) Senecio vulgaris 2.3 Phatak (1974) (continued) 20 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References Lettuce yellow mosaic L. sativa 30 Mandahar (1978) Lilac ring mottle C. amaranticolor 13 Van der Meer et al. (1976) C. quinoa 91 Van der Meer et al. (1976) Celosia argentea 24 Van der Meer et al. (1976) Lima bean mosaic Phaseolus limensis 25 Mandahar (1978) P. limensis 2.2 Sawant and Capoor (1983) P. lunatus 0.3 Gay (1972) Lucerne (Australian) symptomless Chenopodium quinoa 6 Remah et al. (1986) Lucerne Australian latent Medicago sativa 8 Taylor and Smith (1971); Blackstock (1978); Jones et al. (1979) C. amaranticolor Ð Taylor and Smith (1971); Blackstock (1978); Jones et al. (1979) C. quinoa 9 Taylor and Smith (1971); Blackstock (1978); Jones et al. (1979) Lucerne transient streak Melilotus albus 2.5 Paliwal (1983) Lychnis ring spot Beta vulgaris 9.5 Bennett (1959) Capsella bursa-pastoris 9.4 Bennett (1959) Cerastium viscosum 27 Bennett (1959) Lychnis divaricata 58 Bennett (1959) Silene gallica 28 Bennett (1959) S. nodiflora 41 Bennett (1959) Maize chlorotic mottle machlomovirus Zea mays ÐVIDE(1996) Maize dwarf mosaic Zea mays 0.02Ð1.65 Williams et al. (1968); Hill et al. (1974); Tosic and Sutic (1977) Maize leaf spot Zea mays Ð Tsvet kov (1967) Maize mosaic Z. mays Ð Tosic and Sutic (1977) Melon rugose mosaic Cucumis melo 0.9 Mahgoub et al. (1997) Cucumis melo var. 3.8 Mahgoub et al. (1997) flexuosus Melon necrotic spot Cucumis melo 20Ð22.5 Kishi (1966) ; Gonzalez-Garza et al. (1979); Avegelis (1985); Campbell et al. (1996) Mibuna temperate Brassica rapa var. Ð Natsuaki et al. (1983a, b) laciniifolia Mung bean isometric yellow mosaic Vigna radiata Ð Benigno and Favali-Hedayat (1977) Mung bean mosaic potyvirus ÐÐMink(1993) Mulberry ring spot Glycine max 10 Tsuchizaki et al. (1971) Muskmelon mosaic (Syn. Squash Citrullus vulgaris 1.5 Nelson and Knuhtsen (1969) mosaic) Cucumis melo 1Ð27 Mahoney (1935) Cucumis melo 12Ð93 Rader et al. (1947); Seth et al. (1980) C. melo 12Ð93 Rader et al. (1947); Mukhayyish and Makkouk (1983) C. melo 7Ð21 Grogan et al. (1959) C. melo 9Ð10 Kemp et al. (1972) C. melo 3 Nelson and Knuhtsen (1973) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References C. melo 4Phatak(1974) C. melo 0Ð34.6 Alvarez and Campbell (1978) C. melo 30 Lange et al. (1983) C. melo Ð Izadpanah (1987); Avegelis and Katis (1989a, b) Cucurbita flexuous Ð Rader et al. (1947) C. maxima 0.2Ð1.5 Grogan et al. (1959) Cucumis melo 6.6Ð20 Grogan et al. (1959) C. melo 3 Nelson and Knuhtsen (1973) C. mixta 0.3 Grogan et al. (1959) C. moschata Ð Rader et al. (1947); Campbell (1971) C. pepo 2.2 Middleton (1944) C. pepo Ð Rader et al. (1947) C. pepo 5 Grogan et al. (1959); Knuhtsen and Nelson (1968) Chenopodium murale 23 Lockhart et al. (1985) C. quinoa 20 Lockhart et al. (1985) Muskmelon necrotic spot Cucumis melo Ð Gonzalez-Garza et al. (1979); Avegelis (1985) C. melo 22.5 Avegelis (1985) Sechium edule Ð Singh (1981) Nicotiana velutina mosaic <72 Randles et al. (1976) Nicotiana spp. Ð Randles et al. (1976) Oat mosaic Avena sativa ÐCarr(1971) Olive latent virus-1 Olive 35Ð82 Saponari et al. (2002) Onion mosaic Allium cepa ÐCheremuskina(1977) Onion yellow dwarf A. cepa 6Ð29 Hardtl (1964, 1972) Panicum mosaic sobemo Panicum spp. Ð VIDE (1996) Papaya ring spot Carica papaya 0.15 Bayot et al. (1990) Paprika mild mottle tobamovirus Capsicum spp. Ð VIDE (1996) Parsley latent Petroselinum crispum Ð Bos et al. (1979) Pea early browning Pisum sativum 37 Bos and van der Want (1962) P. sativum 1Ð2 Harrison (1973) P. sativum 30 Lockhart and Fischer (1976) P. sativum 61 Fiedorow (1983) P. sativum 25 Pospieszny and Frencel (1985) Vicia faba 5 Cockbain et al. (1983) V. faba 0.3Ð8 Fiedorow (1983) V. faba 9Ð45 Mahir et al. (1992) Pea enation mosaic Lathyrus ochrus Ð Kovachevski (1978) P. sativum 1.5 Blattny (1956) P. sativum Ð Kovachevski (1978) P. sativum 4Ð5 Kheder and Eppler (1988) Pea false leaf roll P. sativum 40 Thottappilly and Schmutterer (1968) Pea mild mosaic P. sativum 15 Clark (1972) Pea mosaic (Syn. Bean yellow mosaic) P. sativum Ð Quantz (1958) Lathyrus Ð Mandahar (1978) Trifolium hybridum 0.7 Mandahar (1978) T. pratense 47 Mandahar (1978) (continued) 22 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References Pea seed-borne mosaic (Syn. Pea Cicer arietinum Ð Makkouk et al. (2001) fizzle top and Pea leaf rolling Lathyrus clymenum 5 Latham and Jones (2001a) virus) Lens culinaris 0.8 AlÐMabrouk and Mansour (1998) Lens culinaris 32Ð44 Hampton and Muehlbauer (1977); Eppler et al. (1988) L. culinaris 0.5Ð5 Hampton (1982); Goodell and Hampton (1984); Bayaa et al. (1998) L. culinaris Ð Erdiller and Akbas (1996); Makkouk et al. (2001) L. culinaris 6 Coutts et al. (2008) Pisum arvense 9 Zimmer and Ali-Khan (1976) Pisum sativum 8Ð30 Inouye (1967) P. sativum 0Ð88 Stevenson and Hagedorn (1969, 1973) P. sativum 20Ð80 Hampton (1969) P. sativum 65Ð90 Mink et al. (1969); Cockbain (1988); Alconero and Hoch (1989) P. sativum 2Ð55 Musil (1970) P. sativum 4Ð32 Hampton (1972) P. sativum 0.5Ð5.8 Chiko and Zimmer (1978) P. sativum 30Ð60 Thakur et al. (1984); Rishi and Singh (1987) P. sativum 23Ð24 Kheder and Eppler (1988); Johansen et al. (1996) P. sativum 10 Kumar et al. (1991) P. sativum 58 McKeown and Biddle (1991) P. sativum 10 Zimmer and Lamb (1993); Sontakke and Chavan (2007) P. sativum 1Ð18 Latham and Jones (2001a); Deepti Anand et al. (2006) P. sativum 1.9Ð32.7 Gallo and Jurik (1995) P. sativum 5Ð30 Coutts et al. (2008) Vicia spp. 0.11Ð3 Hampton and Mink (1975); Musil (1980); Boulton et al. (1996) Vicia faba 0.2Ð2 Latham and Jones (2001b); Coutts et al. (2008) Pea stem necrosis Pisum sativum ÐVIDE(1996) Pea streak Pisum sativum 1.7 Kheder and Eppler (1988) Peach latent Prunus sp. 2.4 Mandahar (1978) Peach rosette mosaic Chenopodium quinoa 90 Dias and Cation (1976) Taraxacum officinale 3.6 Ramsdell and Myers (1978) Vitis vinifera 9.5 Ramsdell and Myers (1978) Peanut clump (African) A. hypogaea 24Ð48 Thouvenel et al. (1978) Eleusine coracana 5.2 Dieryck et al. (2009) Pennisetum glaucum 0.9 Dieryck et al. (2009) Peanut clump (Indian) A. hypogaea 3.5Ð17 Reddy et al. (1998a, b) A. hypogaea 9Ð11 Reddy et al. (1988, 1989) Eleusine coracana 5.2Ð6.5 Reddy et al. (1989, 1998a, b) Pennisetum glauca 0.93 Reddy et al. (1989, 1998a, b) Setaria italica 9.7Ð10.2 Reddy et al. (1989, 1998a, b) Triticum aestivum 0.5Ð1.3 Delfosse et al. (1999) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Peanut marginal chlorosis A. hypogaea 30Ð100 van Velsen (1961) Peanut mottle A. hypogaea 0.02Ð2 Kuhn (1965); Demski et al. (1983) A. hypogaea 20 Bock (1973) A. hypogaea 3.7 Paguio and Kuhn (1974) A. hypogaea 0Ð8.5 Adams and Kuhn (1977) A. hypogaea 1.3 Bharatan et al. (1984); Iizuka and Reddy (1986) A. hypogaea 1Ð7 Puttaraju et al. (2001) Glycine max 0.22 Iwaki et al. (1986) Lupinus albus (white lupin) 0.37 Demskietal.(1983) Phaseolus vulgaris 1.0 Demskietal.(1983) Vigna unguiculata 0.8 Demskietal.(1983) Peanut stunt A. hypogaea 3Ð4 Iizuka and Yunoki (1974) Glycine max 0.2 Troutman et al. (1967); Kuhn (1969) Peanut stripe (Syn. Peanut mild A. hypogaea 5Ð20 Xu et al. (1991) mottle) A. hypogaea 1.3Ð4.8 Xu et al. (1983) A. hypogaea 19.3Ð37.6 Demski et al. (1984a, b) A. hypogaea 28.8 Prasada Rao et al. (1988) A. hypogaea 43 Okhi et al. (1989) A. hypogaea 12.5 Chang et al. (1990) A. hypogaea 60.0 Matsumoto et al. (1991) Glycine max 28.0 Warwick and Demski (1988) Glycine max 3 Vetten et al. (1992) Vigna unguiculata 17.8 Vetten et al. (1992) Pelargonium zonate spot Nicotiana glutinosa 5 Gallitelli (1982) Pepino mosaic Lycopersicon esculentum 1.84 Cordoba-Selles et al. (2007) Pepper chat fruit viroid Capsicum annuum 19 Verhoeven et al. (2009) Piper yellow mottle Piper nigrum 30 Hareesh and Bhat (2010) Plum pox virus Apricot 24Ð92 Nemeth and Kolber (1982); Pasquini et al. (1998) Peach 15Ð84 Nemeth and Kolber (1983) Plum 64 Nemeth and Kolber (1983) Potato Andean latent tymovirus Solanum tuberosum <1VIDE(1996); Jones and Fribourg (1977) Potato spindle tuber Lycopersicon esculentum 2Ð11 Singh (1970); Kryczynski et al. (1988) Physalis peruviana 29 McClean (1948) Scopolia sinensis 71 Singh and Finnie (1973) Solanum incanum 53 McClean (1948) S. tuberosum 6Ð66 Singh (1970); Singh et al. (1992a, b) S. tuberosum 87Ð100 Hunter et al. (1969); Fernow et al. (1970); Grasmick and Slack (1986) Potato virus T 5Ð72 Salazar and Harrison (1978) Nicandra physalodes 28 Salazar and Harrison (1978) Solanum demissum-A 39 Salazar and Harrison (1978) Solanum tuberosum cv. Cara 33Ð59 Jones (1982) Solanum tuberosum cv. D42/8 0Ð2 Jones (1982) (continued) 24 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References Potato virus U Chenopodium quinoa Ð Jones et al. (1983) C. amaranticolor Ð Jones et al. (1983) Nicotiana debneyi Ð Jones et al. (1983) N. tabacum cv. Xanthi Ð Jones et al. (1983) Potato virus X Solanum tuberosum 0.6Ð2.3 Darozhkin and Chykava (1974) S. tuberosum 14Ð16 Mandahar (1978) Potato virus Y (Syn. Brinjal mosaic) Solanum nigrum Ð Eskarous et al. (1983) Solanum melongena 9Ð41 Mayee and Khatri (1975) Potato yellowing Solanum brevidens 20 Valkonen et al. (1992) Prune dwarf (Syn. Cherry ring Prunus spp. 33.3 Ramaswamy and Posnette (1971) mottle, Syn. Cherry yellows) Prunus spp. 17Ð77.7 Mandahar (1978) Prunus avium 0Ð58 Mink and Aichele (1984) Prunus cerasus 3Ð30 Cation (1952); Gilmer and Way (1960) P. mahaleb 9 Cation (1949, 1952) P. mahaleb 1.5Ð21.4 Schimanski and Schade (1974) P. persica Ð Cochran (1950) P. persica 10 Mink and Aichele (1984) Prunus necrotic ring spot (Syn. Cucurbita maxima 2.7 Das et al. (1961) Peach necrotic leaf spot, Syn. Prunus americana 1.1 Hobart (1956); Schimanski and Fuchs Peach ring spot) (1984) P. amygdalus Ð Williams et al. (1970); Barba (1986) P. avium 6.0 Cochran (1946) P. avium 37 Megahed and Moore (1967) P. cerasus 20Ð56 Cation (1949, 1952) P. cerasus 91 Megahed and Moore (1967) P. cerasus ÐDavidson(1976) P. mahaleb 10 Cation (1949, 1952) P. mahaleb 70 Megahed and Moore (1967) P. pensylvanica 37 Megahed and Moore (1967) P. persica 3Ð9 Cochran (1950) P. persica 16 Millikan (1959); Wagnon et al. (1960) P. persica 1.1Ð11.7 Wagnon et al. (1960); Mandahar (1978) P. persica 16 Millikan (1959); Wagnon et al. (1960) P. persica 17 Mink and Aichele (1984) Rubus idaeus 40Ð50 Cadman (1965) Radish yellow edge Brassica vulgaris Ð Natsuaki et al. (1979) Raphanus sativus 80Ð100 Natsuaki et al. (1979, 1983a, b) Loganberry degeneration Rubus longanobaccus ÐOrmerod(1970) Raspberry bushy dwarf (Syn. Rubus idaeus 22Ð60 Cadman (1965); Converse (1973) Loganberry degeneration) Rubus longanobaccus ÐOrmerod(1970) Raspberry latent (Black raspberry Rubus idaeus 10 Converse and Lister (1969) latent) Stellaria media 8Ð26 Murant et al. (1968) Raspberry ring spot Beta vulgaris 50Ð55 Lister and Murant (1967) Capsella 2Ð10 Lister and Murant (1967) bursa-pastoris Fragaria x ananassa 35Ð49 Lister (1960); Lister and Murant (1967) Fragaria spp. 35 Lister and Murant (1967) Glycine max 7.2 Lister (1960); Lister and Murant (1967) G. soja 20 Mandahar (1978) Petunia violacea 16.5 Phatak (1974) Rubus idaeus 18 Lister and Murant (1967) Stellaria media 29 Lister and Murant (1967) Red clover cryptic Trifolium pratense Ð Boccardo et al. (1983) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Red clover mottle Trifolium spp.ÐMink(1993) Red clover vein mosaic carlavirus Trifolium pratense ÐVIDE(1996) Vicia faba ÐVIDE(1996) Red pepper cryptic-1 Capsicum spp. Ð Boccardo et al. (1985); Natsuaki et al. (1987); Vide (1996) Red pepper cryptic-2 Capsicum spp. Ð Boccardo et al. (1985); Natsuaki et al. (1987); VIDE (1996) Rhubarb temperate Rheum rhaponticum Ð Natsuaki et al. (1983a, b) Rubus Chinese seed-borne nepovirus Rubus spp. Ð VIDE (1996) Runner bean mosaic Phaseolus coccineus 42 Vashisth and Nagaich (1965) Rye grass cryptic Lolium multiflorum 82 Plumb (1973); Plumb and Misari (1974); Boccardo et al. (1983) Safflower mosaic Carthamus tinctorius 2.2Ð5.0 Chauhan and Singh (1979) Santosai temperate Brassica rapa var. Ð Natsuaki et al. (1983a, b) amplexicaulis sub var. dentata Satsuma dwarf Phaseolus vulgaris 8.6 Kishi (1967) Sincomas mosaic Pachyrhizus erosus 40Ð80 Fajardo and Maranon (1932) Sowbane mosaic Atriplex pacifica 21 Bennett and Costa (1961) Chenopodium album 30 Bennett and Costa (1961) C. amaranticolor 14Ð62 Kado (1967); Dias and Waterworth (1967) C. murale 45Ð70 Bennett and Costa (1961) C. quinoa 2Ð46 Bancroft and Tolin (1967); Dias and Waterworth (1967) Soybean mild mosaic Glycine max 22Ð70 Takahashi et al. (1974, 1980) Soybean mosaic Glycine max 0Ð68 Kendrick and Gardner (1924); Ganesha Naik and Keshava Murthy (1997) G. max 40 Heinze and Kohler (1941) G. max 1Ð18 Ross (1963) G. max 1Ð24 Kennedy and Cooper (1967) G. max 34 Iizuka (1973) G. max 55.9 Phatak (1974) G. max 10.6Ð29.1 Suteri (1981) G. max 20.5Ð29.5 Kim and Lee (1986) G. max 64 Edwardson and Christie (1986) G. max 10 Nakano et al. (1988) G. max 11.2Ð41.1 Tu (1989) G. max 25.7Ð91.7 Pacumbaba (1995) G. max 43 Domier et al. (2007) G. max 32.9 Patil and Byadgi (2005) G. max 5.3 Golnaraghi et al. (2004) G. soja 10Ð25 Mandahar (1978) Lupinus albus 1.2 Vroon et al. (1988) Phaseolus vulgaris 1.6 Castano and Morales (1983) Soybean streak Glycine max 95 Iizuka (1973) Spinach latent virus Celosia cristata 53 Bos et al. (1980) Chenopodium quinoa 90 Bos et al. (1980) C. quinoa 60 Stefanac and Wrischer (1983) Nicotiana clevelandii 90 Stefanac and Wrischer (1983) (continued) 26 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References N. megalosiphon 95 Stefanac and Wrischer (1983) Nicotiana rustica 30 Bos et al. (1980) N. tabacum white Burley 90 Bos et al. (1980) N. tabacum Xanthi 94 Bos et al. (1980) Spinacia oleracea 50 Bos et al. (1980) S. oleracea 56 Stefanac and Wrischer (1983) Spinach temperate crypto Spinacia oleracea Ð Natsuaki et al. (1983a, b); VIDE (1996) Stone fruit ring spot Ð Mandahar (1978) Strawberry latent ring spot Amaranthus viridis 96 Van Hoof (1976) Apium graveolens 98Ð100 Walkey and Whittingham-Jones (1970) Capsella bursa-pastoris 4 Schmelzer (1969); Allen et al. (1970); Van Hoof (1976) Chenopodium quinoa 63Ð100 Schmelzer (1969); Allen et al. (1970) C. quinoa 74 Hanson and Campbell (1979) Lamium amplexicaule Ð Murant and Goold (1969) Mentha arvensis 6 Taylor and Thomas (1968) Pastinaca sativa 22.4 Hicks et al. (1986) Petroselinum crispum 6.6 Hanson and Campbell (1979) var. neapolitanum Petroselinum hortense Ð Bellardi and Bertaccini (1991, 1992) Rosa multiflora Ð Thomas (1981) Rosa rugosa Ð Thomas (1981) Rubus idaeus 75 Anon (1968); Murant and Goold (1969) Senecio vulgaris 20 Murant and Goold (1969); Van Hoof (1976) Solanum nigrum 0.1 Van Hoof (1976) Stellaria media < 60 Van Hoof (1976) Stellaria media 97 Murant and Goold (1969) Strawberry pallidosis Fragaria vesca Ð Yoshikawa and Converse (1990) Subterranean clover mottle Trifolium subterraneum 0.5Ð3 Francki et al. (1988); Njeru et al. (1997) Sugarcane mosaic Zea mays 0.1Ð0.4 Shepherd and Holdeman (1965); Williams et al. (1968); Baudin (1969); Mikel et al. (1984); Von Wechmar et al. (1984) Zea mays 4.81 Li et al. (2007) Sunflower mosaic potyvirus Helianthus annuus ÐMink(1993); VIDE (1996) Sunflower rugose mosaic Helianthus annuus 5.6 Singh (1979) Sunflower ring spot ilarvirus Helianthus annuus ?? VIDE (1996) Sunn-hemp mosaic (Syn. Vigna unguiculata 2.5Ð17.5 Mali et al. (1989) Sunn-hemp rosette) Crotalaria juncea 10Ð20 Verma and Awasthi (1978) Sweet potato ring spot Ipomoea batatas ÐVIDE(1996) Telfairia mosaic Telfairia occientatis 6Ð20 Anno-Nyako (1988) Tobacco mosaic Arabidopsis thaliana High de Assis Filho and Sherwood (2000) Capsicum annuum 45 Glaeser (1976); Demski (1981) Capsicum annuum 13.5Ð29.6 Tosic et al. (1980); Chitra et al. (1998, 2002) Capsicum frutescens 22 McKinney (1952); Sakamoto and Matsuo (1972) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Capsicum frutescens 18.4 Cicek and Yorganci (1991) Carthamus tinctorius Ð Lockhart and Goethals (1977) Lycopersicon 2Ð6 Doolittle and Beecher (1937); Taylor esculentum et al. (1961); Chitra et al. (1998, 1999, 2002) Lycopersicon 98.1 Cicek and Yorganci (1991) esculentum Malus platycarpa 38 Gilmer and Wilks (1967) Malus pumila 21 Allen (1969) M. sylvestris 3Ð37 Gilmer and Wilks (1967) Plantago major Ð Prochazkova (1977) Pyrus communis 35 Gilmer and Wilks (1967) Vigna unguiculata 1Ð4 Phatak (1974) V. unguiculata 14.6Ð22.6 Mali et al. (1987) Vitis vinifera 20 Gilmer and Kelts (1968) Tobacco necrosis Zea mays Ð Von Wechmar et al. (1992) Tobacco rattle Beta vulgaris 15Ð20 Dikova (2005) Capsella bursa-pastoris 1.9 Lister and Murant (1967) Lamium amplexicaule 2.2 Lister and Murant (1967) Myosotis arvensis 6.0 Lister and Murant (1967) Papaver rhoeas 1.1 Lister and Murant (1967) Senecio vulgaris 1 Cooper and Harrison (1973) Viola arvensis 3 Lister and Murant (1967); Cooper and Harrison (1973) Tobacco ring spot Amaranthus hybridus 14Ð21 Sammons and Barnett (1987) Cucumis melo 3Ð7 McLean (1962) Cucumis sativus Ð Stace-Smith (1970) Gladiolus spp. 4 Sushak (1976) Glycine max 54Ð78 Desjardins et al. (1954) G. max 78Ð82 Kahn (1956) G. max 100 Athow and Bancroft (1959) G. max 40 Kahn et al. (1962) G. max 100 Owusu et al. (1968) G. max 100 Iizuka (1973) G. max 94Ð97 Yang and Hamilton (1974); Hamilton (1985a, b) G. max 2.1 Golnaraghi et al. (2004) 25Ð50 Kahn et al. (1962) G. globosa 46 Iizuka (1973) Lactuca sativa 3 Grogan and Schnathorst (1955) L. sativa 21 Iizuka (1973) Nicotiana glutinosa Ð Stace-Smith (1970) N. tabacum 4.9Ð17 Valleau (1941) Pelargonium hortorum 4Ð6 Scarborough and Smith (1975) Petunia violacea 20 Henderson (1931) Phaseolus aureus 68Ð91 Shivanathan (1977) Senecio vulgaris 52 Tomilson and Carter (1971) Solanum melongena 3.2Ð9.8 Sastry and Nayudu (1976) Solanum tuberosum 2Ð9 Jones (1982) Taraxacum officinale 9Ð36 Tuite (1960) Vigna sinensis 82 Kahn (1956) Zinnia elegans 5 Iizuka (1973) (continued) 28 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References Tobacco streak (Syn. Asparagus A. officinalis Ð Van Hoof (1970) stunt, Syn. Bean red node, Syn. Chenopodium quinoa Ð Brunt (1968); Shukla and Gough (1983) Datura quercina) Datura stramonium 79 Blakeslee (1921); Brunt (1968) D. stramonium 94 Edwardson and Purcifull (1974) Fragaria vesca 0Ð35 Johnson et al. (1984) Glycine max 2.6Ð30 Ghanekar and Schwenk (1974) G. max 90 Kaiser et al. (1982) G. max 30Ð80 Truol et al. (1987) G. max 2.3 Golnaraghi et al. (2004) Lycopersicon esculentum 40Ð76 Sdoodee and Teakle (1988) Melilotus albus ÐKaiseretal.(1982) Parthenium hysterophorus 6.8Ð48 Sharman et al. (2009) Phaseolus vulgaris 1Ð26 Thomas and Graham (1951); Kaiser et al. (1991) Phaseolus vulgaris 27 Thomas and Graham (1951) Vigna unguiculata 1Kaiseretal.(1982); Shukla and Gough (1983) Tomato apical stunt viroid Lycopersicon esculentum 80 Antignus et al. (2007) Tomato aspermy Phaseolus vulgaris 18.7 Wang (1982) Stellaria media Ð Noordam et al. (1965) Tomato black ring Beta vulgaris 3Ð27 Gibbs and Harrison (1964) B. vulgaris 56 Lister and Murant (1967) Capsella bursa-pastoris 90 Lister and Murant (1967) Cerastium vulgatum 33Ð100 Lister and Murant (1967) Chenopodium album 84 Lister and Murant (1967) C. quinoa 78 Hanada and Harrison (1977) Fragaria x ananassa 40 Lister (1960) Fumaria officinalis 100 Lister and Murant (1967) Geranium dissectum Ð Murant and Lister (1967) Glycine max 83 Lister (1960) Lactuca sativa 3 Morand and Poutier (1978) Lamium amplexicaule 10Ð48 Lister and Murant (1967) Ligustrum vulgare 5.7Ð8.3 Lister and Murant (1967) Lycopersicon esculentum 19 Lister and Murant (1967) Myosotis arvensis 100 Lister and Murant (1967) Nicotiana clevelandii Ð Hanada and Harrison (1977) N. rustica 4.4Ð8.8 Lister and Murant (1967) N. tabacum 6 Hanada and Harrison (1977) Petunia violacea 29.1 Phatak (1974) Poa annua 2.7 Lister and Murant (1967) Polygonum aviculare Ð Murant and Lister (1967) P. convolvulus Ð Murant and Lister (1967) P. persicaria 21Ð100 Lister and Murant (1967) Rubus idaeus 1Ð6 Lister and Murant (1967) Rubus spp. 5Ð19 Lister and Murant (1967) Sambucus nigra 0.1Ð1 Schimanski (1987) Senecio vulgaris 3Ð40 Lister (1960); Lister and Murant (1967) Spergula arvensis 63 Lister and Murant (1967) Stellaria media 65 Lister and Murant (1967) Vigna sinensis 23 Lister (1960) (continued) Table 1.2 (continued) Virus/viroid Host Per cent References Tomato bushy stunt Lycopersicon esculentum 50Ð65 Tomilson and Faithfull (1984) Malus pumila 17 Allen (1969) M. pumila 1.7Ð6.9 Kegler and Schimanski (1982) Prunus avium High Allen and Davidson (1967) Tomato chlorotic dwarf viroid Lycopersicon esculentum 85.5Ð94.4 Singh and Dilworth (2009) Tomato mosaic Lycopersicon esculentum 16.7 Chitra et al. (2002); Ismaeil et al. (2011); Hadas et al. (2004) Tomato planta macho viroid Lycopersicon esculentum Ð Galindo et al. (1982) Tomato ring spot (Syn. Chenopodium amaranticolor 57 Cory and Hewitt (1968) Grapevine yellow vein) Fragaria vesca 68 Kahn (1956); Mellor and Stace-Smith (1963) Gladiolus spp. 7 Mellor and Stace-Smith (1963); Sushak (1976) Fragaria x ananassa 26 Mellor and Stace-Smith (1963) Glycine max 76Ð80 Kahn (1956); Lister and Murant (1967) Glycine max 58 Cory and Hewitt (1968) G. max 1.4 Golnaraghi et al. (2004) Gomphrena globosa 76 Keplinger and Braun (1973) Lycopersicon esculentum 3 Hollings et al. (1972) Nicotiana tabacum 11 Hollings et al. (1972) Pelargonium hortorum 11 Hollings et al. (1972) Rubus idaeus 2 Braun and Keplinger (1973) Sambucus spp. 11 Uyemoto et al. (1971) Taraxacum officinale 23.8 Mountain et al. (1983) Trifolium pratense 3Ð7 Hampton (1967) Tomato spotted wilt Lycopersicon esculentum Ð Jones (1944) Senecio cruentus 96 Jones (1944) Tomato streak Lycopersicon esculentum 66 Berkeley and Madden (1932) Turnip mosaic Raphanus raphanistrum 4 Tomilson and Walker (1973) Turnip yellow mosaic Arabidopsis thaliana 72.6 de Assis Filho and Sherwood (2000) Alliaria petiolata 2.3 Pelikanova (1990) Brassica chinensis var. 9.5 Benetti and Kaswalder (1982/83) parachinensis Brassica napus var. silvestris 1.6Ð8.3 Spak et al. (1993) Camelina sativa 20 Hein (1984) Crambe hispanica 2.7 Benetti and Kaswalder (1982/83) Urdbeanleafcrinkle(Syn. Vigna mungo 18.3 Kolte and Nene (1972); Beniwal and Blackgram leaf crinkle, Syn. Chaubey (1984);Beniwaletal. Bean urd leaf crinkle virus) (1984); Makwana et al. (2001) V. mungo 20.3Ð41.8 Narayanaswamy and Jaganathan (1975); Patel et al. (1999); Mahajan and Joi (1999); Prasad et al. (1998) V. mungo 17.6 Dubey and Sharma (1985) V. mungo 8.6 Ravinder and Jeyarajan (1989); Ravinder Reddy et al. (2005a, b) V. mungo 1Ð83 Pushpalatha et al. (1999) V. mungo 2.2Ð28.7 Sharma et al. (2007) Vigna unguiculata 6Ð15 Beniwal et al. (1980) (continued) 30 1Introduction

Table 1.2 (continued) Virus/viroid Host Per cent References Vicia cryptic Vicia faba High Kenten et al. (1978, 1979, 1980a, b, 1981); Abou-Elnasr et al. (1985) Watermelon mosaic Cucumis pepo Ð Bhargava and Joshi (1960) Echinocystis lobata 2 Lindberg et al. (1956) Wheat mosaic Triticum spp. 0.01 Panarin and Zabavina (1978) Zea mays 0.03 Panarin and Zabavina (1978) Wheat soil-borne mosaic Secale cereale 3 Jezewska (1995) Wheat streak mosaic Triticum aestivum 0.22Ð0.4 Lanoiselet et al. (2008) Zea mays 0.01Ð0.1 Hill et al. (1974); Panarin and Zabavina (1978); Jones et al. (2005) Z. mays 0.2Ð1.5 Jones et al. (2005) Wheat striate mosaic Triticum aestivum Ð Abdel Hak et al. (1977) White clover cryptic 1 Trifolium repens High Boccardo et al. (1985); Natsuaki et al. (1986) White clover cryptic 2 T. repens High Boccardo et al. (1985); Natsuaki et al. (1986) White clover cryptic 3 T. repens Low Boccardo et al. (1985); Natsuaki et al. (1986) White clover mosaic T. pretense 6 Hampton (1963); Lee et al. (2004) White clover temperate T. repens High Natsuaki et al. (1986) Winged bean ring spot Psophocarpus tetragonolobus Ð Fauquet et al. (1979) Zucchini yellow mosaic Cucumis pepo 1.4Ð18.95 Davis and Mizuki (1986); Schrijnwerkers et al. (1991); RiedleÐBauer et al. (2002); Tobias et al. (2008) Cucurbita pepo 1.6 Simmons et al. (2011) Note: Per cent seed transmission has not been given or doubtful in some cases because of inadequate studies or has not been reported or due to non-availability of original article. Fig. 1.2 Symptoms on foliage and seeds due to some seed-borne viruses 32 1Introduction

References Allen WR, Davidson TR (1967) Tomato bushy stunt virus from Prunus avium L. I. Field studies and virus Abdel Hak T, Ghobrial E, Kamel AH (1977) Studies on characteristics. Can J Bot 45:2375Ð2383 striate mosaic of wheat in Egypt. Agric Res Rev 55:1 Allen WR, Davidson TR, Briscoe MR (1970) Properties Abdel-Salam AM, Amin AH (1990) An Egyptian isolate of a strain of strawberry latent ringspot virus isolated of beet curly top virus: new differential hosts, physical from sweet cherry growing in Ontario. Phytopathology properties, seed transmission and serologic studies. 60:1262Ð1265 Bull Fac Agric Univ Cairo 41:843Ð858 Allen DJ, Thottappilly G, Rossel HW (1982) Cow- Abdullahi I, Ikotin T, Winter S, Thottappilly G, Atiri GI pea mottle virus: field resistance and seed trans- (2001) Investigation on seed transmission of cucumber mission in virus tolerant cowpea. Ann Appl Biol mosaic virus in cowpea. 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J Virol Methods Seed biology, vol II. Academic, London, pp 318Ð416, 163:234Ð237 447 pp Allarangaye MD, Traore O, Traore EVS, Millogo RJ, Baker KF, Smith SH (1966) Dynamics of seed trans- Konate G (2006) Evidence of non-transmission of rice mission of plant pathogens. Annu Rev Phytopathol yellow mottle virus through seeds of wild host species. 4:311Ð334 J Plant Pathol 88:309Ð315 Bancroft JB, Tolin SA (1967) Apple latent virus 2 is Allard HA (1914) The mosaic disease of tobacco. Bull. sowbane mosaic virus. Phytopathology 57:639Ð640 No. 40. USDA, Bur. Plant Ind, Washington, DC, 33 pp Barba M (1986) Detection of apple mosaic and prunus Allen WR (1969) Occurrence and seed transmission of necrotic ring spot viruses in almond by ELISA. tomato bushy stunt virus in apple. Can J Plant Sci Archiev fur Phytopathologie and Pflanzenschutz 49:797Ð799 22:279Ð282 References 33

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Phytopathol- mosaic virus, investigations on aphid transmission and ogy 78:860 seed transmission. Kulonlenyomat A Novenyvedelmi Blackstock JM (1978) Lucerne transient streak and Kutato Intezet Evkonyve 13:167Ð176 lucerne latent, two new viruses of lucerne. Aust J Agric Behncken GM (1983) Guar symptomless virus. In: Res 29:291Ð304 Boswell KF, Gibbs AJ (eds) Descriptions and keys Blakeslee AF (1921) A graft infectious disease of datura from VIDE. Austral. Natl. Univ, Canberra resembling a vegetative mutation. J Genet 11:17Ð36 Bellardi MG, Bertaccini A (1991) Parsley seeds infected Blaszczak W (1963) Seed transmission of narrowleaved- by strawberry latent ring spot virus (SLRV). Phy- ness of yellow lupin (NYL). Genet Pol 4:65Ð77 topathol Mediterr 30:198Ð199 Blaszczak W (1965) Severe strain of yellow bean mosaic Bellardi MG, Bertaccini A (1992) Strawberry latent ring virus found on Trifolium pratense L. 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Shepherd RJ, Fulton RW (1962) Identity of a seed-borne Singh RP, Boucher A, Wang RG (1992b) Detection of virus of cowpea. Phytopathology 52:489Ð493 potato spindle tuber viroid in the pollen and various Shepherd RJ, Holdeman QL (1965) Seed transmission of parts of potato plant pollinated with viroid-infected the Johnson grass strain of the sugarcane mosaic virus pollen. Plant Dis 76:951Ð953 in Corn. Plant Dis Rep 49:468Ð469 Singh RP, Dilworth AD, Xiaoping AO, Singh M, Baran- Shilpashree K (2006). Studies on blackeye cowpea mo- wal VK (2009) Citrus exocortis viroid transmis- saic viral disease on cowpea (Vigna unguiculata (L.) sion through commercially distributed seeds of Im- Walp). M.Sc. thesis UAS, Dharwad, India patiens and Verbena plants. Eur J Plant Pathol Shivanathan P (1977) A seed-borne virus of Phaseolus 124:691Ð694 aureus (Roxb). 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Tsuchizaki T, Yora K, Asuyama H (1970b) The viruses Van der Meer FA, Huttinger H, Maat DZ (1976) Lilac ring causing mosaic of cowpea and Azuki bean and their mottle virus: isolation from lilac, some properties, and transmissibility through seeds. Ann Phytopathol Soc relation to lilac ringspot disease. Neth J Plant Pathol Japan 36:112Ð120 82:67Ð80 Tsuchizaki T, Hibino H, Saito Y (1971) Mulberry ringspot Van Hoof HA (1959) Seed transmission of lettuce mosaic virus isolated from mulberry showing ringspot symp- virus in Lactuca serriola Tijdschr. PIZiekt 65:44Ð46 toms. Ann Phytopathol Soc Japan 37:266Ð271 Van Hoof HA (1970) Some observations on retention of Tsuchizaki T, Senboku T, Iwaki M, Pholauporn S, tobacco rattle virus in nematodes. Neth J Plant Pathol Srithongchi W, Deema N, Ong CA (1984) Black- 77:30Ð31 eye cowpea mosaic virus from asparagus bean Vigna Van Hoof HA (1976) Seed transmission of strawberry sesquipedalis. in Thailand and Malaysia and their latent ring spot virus. Med Fac Landbonww Rijksunib relationships to a Japanese isolate. Annu Phytopathol Gent 41:791Ð794 Soc Japan 50:461Ð468 Van Koot Y (1949) Enkele nieuwe gezichtspunten betr- Tsuchizaki T, Iwaki M, Thongmeearkom P, Sarindu N, effende het virus van het tomatenmosaiek. Tijdschr Deema N (1986) Bean common mosaic virus isolated PIZiekt 55:152Ð166 from mungbean (Vigna radiata) in Thailand. In: Virus Van Velsen RJ (1961) Marginal chlorosis, a seedborne diseases of rice and legumes in tropics. Tech. Bull. No. virus of Arachis hypogaea variety ‘Schwarz 21’ in 21. TARC, Japan, pp 84Ð188, p 238 New Guinea. P N G Agric J 14:38Ð40 Tsuchizaki T, Senboku T, Iwaki M, Kiratiya- ngul, Varma P, Gibbs AJ (1967) Preliminary studies on sap- Srithongchai W, Deema N, Ong CA (1986) Blackeye transmissible viruses of red clover (Trifolium pratense cowpea mosaic virus from asparagus bean (Vigna L.) in England and Wales. Ann Appl Biol 59:23Ð30 sesquipedalis) in Thailand and Malaysia. In: Virus Varma A, Krishna Reddy M, Malathi VG (1992) Influence diseases of rice and legumes in tropics. Tech. Bull No. of the amount of the blackgram mottle virus in differ- 21. TARC, Japan, pp 213Ð28, p 238 ent tissues on transmission through the seeds of Vigna Tsvet kov D (1967) Listno petnosvane po zhitnite kulturi mungo. Plant Pathol 41:274Ð281 (leafspot of cereals). Rastilt Zasht 15:29Ð31 Vashisth KS, Nagaich BB (1965) A mosaic disease of Tu JC (1989) Effect of different strains of soybean mo- runner bean. Indian Phytopathol 18:311 saic virus on growth, maturity, yield, seed mottling Vemana K, Jain RK (2010) New experimental hosts of and seed transmission in several soybean cultivars. J tobacco streak virus and absence of true seed trans- Phytopathol 126:231Ð236 mission in leguminous hosts. Indian J Virol 21(2): Tuite J (1960) The natural occurrence of tobacco ringspot 117Ð127 virus. Phytopathology 50:296Ð298 Verhoeven JKJ, Jansen CCC, Roenhorst JW, Flores R, de Udayashankar AC, Nayaka CS, Kumar BH, Shetty HS, la Pena M (2009) Pepper chat fruit viroid: biological Prakash HS (2009) Detection and identification of and molecular properties of a proposed new species of the Black eye cowpea mosaic strain of Bean common the genus Pospiviroid. Virus Res 144:209Ð214 mosaic virus in seeds of cowpea from Southern India. Verma HN, Awasthi LP (1978) Further studies on a rosette Phytoparasitica 37:283Ð293 virus of Crotalaria juncea. Phytopathol Z 92:83Ð87 Uyeda I, Mink GI (1981) Properties of asparagus virus II, Vetten HJ, Green SK, Lesemann DE (1992) Characteri- a new member of the Ilarvirus group. Phytopathology zation of peanut stripe virus isolates from soybean in 71:1264Ð1269 Taiwan. J Phytopathol 135:107Ð124 Uyemoto JK, Grogan RG (1977) Southern bean mosaic Vide 1996 :- Brunt AA, Crabtree K, Dallwitz MJ, Gibbs virus: evidence for seed transmission in bean embryos. AJ, Watson L, Zurcher EJ (eds) (1996 onwards). Phytopathology 67:1190Ð1196 Plant viruses online: descriptions and lists from the Uyemoto JK, Gilmer RM, Williams E (1971) Sap- VIDE database. Version: 20 August 1996. URL http:// transmissible viruses of elderberry in New York. Plant biology.anu.edu.au/Groups/MES/vide/ Dis Rep 53:913Ð916 Vishnichenko VK, Konareva TN, Zavriev SK (1990) De- Uyemoto JK, Tascheberg EF, Hummer DK (1977) Iso- tection of the coat protein antigen of Leak yellow stripe lation and identification of a strain of grapevine Bul- virus in shoots of the resistant species Allium cepa by garian latent virus in Concord grapevine in New York a method of immunoblotting. Doklady Vsesoyuznoi State. Plant Dis Rep 61:949Ð953 ordena Lenina No: 10, pp 19Ð21 Valkonen JPT, Pehu E, Watanabe K (1992) Symptom Von Wechmar MB, Lindsey S, Buckton P (1992) Dis- expression and seed transmission of alfalfa mosaic covery of a new and potentially serious disease of virus and potato yellowing virus and Potato yellowing maize. Technical communication; Dept. Agric. Devel- virus (SB-22) in Solanum brevidens and S. tuberosum. opt; South Africa; No: 232, pp 139Ð142 Potato Res 35:403Ð410 Von Wechmar MBD, Kaufmann A, Desmarais F, Rybicki Valleau WD (1941) Seed transmission and sterility studies EP (1984) Detection of seed transmitted brome mosaic of two strains of tobacco ringspot. Res Bull Kentucky virus by ELISA, radial immunodiffusion and immuno- Agric Exp Stn 327 electroblotting tests. Pytopathol Z 109(4):341Ð352 References 53

Vorra-urai S, Cockbain AJ (1977) Further studies on seed Watson MA, Serjeant EP (1962) Carrot motley dwarf. transmission of broad bean stain virus and Echtes Report Rothamsted Experimental Station 1961, pp Ackerbohnenmosaik virus in field beans (Vicia faba). 106Ð107 Ann Appl Biol 87(3):365Ð374 Westerdijk J (1910) Die Mosaikkrankheit der To- Vovlas C (1973) Seed transmission of chicory yellow maten. Mededelingen Phytopathologisch Laborato- mottle virus. Transmission per seme del virus deka rium Willie Comelin Scholten, Amsterdam, 1, 20 maculatura gialla della cicoria. Phytopathol Mediterr White RF, Woods RD (1978) Beet cryptic virus in some 12:104Ð105 varieties of Beta vulgaris. Phytopathol Z 91:91Ð93 Vroon CW, Pietersen C, Van Tonder HJ (1988) Seed Williams LE, Findley WR, Dollinger EJ, Ritter RM transmission of soybean mosaic virus in Lupinus albus (1968) Seed transmission studies of maize dwarf mo- L. Phytoparasitica 20:169Ð175 saic virus in corn. Plant Dis Rep 52:863Ð864 Wagnon K II, Traylor JA, Williams HE, Weinberger JH Williams HE, Jones RW, Traylor JA, Wagnon HK (1970) (1960) Observations on the passage of peach necrotic Passage of necrotic ringspot virus through almond leaf spot and peach ringspot viruses through peach seeds. Plant Dis Rep 54:822Ð824 and nectarine seeds and their effects on the resulting Xu Z, Barnett OW (1984) Identification of a cucumber seedlings. Plant Dis Rep 44:117Ð119 mosaic virus strain from naturally infected peanuts in Wah Y, Symons RH (1999) Transmission of viroids via China. Plant Dis 68:386Ð389 grape seeds. J Phytopathol 147:285Ð291 Xu Z, Yu Z, Liu J, Barnett OW (1983) A virus causing Walkey DGA (1967) Seed transmission of arabis mo- peanut mild mottle in Hubei province, China. Plant Dis saic virus in lettuce (Lactuca sativa). Plant Dis Rep 67:1029Ð1032 51:883Ð884E Xu Z, Chen K, Zhang Z, Chen J (1991) Seed trans- Walkey DGA, Whittingham-Jones SG (1970) Seed trans- mission of peanut stripe virus in peanut. Plant Dis mission of strawberry latent ringspot virus in cel- 75:723Ð726 ery (Apium graveolens var. duice). Plant Dis Rep Yakovleva N (1965) Borba s zelenoi mazaikoi Ogurtsov 54:802Ð803 (Control of green mosaic of cucumber). Zashch Rast Wallace JM (1957) Virus-strain interference in relation Vredit Bolez 10:50Ð51 to symptoms of psorosis disease of citrus. Hilgardia Yang AF, Hamilton RI (1974) The mechanism of seed 27:223Ð246 transmission of tobacco ringspot virus in soybean. Wallace JM, Drake RJ (1953) Seed transmission of avo- Virology 62:26Ð37 cado sun-blotch virus. Citrus Leaves, December 1953. Yang Y, Kim KS, Anderson EJ (1997) Seed transmission Reprint. 2 pp of cucumber mosaic virus in spinach. Phytopathology Wallace JM, Drake RJ (1962) A high rate of seed trans- 87:924Ð931 mission of avocado sun-blotch virus from symptom- Yoshikawa N, Converse RH (1990) Strawberry pallidosis less trees and the origin of such trees. Phytopathology disease: distinctive dsRNA species associated with la- 52:237Ð241 tent infections in indicators and in diseased strawberry Walter MH, Wyatt SD, Kaiser WJ (1995) Comparison of cultivars. Phytopathology 80:543Ð548 the RNAs and some physico-chemical properties of Zettler FW, Evans IR (1972) Blackeye cowpea mo- the seed transmitted Tobacco streak virus isolate Mel saic virus in Florida: host range and incidence in 40 and the infrequently seed transmitted isolate Mel F. certified cowpea seed. Proc Fla Statc Hortic Soc Phytopathology 85:1394Ð1399 85:99Ð101 Walters EC Jr (1962a) Thirty eight years behind the times Zimmer RC, Ali-khan ST (1976) New seed-borne virus of and still they germinate. Assoc Oft Seed Anal Newsl field peas. Can Agric 21:6Ð7 36:8 Zimmer RC, Lamb RJ (1993) Amplification and spread Walters HJ (1962b) Variation in isolates of tobacco of pea seed-borne mosaic-virus in field grown peas. ringspot virus from soybean. Phytopathology Can J Plant Pathol Rev Canadienne De Phytopathol 52:31Ð32 15:17Ð22 Wan Chow Wah VF, Symons RH (1999) Transmission of Zschau K (1962) Versuche and Beobachtungen Zur viroids via grape seeds. J Phytopathol 147:285Ð291 Samenubentragurg der Mosaikkrankheit der lupinen, Wang WY (1982) Tech. Bull. Plant Quarantine Res. No. insbesondere der Gelblupine. Nachr Bl.dt Pflschutzdi- 3., Plant Quarantine, Dong San Huan, Beijing enst 16:1Ð7 Warwick D, Demski JW (1988) Susceptibility and re- Zschau K, Janke C (1962) Samenubertragung des sistance of soybean to peanut stripe virus. Plant Dis Luzernemosaik Virus and Luzerne. NachBl dt Pf- 72:19Ð21 schutzdienst 16:94Ð96 Identification and Taxonomic Groups 2

Abstract In the early period of plant virus research work, inadequate identification has led to lot of confusion due to lack of an internationally accepted classification of plant viruses. The discriminatory criteria are established by ICTV study groups, who have followed the rules laid out in the International Code of Virus Classification and Nomenclature (ICVCN). As new types of viruses continue to be discovered, new names must be created for taxa; several rules in the ICVCN govern the construction of these names. Till now, nearly 231 virus and viroid diseases are reported to be seed transmitted in different crop plants. As per the latest ICTV clas- sification by King et al. (Virus Taxonomy: 9th report of the interna- tional committee on taxonomy of viruses. Elsevier, San Diego, 2011), the seed-transmitted viruses were concerned to 231 virus families/plant virus groups. Among these plant virus groups, seed-transmitted viruses are distributed in 24 virus groups including Alfamovirus, Bromovirus, Capillovirus, Carlavirus, Carmovirus, Caulimovirus, Comovirus, Cryp- tovirus, Cucumovirus, Enamovirus, Fabavirus, Furovirus, Hordeivirus, Ilarvirus, Necrovirus, Nepovirus, Potexvirus, Potyvirus, Sobemovirus, To- bamovirus, Tobravirus, Tombusvirus, Tospovirus and Tymovirus groups. Greater number of seed-transmitted viruses are found in Poty (35), Nepo (28), Crypto (28), Ilar (14), Tobamo (7), Potex (7), Como (6), Carla (5), Carmo (5), Cucumo (5), Sobemo (5), Furo (4), Bromo (3) and Tymo (3) virus groups. On the other hand, in some groups such as Alfamo, Capillo, Caulimo, Enamo, Faba, Hordei, Necro, Tobra and Tombus groups, the seed-transmitted viruses recorded were very few. No seed transmission was noticed in Clostero, Diantho, Gemini, Luteo, Marafi, Parsnip yellow fleck, Reo, Rhabdo, Tenui and Waika groups.

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 2, 55 © Springer India 2013 56 2 Identification and Taxonomic Groups

problem, Bos (1964) suggested the use of stan- 2.1 Identification dardised vernacular names while listing out the viruses from legume crops. Subsequent schemes In order to detect and identify a seed-transmitted of nomenclature (Gibbs and Harrison (1976); virus, it is imperative to understand the Matthews (1979, 1982); Fauquet et al. (2005); characteristics of seed-transmitted viruses for King et al. (2011) and grouping Harrison et al. comparison with the available information on (1971); Francki (1981)) of viruses and the recent previously described viruses. Diagnosis of a CMI/AAB descriptions have contributed greatly virus disease is never unequivocal unless the to the knowledge of plant viruses. During 1996, virus is isolated, studied outside its host and Brunt and his associates’ compiled publication demonstrated by Koch’s postulates. By following is one of the most important sources of descrip- the ‘ten commandments of the plant viruses’ tions of viruses of tropical plants. Information on proposed by Bos (Bos 1976a), a reasonable the identification of virus diseases in the form diagnosis of the viral disease could be made. of Virus Information Data Exchange (VIDE) is The diagnosis begins with particulars of the published by Boswell and Gibbs 1986 and Gibbs diseased plant and then deals with the virus 1989. A system called ‘DELTA’ which is specif- itself, indicating a gradual shift from clinical ically designed to handle all forms of taxonomic observations to etiological diagnosis. information has been used to store the informa- The pragmatic approach depends on the fact tion in computers (Brunt et al. 1996). that each virus has a definite host range that is The taxonomic approach assesses the group often confined to a wide or limited number of to which a virus belongs by determining some host plants. The natural hosts of the virus are of its group-specific characteristics such as the first identified and a comparison is then made shape and size of its particles. Then, more spe- with previously isolated species or their close cific tests including host range are used to check relatives, and their properties are also compared whether the unknown virus is an already de- with those of the unknown virus. Based on the scribed member of the likely group or not. This morphology of the virus involved, they can easily approach requires some understanding in virus be differentiated by their shape and size. Serology classification. Modern taxonomic classification is and nucleic acid hybridisation tests are being also taken into the consideration of molecular widely used for the identification of different characterisation of viruses which has become plant viruses and their strains. At present the reliable and authentic (Van Regenmortal et al. virus identification is being done in scientific 2000; Fauquet et al. 2005; King et al. 2011). The laboratories through specialised techniques. Dif- ICTV 9th report of 2009 which was published by ferent steps involve are seed morphology, indi- King et al. (2011) has 6 orders, 87 families, 19 cator hosts, transmission, electron microscopy, subfamilies, 349 genera and 2,284 species. serology, molecular methods, etc., which can help Based on a set of characteristics, more than in detection and identification of seed-transmitted 975 plant viruses have been described and clas- viruses. The total list of conventional viruses, sified by International Committee on Taxonomy cryptic viruses and viroids which are seed trans- of Viruses (ICTV) into 34 well-defined groups mitted and reported from different parts of the and a few less well-defined groups or subgroups world is presented in Table 1.2. (Hamilton et al. 1981;Brown1989; Martelli 1992; Brunt et al. 1996; Fauquet et al. 2005;Van Regenmortel et al. 2005; King et al. 2011). 2.2 Classification of Viruses In the Table 1.2, available information on seed-transmitted virus diseases of fruits, In the early period of plant virus research work, vegetable and ornamental crops, including in inadequate identification has lead to lot of con- collateral hosts, was listed along with percentage fusion due to lack of an internationally accepted of seed transmission. In Table 2.1 taxonomic classification of plant viruses. Looking into this position of seed-transmitted plant viruses is 2.2 Classification of Viruses 57

Table 2.1 Taxonomic position of seed-transmitted viruses S. no Species (virus/viroid) Order Family Genus 1 Alfalfa mosaic (Syn.) Berseem mosaic Ð Alfamovirus 2 Potato Yellowing Ð Bromoviridae Alfamovirus 3 Alfalfa cryptic Ð Partitiviridae Alphacryptovirus 4 Alfalfa temperate Ð Partitiviridae Alphacryptovirus 5 Avocado viruses 1,2,3 Ð Partitiviridae Alphacryptovirus 6 Beet 1 alpha crypto (Syn.) Beet temperate Ð Partitiviridae Alphacryptovirus 7 Beet 2 alpha crypto Ð Partitiviridae Alphacryptovirus 8 Beet 3 alpha crypto Ð Partitiviridae Alphacryptovirus 9 Carrot temperate 1 Ð Partitiviridae Alphacryptovirus 10 Carrot temperate 3 Ð Partitiviridae Alphacryptovirus 11 Carrot temperate 4 Ð Partitiviridae Alphacryptovirus 12 Fescue cryptic Ð Partitiviridae Alphacryptovirus 13 Garland chrysanthemum temperate Ð Partitiviridae Alphacryptovirus 14 Hop trefoil cryptic 1 Ð Partitiviridae Alphacryptovirus 15 Hop trefoil cryptic 3 Ð Partitiviridae Alphacryptovirus 16 Mibuna temperate Ð Partitiviridae Alphacryptovirus 17 Radish yellow edge Ð Partitiviridae Alphacryptovirus 18 Red clover cryptic Ð Partitiviridae Alphacryptovirus 19 Red pepper cryptic-1 Ð Partitiviridae Alphacryptovirus 20 Red pepper cryptic-2 Ð Partitiviridae Alphacryptovirus 21 Rhubarb temperate Ð Partitiviridae Alphacryptovirus 22 Rye grass cryptic Ð Partitiviridae Alphacryptovirus 23 Santosai temperate Ð Partitiviridae Alphacryptovirus 24 Spinach temperate crypto Ð Partitiviridae Alphacryptovirus 25 Vicia cryptic Ð Partitiviridae Alphacryptovirus 26 White clover cryptic 1 Ð Partitiviridae Alphacryptovirus 27 White clover cryptic 3 Ð Partitiviridae Alphacryptovirus 28 Pelargonium zonate spot Ð Bromoviridae Anulavirus 29 Apple dapple viroid (Syn.) Apple scar skin viroid Ð Pospiviroidae Apscaviroid 30 Grapevine yellow speckle Ð Pospiviroidae Apscaviroid 31 Cucumber leaf spot carmovirus Ð Tombusviridae Aureusvirus 32 Avocado sunblotch Ð Avsunviroidae Avsunviroid 33 Banana streak Ð Caulimoviridae Badnavirus 34 Cacao swollen shoot Ð Caulimoviridae Badnavirus 35 Citrus mosaic Ð Caulimoviridae Badnavirus 36 Citrus yellow mosaic Ð Caulimoviridae Badnavirus 37 Kalanchoe top-spotting Ð Caulimoviridae Badnavirus 38 Piper yellow mottle Ð Caulimoviridae Badnavirus 39 Abutilon mosaic Ð Geminiviridae Begomovirus 40 Carrot temperate 2 Ð Partitiviridae Betacryptovirus 41 Hop trefoil cryptic 2 Ð Partitiviridae Betacryptovirus 42 White clover cryptic 2 Ð Partitiviridae Betacryptovirus 43 Broad bean mottle Ð Bromoviridae Bromovirus 44 Brome mosaic Ð Bromoviridae Bromovirus 45 Cowpea chlorotic spot virus Ð Bromoviridae Bromovirus 46 Oat mosaic Ð Bymovirus 47 Citrus tatter leaf Ð Flexiviridae Capillovirus (continued) 58 2 Identification and Taxonomic Groups

Table 2.1 (continued) S. no Species (virus/viroid) Order Family Genus 48 Carlavirus Tymovirales Betaflexiviridae Carlavirus 49 Clover (red) vein mosaic Tymovirales Betaflexiviridae Carlavirus 50 Cowpea mild mottle Tymovirales Betaflexiviridae Carlavirus 51 Pea streak Tymovirales Betaflexiviridae Carlavirus 52 Red clover vein mosaic Tymovirales Betaflexiviridae Carlavirus 53 Blackgram mottle Ð Tombusviridae Carmovirus 54 Cowpea mottle Ð Tombusviridae Carmovirus 55 Melon necrotic spot Ð Tombusviridae Carmovirus 56 Muskmelon necrotic spot Ð Tombusviridae Carmovirus 57 Pea stem necrosis Ð Tombusviridae Carmovirus 58 Cauliflower mosaic Ð Caulimoviridae Caulimovirus 59 Dahlia mosaic Ð Caulimoviridae Caulimovirus 60 Citrus leaf blotch Tymovirales Betaflexiviridae Citrivirus 61 Coconut cadang-cadang viroid Ð Pospiviroidae Cocadviroid 62 Coleus blumei viroid 1 Ð Pospiviroidae Coleviroid 63 Coleus blumei viroid 2 Ð Pospiviroidae Coleviroid 64 Coleus blumei viroid 3 Ð Pospiviroidae Coleviroid 65 Bean pod mottle Picornavirales Comovirus 66 Broad bean true mosaic Picornavirales Secoviridae Comovirus 67 Broad bean stain Picornavirales Secoviridae Comovirus 68 Cowpea mosaic Picornavirales Secoviridae Comovirus 69 Cowpea severe mosaic Picornavirales Secoviridae Comovirus 70 Echtes Ackerbohnen mosaic Picornavirales Secoviridae Comovirus (Syn. Broad bean true mosaic) 71 Muskmelon mosaic (Syn.) Picornavirales Secoviridae Comovirus Squash mosaic 72 Pea mild mosaic Picornavirales Secoviridae Comovirus 73 Red clover mottle comovirus Picornavirales Secoviridae Comovirus 74 Strawberry pallidosis Ð Closteroviridae Crinivirus 75 Banana viruses Ð Bromoviridae Cucumovirus 76 Cucumber mosaic (Syn.) Ð Bromoviridae Cucumovirus Cowpea banding mosaic (Syn.) Cowpea ring spot (Syn.) Soybean stunt 77 Peanut stunt Ð Bromoviridae Cucumovirus 78 Tomato aspermy Ð Bromoviridae Cucumovirus 79 Winged bean ring spot Ð Bromoviridae Cucumovirus 80 Beet curly top Ð Geminiviridae Curtovirus 81 Pea enation mosaic Ð Luteoviridae Enamovirus 82 Broad bean wilt Picornavirales Secoviridae Fabavirus 83 Nicotiana velutina mosaic Ð Virgaviridae Furovirus 84 Peanut clump (Indian)Ð Virgaviridae Furovirus 85 Wheat mosaic Ð Virgaviridae Furovirus 86 Wheat soil-borne mosaic Ð Virgaviridae Furovirus 87 Barley stripe mosaic(Syn. Ð Virgaviridae Hordeivirus Barley false stripe) 88 Lychnis ring spot Ð Virgaviridae Hordeivirus (continued) 2.2 Classification of Viruses 59

Table 2.1 (continued) S. no Species (virus/viroid) Order Family Genus 89 Hop stunt viroid (Syn.) Ð Pospiviroidae Hostuviroid Cucumber pale fruit viroid (Syn.) Grapevine viroid 90 Raspberry bushy dwarf (Syn.) ÐÐIdaeovirus Loganberry degeneration 91 Apple mosaic Ð Bromoviridae Ilarvirus 92 Asparagus latent (Syn.) Ð Bromoviridae Ilarvirus Asparagus virus II 93 Black raspberry latent Ð Bromoviridae Ilarvirus 94 Elm mottle Ð Bromoviridae Ilarvirus 95 Fragaria Chiloensis Ilarvirus Ð Bromoviridae Ilarvirus 96 Humulus Japonicas Ð Bromoviridae Ilarvirus 97 Hydrangea mosaic Ð Bromoviridae Ilarvirus 98 Lilac ring mottle Ð Bromoviridae Ilarvirus 99 Prune dwarf (Syn.) Cherry ring Ð Bromoviridae Ilarvirus mottle (Syn.) Cherry yellows 100 Prunus necrotic ring spot Ð Bromoviridae Ilarvirus (Syn.) Peach necrotic leaf spot (Syn.) Peach ring spot 101 Raspberry latent (Black Ð Bromoviridae Ilarvirus raspberry latent) 102 Spinach latent virus Ð Bromoviridae Ilarvirus 103 Sunflower ring spot Ð Bromoviridae Ilarvirus 104 Tobacco streak (Syn.) Ð Bromoviridae Ilarvirus Asparagus stunt (Syn.) Bean red node (Syn.) Datura quercina 105 Maize chlorotic mottle Ð Tombusviridae Machlomovirus machlomovirus 106 Olive latent virus-1 Ð Tombusviridae Necrovirus 107 Tobacco necrosis Ð Tombusviridae Necrovirus 108 Arabis mosaic Picornavirales Secoviridae Nepovirus 109 Arracacha virus A Picornavirales Secoviridae Nepovirus 110 Arracacha virus B Picornavirales Secoviridae Nepovirus 111 Artichoke Italian Latent Picornavirales Secoviridae Nepovirus 112 Artichoke yellow ring spot Picornavirales Secoviridae Nepovirus 113 Australian Lucerne latent Ð Secoviridae Nepovirus 114 Cacao necrosis Picornavirales Secoviridae Nepovirus 115 Cherry leaf roll Picornavirales Secoviridae Nepovirus 116 Cherry rasp leaf Picornavirales Secoviridae Nepovirus 117 Chicory yellow mottle Picornavirales Secoviridae Nepovirus 118 Crimson clover latent Picornavirales Secoviridae Nepovirus 119 Cycas necrotic stunt Picornavirales Secoviridae Nepovirus 120 Eucharis mottle Picornavirales Secoviridae Nepovirus 121 Grapevine Bulgarian latent Picornavirales Secoviridae Nepovirus 122 Grapevine fanleaf Picornavirales Secoviridae Nepovirus 123 Hibiscus latent ring spot Picornavirales Secoviridae Nepovirus 124 Lucerne (Australian) Picornavirales Secoviridae Nepovirus symptomless (continued) 60 2 Identification and Taxonomic Groups

Table 2.1 (continued) S. no Species (virus/viroid) Order Family Genus 125 Lucerne Australian latent Picornavirales Secoviridae Nepovirus 126 Mulberry ring spot Picornavirales Secoviridae Nepovirus 127 Peach rosette mosaic Picornavirales Secoviridae Nepovirus 128 Potato virus U Picornavirales Secoviridae Nepovirus 129 Raspberry ring spot Picornavirales Secoviridae Nepovirus 130 Rubus Chinese seed-borne Picornavirales Secoviridae Nepovirus 131 Satsuma dwarf Picornavirales Secoviridae Nepovirus 132 Sweet potato ring spot Picornavirales Secoviridae Nepovirus 133 Tobacco ring spot Picornavirales Secoviridae Nepovirus 134 Tomato black ring Picornavirales Secoviridae Nepovirus 135 Tomato ring spot (Syn.) Picornavirales Secoviridae Nepovirus Grapevine yellow vein 136 Clover (red) mosaic Mononegavirales Rhabdoviridae Nucleorhabdovirus 137 Coffee ring spot Mononegavirales Rhabdoviridae Nucleorhabdovirus 138 Maize mosaic Mononegavirales Rhabdoviridae Nucleorhabdovirus 139 Wheat striate mosaic Mononegavirales Rhabdoviridae Nucleorhabdovirus 140 Citrus psorosis Ð Ophioviridae Ophiovirus 141 Peach latent Ð Avsunviroidae Pelamoviroid 142 Beet mild yellowing Ð Luteoviridae Polerovirus 143 Carrot red leaf Ð Luteoviridae Polerovirus 144 Chrysanthemum stunt viroid Ð Pospiviroidae Pospiviroid 145 Citrus exocortis viroid Ð Pospiviroidae Pospiviroid 146 Pepper chat fruit viroid Ð Pospiviroidae Pospiviroid 147 Potato spindle tuber Ð Pospiviroidae Pospiviroid 148 Tomato apical stunt viroid Ð Pospiviroidae Pospiviroid 149 Tomato chlorotic dwarf viroid Ð Pospiviroidae Pospiviroid 150 Tomato planta macho viroid Ð Pospiviroidae Pospiviroid 151 Clover (white) mosaic Tymovirales Alphaflexiviridae Potexvirus 152 Clover yellow mosaic Tymovirales Alphaflexiviridae Potexvirus 153 Foxtail mosaic potexvirus Tymovirales Alphaflexiviridae Potexvirus 154 Hosta virus x Tymovirales Alphaflexiviridae Potexvirus 155 Pepino mosaic Tymovirales Alphaflexiviridae Potexvirus 156 Potato virus X Tymovirales Alphaflexiviridae Potexvirus 157 White clover mosaic Tymovirales Alphaflexiviridae Potexvirus 158 Mung bean mosaic Ð Potyviridae Poty virus 159 Artichoke latent Ð Potyviridae Potyvirus 160 Asparagus virus I Ð Potyviridae Potyvirus 161 Bean common mosaic (Syn.) Ð Potyviridae Potyvirus Bean western mosaic (Syn.) Azuki bean mosaic 162 Bean common mosaic necrosis Ð Potyviridae Potyvirus 163 Bean yellow mosaic Ð Potyviridae Potyvirus 164 Blackeye cowpea mosaic Ð Potyviridae Potyvirus 165 Bramble yellow mosaic Ð Potyviridae Potyvirus 166 Broad bean mild mosaic Ð Potyviridae Potyvirus 167 Cassia yellow spot (poty)Ð Potyviridae Potyvirus 168 Celery latent Ð Potyviridae Potyvirus 169 Cowpea aphid-borne mosaic Ð Potyviridae Potyvirus (continued) 2.2 Classification of Viruses 61

Table 2.1 (continued) S. no Species (virus/viroid) Order Family Genus 170 Cowpea green vein-banding Ð Potyviridae Potyvirus 171 Cowpea moroccan aphid-borne mosaic Ð Potyviridae Potyvirus 172 Desmodium mosaic Ð Potyviridae Potyvirus 173 Guar symptomless Ð Potyviridae Potyvirus 174 Hippeastrum mosaic Ð Potyviridae Potyvirus 175 Leek yellow stripe Ð Potyviridae Potyvirus 176 Lettuce mosaic Ð Potyviridae Potyvirus 177 Maize dwarf mosaic Ð Potyviridae Potyvirus 178 Onion yellow dwarf Ð Potyviridae Potyvirus 179 Papaya ring spot Ð Potyviridae Potyvirus 180 Pea mosaic (Syn. Bean yellow mosaic)Ð Potyviridae Potyvirus 181 Pea seed-borne mosaic (syn. pea fizzle top Ð Potyviridae Potyvirus and Pea leaf rolling virus) 182 Peanut mottle Ð Potyviridae Potyvirus 183 Peanut stripe (Syn. Peanut mild mottle)Ð Potyviridae Potyvirus 184 Plum pox virus Ð Potyviridae Potyvirus 185 Potato virus Y (Syn.) Brinjal mosaic Ð Potyviridae Potyvirus 186 Soybean mosaic Ð Potyviridae Potyvirus 187 Sugarcane mosaic Ð Potyviridae Potyvirus 188 Sunflower mosaic potyvirus Ð Potyviridae Potyvirus 189 Telfairia mosaic Ð Potyviridae Potyvirus 190 Turnip mosaic Ð Potyviridae Potyvirus 191 Watermelon mosaic Ð Potyviridae Potyvirus 192 Zucchini yellow mosaic Ð Potyviridae Potyvirus 193 Bean southern mosaic (Syn. Southern ÐÐ Sobemovirus bean mosaic) 194 Lucerne transient streak ÐÐ Sobemovirus 195 Panicum mosaic ÐÐ Sobemovirus 196 Sowbane mosaic ÐÐ Sobemovirus 197 Subterranean clover mottle ÐÐ Sobemovirus 198 Cucumber green mottle mosaic Ð Virgaviridae Tobamovirus 199 Paprika mild mottle tobamovirus Ð Virgaviridae Tobamovirus 200 Pepper mild mottle Ð Virgaviridae Tobamovirus 201 Pepper mild mottle tobamovirus Ð Virgaviridae Tobamovirus 202 Sunn-hemp mosaic (Syn.) Sunn-hemp Ð Virgaviridae Tobamovirus rosette 203 Tobacco mosaic Ð Virgaviridae Tobamovirus 204 Tomato mosaic Ð Virgaviridae Tobamovirus 205 Pea early browning Ð Virgaviridae Tobravirus 206 Tobacco rattle Ð Virgaviridae Tobravirus 207 Tomato bushy stunt Ð Tombusviridae Tombusvirus 208 Tomato spotted wilt Ð Bunyaviridae Tospovirus 209 Apple chlorotic leaf spot Tymovirales Betaflexiviridae Trichovirus 210 Wheat streak mosaic Ð Potyviridae Tritimovirus 211 Dulcamara mottle Tymovirales Tymoviridae Tymovirus 212 Eggplant mosaic (Syn. Andean Tymovirales Tymoviridae Tymovirus potato latent) (continued) 62 2 Identification and Taxonomic Groups

Table 2.1 (continued) S. no Species (virus/viroid) Order Family Genus 213 Melon rugose mosaic Tymovirales Tymoviridae Tymovirus 214 Potato Andean latent Tymovirales Tymoviridae Tymovirus 215 Turnip yellow mosaic Tymovirales Tymoviridae Tymovirus 216 Sunflower rugose mosaic Ð Luteoviridae Umbravirus 217 Cherry necrotic rusty mottle Tymovirales Betaflexiviridae Unassigned 218 Potato virus T Tymovirales Betaflexiviridae Unassigned 219 Strawberry latent ring spot Picornavirales Secoviridae Unassigned 220 High plains virus Ð Ð Unassigned 221 Urdbean leaf crinkle (Syn.) Ð Ð Unassigned Black gram leaf crinkle (Syn.) bean urd leaf crinkle virus 222 Barley mottle mosaic Ð Ð Unassigned 223 Brinjal ring mosaic Ð Ð Unassigned 224 Brinjal severe mosaic Ð Ð Unassigned 225 Carrot motley leaf Ð Ð Unassigned 226 Cherry ring spot Ð Ð Unassigned 227 Cineraria mosaic Ð Ð Unassigned 228 Mung bean isometric yellow Ð Ð Unassigned mosaic 229 Parsley latent Ð Ð Unassigned 230 Peanut marginal chlorosis Ð Ð Unassigned 231 Soybean mild mosaic Ð Ð Unassigned

presented by taking the information from Fauquet (3) groups (Tables 2.1 and 2.2). On the other et al. (2005) and King et al. (2011) classification hand, in some groups such as Alfamovirus, tables. The seed-transmitted viruses are confined Capillovirus, Caulimovirus, Enamovirus, to 34 virus groups, which are in Table 2.2.To Fabavirus, Hordeivirus, Necrovirus, Tobravirus, have more clarity, the information on a particle Tombusvirus and Tospovirus groups, the seed- morphology and genome and vector is also transmitted viruses recorded were very few. No provided. seed transmission was noticed in Closterovirus, Among these plant virus groups, 231 well- Dianthovirus, Geminivirus, Luteovirus, Parsnip characterised seed-transmitted viruses are yellow fleck virus, Reovirus, Rhabdovirus, distributed in 24 virus groups including Al- Tenuivirus and Waikavirus groups. famovirus, Bromovirus, Capillovirus, Carlavirus, Carmovirus, Caulimovirus, Comovirus, Cryp- tovirus, Cucumovirus, Enamovirus, Fabavirus, 2.3 Variability in Certain Furovirus, Hordeivirus, Ilarvirus, Necrovirus, Seed-Transmitted Viruses Nepovirus, Potexvirus, Potyvirus, Sobe- movirus, Tobamovirus, Tobravirus, Tombusvirus, From the above discussed aspects on plant virus Tospovirus and Tymovirus groups. Greater taxonomy, it is clear that the viruses resembling number of seed-transmitted viruses are found each other in genome properties and virion mor- in Potyvirus (35), Nepovirus(28), Cryptovirus phology are grouped into genera and given genus (28), Ilarvirus (14), Tobamovirus (7), Po- names. The phylogenic analysis of some of the texvirus (7), Comovirus (6), Carlavirus (5), viruses indicates that recently described viruses Carmovirus (5), Cucumovirus (5), Sobemovirus are established based on molecular studies as (5), Furovirus (4), Bromovirus (3) and Tymovirus strains of the earlier described viruses. More 2.3 Variability in Certain Seed-Transmitted Viruses 63 (continued) 0 No. of Seed- transmitted viruses 10 ssRNA Aphid 0 11 ssRNA Aphid, whitefly 5 20 ssRNA20 Fungus ssRNA Ð 4 2 20 18 ssRNA Aphid 2 12 ssRNA Ð 1 30 ssRNA Leaf- hopper Ð 30 ssRNA Whitefly Ð 92Ð160 orm 28Ð58 Particle morphology Particle size (nm) Genome Vector Bacillif IsometricFlexuousFlexuousIsometric 26 640 Flexuous rods 620Ð700 Isometric 28Ð30 600Ð2,000 28 ssRNAIsometricIsometric ssRNA BeetleIsometricIsometric 28 BeetleRigid rods 31Ð34 28 ssRNA 30 3 280Ð330 BeetleRigid 5 rods ssRNA dsRNAIsometricIsometric ssRNA Aphid ÐIsometric 100Ð150 ssRNAIsometric 26Ð35 6 Aphid 25 Beetle 31 28 5 ssRNA 0 1 1 ssRNA Thrips ssRNA ssRNA Aphid Leaf- hopper Fungus 14 0 0 2 GeminateGeminate 18 18 Isometric 50 dsDNA Aphid 2 IsometricIsometric 30 38 ssRNA dsRNA Ð Ð 25 3 1 2 - - ent taxonomic groups of plant viruses and their characteristic features borne wheat mosaic - Group A mastroGroup B begomo Maize streak virus African cassava mosaic Subgroup A White clover cryptic virus Subgroup B White clover cryptic virus Alfamo virusBromovirusCapillovirusCarlavirusCarmovirus Alfalfa mosaic Caulimovirus Brome mosaic Closterovirus Apple stem grooving virus Comovirus CarnationCryptovirus latent virus Carnation mottle virus Cauliflower mosaic virus Beet yellows virus CucumovirusDianthovirus Cowpea mosaic virus Enamo virusFabavirusFurovirus Cucumber mosaic virus Carnation ring spot virus Geminivirus Pea enation mosaic virus Broad bean wilt virus Hordeivirus Soil IlarvirusLuteovirusMarafivirusNecrovirus Barley stripe mosaic virus Tobacco streak virus Barley yellow dwarf virus Maize rayadofino virus Tobacco necrosis virus Seed-transmitted viruses in differ Table 2.2 Sl. no.1 2 Genus/group3 4 5 6 Type member 7 8 9 10 11 12 13 14 15 16 17 18 19 20 64 2 Identification and Taxonomic Groups No. of Seed- transmitted viruses 22 ssRNA Nematode 2 1311 ssRNA ssRNA50Ð95 Ð Aphid, whitefly, mite ssRNA 35 Aphid, 0 7 22 22 8 ssRNA Plant- hopper 0 18 ssRNA Ð 7 400 160Ð215 14Ð114 14Ð114 > orm 160Ð380 Particle morphology Particle size (nm) Genome Vector rigid rods Isometric 71 dsRNA Plant- hopper 0 Isometric 70 dsRNA Leaf- hopper 0 IsometricIsometricFlexuous 28 rodsFlexuous 30 rods 470Ð580 680Ð900 Bacillif ssRNAIsometric ssRNAFlexuous NematodeRigid rods AphidShort 28Ð30 & long Isometric 300 Isometric 28 IsometricIsometric 30 ssRNA 0 85 29 Beetle 25 ssRNA ssRNA ssRNA Ð 5 ssRNA Thrips Beetle Leaf- hopper 1 0 1 3 Parsnip yellow fleck virus Fijivirus Fiji disease virus Phytoreo Wound tumour virus ) ) b a ( ( NepovirusParsnip yellow fleck virus Potexvirus TobaccoPotyvirus ring spot virus Reovirus Potato virus X Rhabdovirus Potato virus Y SobemovirusTenuivirus Lettuce necrotic yellows virus TobamovirusTobravirus Southern bean mosaic virus Rice stripeTombusvirus virus Tobacco mosaic virus Tospovirus Tobacco rattle virus TymovirusWaikavirus Tomato bushy stunt virus Tomato spotted wilt virus Turnip yellow mosaic virus Maize chlorotic dwarf (continued) Table 2.2 Sl. no.21 Genus/group22 23 24 Type member 25 26 27 28 29 30 31 32 33 34 References 65 examples one can find are in potyvirus group. and other abstract journals for locating subject Viruses like Blackeye cowpea mosaic and Peanut literature for use in their research work. Since stripe virus are nothing but strains of the Bean 1990, lot of databases and websites are available common mosaic virus with little variation. Vari- which are quite useful for day-to-day plant ability in viruses is noticed in almost all described virus research work, where even the poor virus groups. The molecular structure of viruses library facilities existed and such problems were has the capacity of transferring its characters to solved by certain international organisations. For its duplicates produced in the host cells. example, the ‘Crop Protection Compendium’ (a) Hybridisation: This is one of the means by (CPC), which was available on CD-ROM, which new virus strains are formed. If two was published by CAB International, which strains of virus are inoculated into the same is being updated annually on http://www. host plant, one or more new virus strains cabicompendium.org/cpc. Another valuable may be recovered with properties (virulence, information source for viruses is of AAB symptomatology, etc.) different from those of descriptions on plant viruses and can be accessed either of the original strains introduced into on http://www.dpvweb.net. Even the descriptions the host. These new strains are probably hy- and lists from VIDE Database of the International brids (RNA or DNA recombinants). Albersio Committee on Taxonomy of Viruses are available et al. (1975) reported variability in Squash on http://www.ncbi.nlm.nih.gov/ICTVdb/index. mosaic virus (SqMV) by hybridisation be- htm. ‘The Plant Pathology Internet Guide Book’ tween two strains of virus. They had crossed (http://www.pk.uni-bonn.de/ppigb/ppigb.htm)is strain 1 H and II A of SqMV in pumpkin another source of information for many virology (Cucurbita pepo) and cantaloupe (Cucumis topics. Because of the availability of databases melo) plants and observed the interaction of plant viruses on internet, it has become much between them. easier for the virus research workers to know the (b) Mutations: The evolution of new strains of latest progress that is happening in any corners of viruses may also be due to mutation. These the world (http://www.virology.net). may be a heritable change in the genetic material (RNA or DNA). The production of mutants differing in virulence has also been reported in several viruses, especially TMV, References although they seem to vary mostly in the type of symptoms and severity of disease they Albersio J, Lima A, Nelson MR (1975) Squash mosaic produce rather than in their ability to infect virus variability: nonreciprocal cross-protection be- different host plant varieties. tween strains. Phytopathology 65:837Ð840 Bos L (1964) Tentative list of viruses reported from nat- The previous reports when the advanced iden- urally infected leguminous plants. Neth J Plant Pathol tification techniques were not available have re- 70:161Ð174 ported very negligible (<1%) percentage of seed Bos L (1976a) Problems and prospects in plant virus transmission in certain virusÐhost combinations, identification. EPPO Bull 6:63Ð90 Bos L (1976b) Research on plant virus diseases in the which the later research workers have disproved. developing countries: possible ways for improvement. Without any discrimination based on the avail- FAO Plant Prot Bull 24(4):109Ð118 able literature all the seed-transmitted viruses, Boswell K, Gibbs A (1986) The VIDE data bank for plant viruses. In: Jones RAC, Torrance L Cryptoviruses and viroid diseases were reported (eds) Developments and applications in virus test- in the Tables 1.2, 2.1 and 2.2. ing. Association of Applied Biologists, Warwickshire, In the earlier days, when the computer pp 283Ð287 knowledge was not available, the students, Brown F (1989) The classification and nomenclature of viruses. Summary of results of meetings of Interna- research workers and others used to depend tional Committee on Taxonomy of Viruses. Edmonton, on Review of Plant Pathology, review articles Canada 1987. Intervirology 30:181Ð186 66 2 Identification and Taxonomic Groups

Brunt AA, Crabtree K, Dallwitz MJ, Gibbs AJ, Watson King AMQ, Lefkowitz E, Adams MJ, Carstens EB (2011) L (eds) (1996) Viruses of plants. Description and lists Virus taxonomy: 9th report of the international com- from VIDE data base. CAB International, Wallingford, mittee on taxonomy of viruses. Elsevier, San Diego p 1484 Martelli GP (1992) Classification and nomenclature Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball of plant viruses: state of the art. Plant Dis 76: LA (2005) Virus taxonomy, VIIIth report of the ICTV. 436Ð442 Elsevier Academic Press, London, pp 751Ð756 Matthews REF (1979) Classification and nomenclature Francki RIB (1981) Plant virus taxonomy. In: Kurstak E of viruses. Third report of the International Commit- (ed) Handbook of plant virus infection and compara- tee on the Taxonomy of Viruses. Intervirology 12: tive diagnosis. Elsevier, Amsterdam, 3 131Ð296 Gibbs AJ (1989) A virus database for aiding plant Matthews REF (1982) Classification and nomenclature of pathologists. In: Proceedings of the 2nd meeting of viruses. Fourth report of the International Committee peanut stripe virus coordinators, held at ICRISAT, on the Taxonomy of Viruses. Intervirology 17:1Ð199 Patancheru, India Van Regenmortal MHV, Fauquet CM, Bishop DHL, Gibbs AJ, Harrison BD (1976) Plant virology: the princi- Carstens E, Estes M, S, Maniloff J, Mayo ples. Edward Arnold, London, p 292 M, Mc Geoch D, Pringle C, Wickner R (2000) Virus Hamilton RI, Edwardson JR, Francki RIB, Hsu HT, Hull taxonomy: Seventh report of the International Com- R, Koenig R, Milne RG (1981) Guidelines for iden- mittee on Taxonomy of Viruses. Academic Press, New tification and characterization of plant viruses. J Gen York/San Diego Virol 54:223Ð241 Van Regenmortel HR, Bishop DHL, Van Regenmortel Harrison BD, Finch JT, Gibbs AJ, Hollings M, Shepherd MH, Fauquet C (2005) Virus taxonomy: Eighth re- RJ, Valenta V, Wetter C (1971) Sixteen groups of plant port of the International Committee on Taxonomy of viruses. Virology 45:356Ð363 Viruses. Academic Press, San Diego Economic Significance of Seed-Transmitted Plant Virus 3 Diseases

Abstract The assessment of yield losses due to virus diseases is particularly difficult to estimate accurately both qualitatively and quantitatively. Yield losses are significantly greater when the plants are infected at early stages. Attempts were made to compile the available yield loss data due to seed- transmitted virus diseases in different crop plants. Increased yield losses would also result due to mixed infection of two or more viruses which exhibit synergistic effect. The extent of yield loss varies with the cultivar, stage of infection, virus strain and environmental factors. The yield loss estimates give the necessity of framing suitable virus management measures under field conditions and also stresses to develop resistant cultivars.

crop losses varies considerably in precision as the 3.1 Introduction severity of the disease varies greatly with factors like crop variety, virus strain, locality, the activity Some of the seed-transmitted viral diseases are of the vectors, nutritional status of the infected cosmopolitan in distribution. They greatly influ- crop, etc. ence man’s economic and social facets of life by reducing the yield and quality of plant prod- ucts. The extent of crop loss depends mostly on the disease intensity and its distribution. The 3.2 Assessment of Crop Losses losses caused by the viral and viroid diseases are difficult to estimate unless crops are completely The assessment of losses due to viral diseases destroyed. Farmers and government agencies are is particularly difficult because it is extremely still faced with questions like (1) how damaging hard to prevent contamination of healthy control the viruses are, (2) which areas are the most dam- plants. Similarly, inoculation under vector-proof aging, (3) how yield reductions can be assessed conditions in mesh/glass houses may not reflect on a farm, in a district, a country or a region the true picture of natural field conditions. and (4) how such losses can be predicted. Precise Generally, yield losses are significantly greater answers are not available for virus diseases, and when the plants are infected at early stages. plant virology confronts with the lacunae in the Virus infection causes not only quantitative loss appraisal of losses. The information available on of the harvested product in terms of weight and

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 3, 67 © Springer India 2013 68 3 Economic Significance of Seed-Transmitted Plant Virus Diseases number of units but qualitatively for taste and In cowpea, a yield loss of 13Ð87% and nutritive composition. Estimation of losses based 64Ð75% due to Cowpea mosaic virus was on yield comparisons between plots of inoculated recorded by Kaiser and Mossahebi (1975) and uninoculated plants mostly represents only and Suarez and Gonzalez (1983), respectively. the maximum loss caused by the virus, but rarely While another seed-transmitted virus of cowpea, 100% infection takes place under natural condi- Cowpea banding mosaic virus (CpBMV) reduced tions (Irwin and Schultz 1981; Irwin et al. 2000; the seed yield to 11.5Ð43.5% (Sharma and Varma Ruesink and Irwin 2006). 1981a). Even though in this chapter several examples In Montana, an incidence of 90% Barley stripe of yield losses in different virusÐhost combina- mosaic virus (BSMV) has reduced barley yields tions are furnished, two major crops like barley by 35Ð40% (Eslick 1953). Catherall (1972)has and lettuce have faced very significant enormous reported 62% grain yield loss in barley cultivar yield losses due to Barley stripe mosaic virus Akka. The same virus has caused yield reduction in Montana (USA) and Lettuce mosaic virus in in winter wheat by 19% (Fitzgerald and Timian California (USA), respectively (Grogan 1980; 1960). In currency the estimated total loss in Carroll 1983). barley due to BSMV exceeded $30 million during Some outstanding examples of crop loss 1953Ð1970 (Carroll 1980). Similarly, from West- estimates due to viruses have been reported by ern Australia, Coutts et al. (2008) have reported Bos (1981, 1982) and Waterworth and Hadidi yield losses in lentil cv. Nugget, due to PSbMV; (1998) in their review articles. To quote few the shoot dry weight decrease was 23% and seed examples of crop loss estimates: A 0.1% LMV yield by 96% and individual seed weight by 58%. seed infection resulted in total crop loss of The crop loss estimates are also available for lettuce (Broadbent et al. 1951; Grogan et al. lucerne (Medicago sativa) infected with Alfalfa 1952; Zink et al. 1956). In the USA an estimated mosaic virus (AMV) (Hemmati and McLean annual loss of 4% was caused by the same virus 1977; Bailiss and Ollennu 1986; Jones and during 1951Ð1960 (USDA 1965). On the other Pathipanawat 1989)andVigna angularis and hand, yield loss of 50Ð68% due to BCMV in Broad bean (Latham et al. 2004); asparagus bean crop has been reported by a number of bean infected with Cowpea aphid-borne mosaic workers (Lockhart and Fischer 1974; Hampton (Chang and Kno 1983; Giesler et al. 2002; 1975). Natural aphid transmission of Soybean Latham et al. 2004); soybean with Bean pod mosaic virus (SMV) caused up to 35% loss in mottle virus (Hopkins and Mueller 1984;Ross seed yield in susceptible soybean cultivars (Ross 1986; Giesler et al. 2002) and SMV (Irwin 1977), while mechanical inoculation of soybean and Schultz 1981; Hill et al. 1987;Tu1989); seedlings resulted in yield reductions up to 86% wheat with Indian peanut clump virus (Delfosse (Goodman and Oard 1980). From the USA, yield et al. 1999); barley with BSMV (Nutter et al. losses in certain soybean cultivars were between 1984); French bean with Southern bean mosaic 8 and 25% due to SMV strains (Ross 1969a). virus (Morales and Castano 1985) and BCMV The Peanut mottle virus (PMV) reduced the (Hampton 1975;Omaretal.1978;Sastry peanut seed yield from 31 to 47% (Kuhn et al. et al. 1981; Nakano et al. 1988; Rao et al. 1978), and in Georgia (USA) yield losses ex- 1990; Vishwadhar and Gupta 1990); maize ceeded around $10 million per annum (Paguio with Maize dwarf mosaic virus (Scott et al. and Kuhn 1974; Kuhn et al. 1978). The losses 1988); peanut with Peanut mild mottle virus due to Peanut stripe virus (PStV) were 23 and (PMMV); Peanut mottle virus (Kuhn 1965; 21%, respectively, in peanut cultivars Argentine Idris and Ahmed 1981; Puttaraju et al. 2001); and Florunner (Demski et al. 1984). However, in Cucumber mosaic virus (CMV) (Xu and Zhang Virginia (USA), Peanut stunt virus caused 80% 1986); pea with PSbMV (Kraft and Hampton loss of matured nuts in three commercial peanut 1980; Ovenden and Ashby 1981; Khetrapal fields (Culp and Troutman 1967). et al. 1988; Ali and Randles 1998; Coutts 3.4 Factors Affecting Yield Losses 69 et al. 2009) and lentil with PSbMV (Coutts (Blattny and Osvald 1954). Seeds of spurrey, et al. 2008); urd bean with Urdbean leaf crinkle Spergula arvensis, infected by Tomato black ring virus (ULCV) (Nene 1972; Beniwal et al. 1979; virus germinated more slowly than healthy seeds, Kadian 1982; Indu Sharma and Dubey 1984); urd but there was little or no effect on seedlings bean with BCMV (Agarwal et al. 1976); pepper growth (Lister and Murant 1967). with TMV (Feldman et al. 1969); lettuce with LMV (Ryder and Duffus 1966); subterranean clover with CMV (Jones and Mc Kirdy 1990; 3.4 Factors Affecting Jones 1991); asparagus lettuce with a strain of Yield Losses Lettuce mosaic virus (Xia et al. 1986); Peanut clump virus in wheat (Delfosse et al. 1999); Usually, virus infection at early stages of the sorghum with Sugarcane mosaic virus (Mock crop causes greater loss in yield. For example, et al. 1985); urd and mung bean with Leaf Fletcher et al. (1969) reported 15% yield loss in crinkle disease (Beniwal and Bharathan 1979; with Cucumber green mottle mosaic Chattopadhyay et al. 1986; Manadhare et al. virus (CGMV) when infected at the very early 1999; Negi and Vishunavat 2004, 2006; Ravinder stage. Similarly in soybean, Tobacco ring spot Reddy et al. 2005b;Sharmaetal.2007); lentil virus (TRSV) infection caused a yield reduction with Bean yellow mosaic virus (Kumari et al. of 60% in 80% infected plants, while early in- 1994), Broad bean stain virus (Makkouk and fection resulted in yield loss of 79% (Crittenden Kumari 1990), Alfalfa mosaic virus (Latham and et al. 1966). Puttaraju et al. (2004a, b) observed Jones 2001a), PSbMV (Kumari and Makkouk that early infection in cowpea with BCMVÐICM 1995; Mabrouk and Mansour 1998)andBroad has led to severe yield losses, while infection at bean stain virus (Mabrouk and Mansour 1998); 50 days did not differ much from those of healthy cowpea with Blackeye cowpea mosaic (Puttaraju cowpea plants. Similarly, Bean yellow mosaic et al. 2002; 2004a); lupin with CMV (Bwye virus infection of lentil crop at pre-flowering et al. 1994); chickpea with Tobacco streak virus stage and flowering stage caused yield losses of (Kaiser et al. 1991;Kumarietal.1996); and 96 and 34%, respectively (Kumari et al. 1994). raspberry with Raspberry bushy dwarf disease Increased yield losses would also result due (Jones 1979). It is very difficult to get data on to mixed infection of two or more viruses which exact yield losses in different host and virus exhibit synergistic effect and lead to greater yield infections. There are several obvious reasons for losses than yield loss from either virus alone. not having accurate yield loss data which will Demski and Jellum (1975) recorded average probably remain the foreseeable future. Among yield losses of soybean to be 18, 31, 66 and them are variations in losses by a particular 76% for single infection by PMV, Cowpea virus in a particular crop from year to year, chlorotic mottle virus (CCMV), SMV and different degrees of loss among regions within TRSV, respectively. However, in doubly infected the same year, differences in loss assessment plants, the yield loss percentage was 46 (PMVÐ methodologies and on different agronomical CCMV), 78 (PMVÐSMV), 80 (PMVÐTRSV), practices followed, etc. 82 (CCMVÐTRSV), 87 (CCMVÐSMV) and 98 (SMVÐTRSV) along with decrease in oil content. Similar synergistic reaction and increased yield 3.3 Viruses and Seed Viability losses were recorded with Bean pod mottle and Soybean mosaic viruses in soybean (Ross 1968) Some viruses directly affect the viability of seed. and Pea enation mosaic virus and Pea seed-borne A mild strain of seed-transmitted hop infecting mosaic virus in pea (Bossennec et al. 2000). Even virus (Prunus necrotic ring spot virus) caused a in the case of cowpea stunt (which is a resultant reduction of 20% in germination, while a severe of synergism between Blackeye cowpea mosaic strain reduced germination by not less than 90% and CMV) in cowpea cv. California, the yield 70 3 Economic Significance of Seed-Transmitted Plant Virus Diseases loss was 86.4%, while for the individual viruses, Beniwal SPS, Kolte SJ, Nene YL (1979) Nature and rate it was 2.5 and 14.2%, respectively (Pio-Ribeiro of spread of urd bean leaf crinkle diseases under field et al. 1978). conditions. Indian J Mycol Plant Pathol 9:188Ð192 Blattny C, Osvald V (1954) Pfenos viros chmele (Hu- Losses caused by virus and its vector some- mulus lupulus L.) na potomsto semenem. Preslia, times may cause huge yield losses. For example, (Prague) 26:1Ð26 the yield reduction of broad bean seeds was 6% Bos L (1981) Wild plants in the ecology of virus diseases. due to infection with Pea enation mosaic alone, In: Maramorosch K, Harris KF (eds) Plant diseases and vectors: ecology and epidemiology. Academic, 50% by the infestation of Aphis fabae as pest New York, pp 1Ð33 alone, but 93% due to both virus and aphid vector Bos L (1982) Crop losses caused by viruses. Crop Prot (Hinz and Daebeler 1979). Similar type of studies 1:263Ð282 Bossennec JM, David O, Demange N, Faivre B, Letarnec has to be carried out in some more hostÐvirusÐ B, Palluault M, Taupin P, Maury Y (2000) Incidence vector combinations to get clear understanding on of viruses on yield of field pea. Phytoma 526:21Ð24 these aspects. Broadbent L, Tinsley TW, Buddin W, Roberts ET (1951) The yield loss estimates varied depending on The spread of lettuce mosaic in the field. Ann Appl Biol 38:689Ð706 the cultivar, the virus strain and the stage of Bwye AM, Jones RAC, Proudlove W (1994) Effects of infection. For example, at Georgia (USA), Peanut sowing seed with different levels of infection, plant mottle virus (PMV) strain-N in peanuts reduced density and he growth stage at which plant first de- seed yield by 68% in the cv. Starr and 47% velop symptoms on cucumber mosaic virus infection of narrow leafed lupins lupines angustifolius). Aust in PI-261945, but no losses were reported in PI- Gen Agric Res 45:1395Ð1412 261946. The PMV strain-M2 caused 31% loss in Carroll TW (1980) Barley stripe mosaic virus: its eco- seed yield in the cv. Starr and 20% loss in the nomic importance and control in Montana. Plant Dis above two introductions (Kuhn et al. 1978). Tu 64:135Ð140 Carroll TW (1983) Certification schemes against barley (1989) studied the impact of 7 strains of SMV stripe mosaic. Seed Sci Technol 11:1033Ð1042 on seed yield in 8 soybean cultivars including Catherall PL (1972) Barley stripe mosaic virus. Rept. Oxley 615, which is a resistant line. Similarly, Welsh Plant Breed Stn 1971:62 the extent of yield losses varies with the age of Chang CA, Kno YJ (1983) Cowpea aphid borne mosaic virus and its effect on the yield and quality of aspara- the crop infected. From the examples cited, it is gus bean. J Agric Res China 32(3):270Ð278 clear that loss estimates vary and are influenced Chattopadhyay AK, Bhatttacharya S, Dutta SK (1986) by a number of factors. Practical quantitative Assessment of loss in mungbean cultivars due to leaf assessment methods and the prediction of crop crinkle disease. Int J Trop Plant Dis 4:73Ð76 Coutts BA, Prince RT, Jones RAC (2008) Further studies losses caused by seed-transmitted viruses are still on Pea seed Ð borne mosaic virus in cool Ð season in their infancy. crop legumes: responses to infection and seed quality defects. Aust J Agric Res 59:1130Ð1145 Coutts BA, Prince RT, Jones RAC (2009) Quantifying effects of seedborne inoculum on virus spread, yield losses and seed infection in the Pea seed Ð borne References mosaic virus Ð field pea pathosystem. Phytopathology 99:1156Ð1167 Agarwal VK, Nene YL, Beniwal SPS (1976) Influence Crittenden HW, Hastings KM, Moore DM (1966) Soy- of bean common mosaic virus infection on the flower bean losses caused by tobacco ring spot virus. Plant organelles, seed characters and yield of urd bean. Dis Rep 50:910Ð913 Indian Phytopathol 29:444Ð446 Culp TW, Troutman JL (1967) Reduction in yield and Ali A, Randles JW (1998) The effects of two pathotypes quality of peanuts, Arachis hypogaea by stunt virus. of pea seed-borne mosaic virus on the morphology and Plant Dis Rep 51:856Ð860 yield of pea. Aust Plant Pathol 27:226Ð233 Delfosse P, Reddy AS, Legreve A, Devi PS, Devi KT, Bailiss KW, Ollennu LLA (1986) Effect of alfalfa mosaic Maraite H, Reddy DVR (1999) Indian peanut clump virus isolates on forage yield of lucerne (Medicago virus (IPCV) infection on wheat and barley: symp- sativa) in Britain. Plant Pathol 35(2):162Ð168 toms, yield loss and transmission through seed. Plant Beniwal SPS, Bharathan N (1979) Urd bean leaf crin- Pathol 48:278Ð282 kle virus: effect on yield contributing factors, total Demski JW, Jellum MD (1975) Single and double virus in- yield and seed characters of Urd bean. Seed Res fection of soybeans. Plant characteristics and chemical 7:175Ð181 composition. Phytopathology 75:1154Ð1156 References 71

Demski JW, Reddy DVR, Sowell G Jr (1984) Stripe Jones RAC, Mc Kirdy SJ (1990) Seed Ð borne cucumber disease of groundnuts. FAO Plant Prot Bull 32: mosaic virus infection of Subterranean clover in West- 114Ð115 ern Australia. Ann Appl Biol 115:263Ð277 Eslick RF (1953) Yield reduction in Glacier barley associ- Jones RAC, Pathipanawat W (1989) Seed-borne Alfalfa ated with a virus infection. Plant Dis Rep 37:290Ð291 mosaic virus infecting annual medics (Medicago spp) Feldman JM, Gracia O, Pontis RE, Borinsegna U (1969) in Western Australia. Ann Appl Biol 115:263Ð277 Effect of tobacco mosaic virus on pepper yield. Plant Kadian OP (1982) Yield loss in mungbean and urd- Dis Rep 53:541Ð543 bean due to leaf crinkle diseases. Indian Phytopathol Fitzgerald PJ, Timian RG (1960) Effect of barley stripe 35:642Ð644 mosaic on wheat. Plant Dis Rep 44:359Ð361 Kaiser WJ, Mossahebi GH (1975) Studies with cowpea Fletcher JT, George AJ, Green DE (1969) Cucumber green aphid-borne mosaic virus and its effect on cowpea in mottle mosaic virus, its effect on yield and its control Iran. FAO Plant Prot Bull 23(2):33Ð39 in the Lea Valley, England. Plant Pathol 18:16Ð22 Kaiser WJ, Wyatt SD, Klein RE (1991) Epidemiol- Giesler LJ, Ghabrial SA, Hunt TE, Hill JH (2002) Bean ogy and seed transmission of two tobacco streak pod mottle virus Ð a threat to US soybean production. virus pathotypes associated with seed increases of Plant Dis 86:1280Ð1289 legume germplasm in Eastrus Washington. Plant Dis Goodman RM, Oard JH (1980) Seed transmission and 75:258Ð264 yield losses in tropical soybeans infected by soybean Khetrapal RK, Bossennec JM, Burghofer A, Cousin R, mosaic virus. Plant Dis 64:913Ð914 Maury Y (1988) Effet du pea seed-borne mosaic virus Grogan RG (1980) Control of lettuce mosaic virus with sur le rendement en graines du pois proteagineux. virus-free seed. Plant Dis 64:446Ð449 Agronomic 8(9):811Ð815 Grogan RG, Welch JE, Bardin R (1952) Common lettuce Kraft JM, Hampton RO (1980) Crop losses from pea mosaic and its control by the use of mosaic free seed. seedborne mosaic virus in six processing pea cultivars. Phytopathology 42:573Ð578 Plant Dis 64:922Ð924 Hampton RO (1975) The nature of bean yield reduction Kuhn CW (1965) Symptomatology, host range and effect by bean yellow and bean common mosaic virus. Phy- on yield of seed-transmitted peanut virus. Phytopathol- topathology 65:1342Ð1346 ogy 55:880Ð884 Hemmati K, McLean DL (1977) Gamete-seed transmis- Kuhn CW, Paguio OR, Adams DB (1978) Tolerance sion of alfalfa mosaic virus and its effect on seed ger- in peanuts to peanut mottle virus. Plant Dis Rep mination and yield in alfalfa plants. Phytopathology 62:365Ð368 67:576Ð579 Kumari SG, Makkouk KM (1995) Variability among Hill JH, Bailey TB, Benner HI, Tachibana H, Durand twenty lentil genotypes transmission rates and yield DP (1987) Soybean mosaic virus. Effects of primary loss induced by Pea seed Ð borne mosaic potyvirus disease incidence on yield and seed quality. Plant Dis infection. Phytopathol Mediterr 34:129Ð132 71:237Ð239 Kumari SG, Makkouk KM, Ismail ID (1994) Seed trans- Hinz B, Daebeler F (1979) Untersuchungen uber die mission and yield loss induced in lentil (Lens culi- Verlusthohe bei Ackerbohnen bei gleichzeitigem Be- naris Med) by Bean yellow mosaic poty virus. LENS fall durch das Scharfe Adernmosaik virus der Erbse 21(1):42Ð44 und die Schwarze Bohnenblattlaus (Aphis fabae Kumari SG, Makkouk KM, Ismail ID (1996) Variation Scop.). Arehiv fur Phytopathologie und Pflanzen- among isolates of two viruses affecting lentils: their schutz 15:55Ð62 effect on yield and seed transmissibility. Arab J Plant Hopkins JD, Mueller AJ (1984) Effect of bean pod mottle Prot 14:81Ð85 virus on soybean yield. J Econ Entomol 77(4):943Ð947 Latham LJ, Jones RAC (2001a) Alfalfa mosaic and Idris MO, Ahmed AH (1981) Effect of peanut mottle pea seed-borne mosaic viruses in cool season crop, virus on the yield of groundnut in Sudan. Phytopathol annual pasture and forage legumes: susceptibility, Mediterr 20:174Ð175 sensitivity and seed transmission. Aust J Agric Res Irwin ME, Schultz GA (1981) Soybean mosaic virus. FAO 52:771Ð790 Plant Prot Bull 29:41Ð55 Latham LJ, Jones RAC (2001b) Incidence of virus infec- Irwin ME, Ruesink WG, Isard SA, Kampmeier GE (2000) tion in experimental plots, commercial crops and seed Mitigating epidemics caused by non-persistently trans- stocks of cool season crop legumes. Aust J Agric Res mitted aphid-borne viruses: the role of the pliant envi- 52:397Ð413 ronment. Virus Res 71:185Ð211 Latham LJ, Jones RAC, Coutts BA (2004) Yield losses Jones AT (1979) The effects of black raspberry necrosis caused by virus infection in four combinations of non Ð and raspberry bushy dwarf viruses in Lloyd George persistently aphid transmitted virus and cool Ð season raspberry and their involvement in raspberry bushy crop legume. Aust J Exp Agric 44:57Ð63 dwarf disease. J Hortic Sci 54:267Ð272 Lister RM, Murant AF (1967) Seed-transmission of Jones RAC (1991) Reflective mulch decreases the spread nematode-borne viruses. Ann Appl Biol 59:49Ð62 of two non Ð persistently aphid transmitted viruses to Lockhart BEL, Fischer HU (1974) Chronic infection by narrow leafed lupin (Lupinus angustifolius). Ann Appl seed-borne bean common mosaic virus in Morocco. Biol 118:79Ð85 Plant Dis Rep 58:307Ð308 72 3 Economic Significance of Seed-Transmitted Plant Virus Diseases

Mabrouk O, Mansour AN (1998) Effect of Pea seed-borne potyvirus Ð polyclonal antibody production and its mosaic and broad bean stain viruses on lentil growth application in seed health testing. J Mycol Plant Pathol and yield in Jordan. Sci Hortic 73:175Ð178 14:810Ð815 Makkouk KM, Kumari SG (1990) Variability among 19 Rao GP, Gupta AK, Shukla K, Joshi RD (1990) Nature lentil genotypes in seed transmission rates and yield of yield loss in French Bean (Phaseolus vulgaris L.) loss induced by Broad bean stain virus infection. infected with Bean Common Mosaic Virus. Int Natl J LENS News Lett 17(2):31Ð33 Trop Plant Dis 8:129Ð135 Manadhare VK, Padulo DN, Mahajan PD (1999) Effect of Ravinder Reddy C, Tonapi VA, Varanarasiappan S, Navi leaf crinkle virus infection on seed yield and quality in SS, Jayarajan R (2005a) Studies on seed transmission mungbean. Seed Res 27:128Ð130 of urd bean leaf crinkle virus on Vigna mungo. Indian Mock RG, Stokes IE, Gillaspie AG Jr (1985) Effect of J Plant Prot 33:241Ð245 sugarcane mosaic virus infection in parental stock on Ravinder Reddy C, Tonapi VA, Varanarasiappan S, Navi panicle and seed production of virus free F2 progeny SS, Jayarajan R (2005b) Influence of plant age on in sorghum (Sorghum bicolor). Plant Dis 69:310Ð312 infection and symptomological studies on urd bean Morales FJ, Castano M (1985) Effect of Colombian isolate leaf crinkle virus. Int J Agric Sci 1:1Ð6 of bean southern mosaic virus on selected yield com- Ross JP (1968) Effect of a single and double infections ponents of Phaseolus vulgaris. Plant Dis 69:803Ð804 of soybean mosaic and bean pod mottle viruses on Nakano M, Vsugi T, Shinkai A (1988) Effect of inoc- soybean yield and seed characters. Plant Dis Rep ulation time of soybean mosaic virus on yield and 52:344Ð348 seed quality of soybean. Proc Assoc Plant Prot Kyushu Ross JP (1969a) Effect of time and sequence of inoc- 34:13Ð16 ulation of soybeans with soybean mosaic and bean Negi H, Vishunavat K (2004) Role of Seed borne inocula pod mottle viruses on yields and seed characters. of leaf crinkle virus in disease development and yield Phytopathology 59:1404Ð1408 of urd bean. Ann Plant Prot Sci 12:452Ð453 Ross JP (1969b) Pathogenic variation among isolates of Negi H, Vishunavat K (2006) Urdbean leaf crinkle in- soybean mosaic virus. Phytopathology 59:329Ð832 fection in relation to plant age, seed quality, seed Ross JP (1977) Effect of aphid transmitted soybean mo- transmission and yield in urdbean. Ann Plant Prot Sci saic virus on yields of closely related resistant and 14(1):169Ð172 susceptible soybean lines. Crop Sci 17:869Ð872 Nene YL (1972) A survey of viral diseases of pulse Ross JP (1986) Response of early and late-planted soy- crops in Uttar Pradesh. G.B. Pant Univ. Agric Technol beans to natural infection by bean pod mottle virus. Pantnagar. Res Bull 4:191 pp Plant Dis 70:222Ð224 Nutter FW Jr, Pederson VD, Timian R (1984) Relation- Ruesink WG, Irwin ME (2006) Soybean mosaic virus ship between seed infection by barley stripe mosaic epidemiology: a model and some implications. In: and yield loss. Phytopathology 74:363Ð366 McLean GD, Garrett RG, Ruesink WG (eds) Plant Omar RA, El-Khadem M, Dief AA (1978) Studies on a virus epidemics: monitoring, modeling and predicting seed-borne bean common mosaic virus III. Effect of outbreaks. Academic, Sydney, pp 295Ð313 the virus on yield, biological and chemical characters Ryder EJ, Duffus JE (1966) Effects of beet western of bean seeds. Egyptian J Phytopathol 10:63Ð70 yellows and lettuce mosaic viruses on lettuce seed Ovenden GE, Ashby JW (1981) The effect of pea seed- production, flowering time and other characteristics in borne mosaic virus on yield of peas. Proc Annu Conf the green house. Phytopathology 56:842Ð844 Agron Soc New Zealand 2 (11):61Ð63 Sastry KS, Satyanarayana A, Singh SJ, Rajendran R Paguio OR, Kuhn CW (1974) Incidence and source of (1981) Screening of different bean cultivars against inoculum of peanut mottle virus and its effect on bean common mosaic virus. Pulse Crops Newsl peanut. Phytopathology 64:60Ð64 1(3):30 Pio-Ribeiro G, Wyatt SD, Kuhn CW (1978) Cowpea stunt. Scott GE, Darrah LL, Wallin JR, West DR, Knoke JK, A disease caused by a synergistic interaction of two Louie R, Gudanskas RT, Bockholt AJ, Damsteegt viruses. Phytopathology 68:1260Ð1265 VD, Uyemoto JK (1988) Yield losses caused by Puttaraju HR, Prakash HS, Shetty HS (2001) Detection of Maize dwarf mosaic virus in Maize. Crop Sci 28: peanut mottle poty virus in leaf and seed of peanut and 691Ð694 its effect on yield. Indian Phytopathol 54:479Ð480 Sharma I, Dubey GS (1984) Control of urdbean leaf Puttaraju HR, Prakash HS, Shetty HS (2002) Contribution crinkle virus through heat treatment, chemotherapy of seed borne Black eye cowpea mosaic potyvirus and resistance. Indian Phytopathol 37:26Ð30 to disease dynamics and loss of yield. Trop Sci Sharma SR, Varma A (1981a) Assessment of losses 42:147Ð152 caused by cowpea banding mosaic (CpBMV) and Puttaraju HR, Prakash HS, Shetty HS (2004a) Seed infec- cowpea chlorotic spot (CpCSV) virus in cowpea. In: tion by Black eye cowpea mosaic potyvirus and yield Proceedings of the 3rd international symposium on loss in different cowpea varieties. J Mycol Plant Pathol plant pathology (IPS), New Delhi, p. 95 (Abstr.) 34:41Ð46 Sharma SR, Varma A (1981b) Reaction of some cow- Puttaraju HR, Shylaja H, Dharmesh M, Prakash HS, pea cultivars and lines against three sap transmissible Shetty HS (2004b) Black eye cowpea mosaic viruses. 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Sharma RB, Prasad SM, Kudada N (2007) Leaf crinkle Waterworth HE, Hadidi A (1998) Economic losses due virus disease in urd bean (Vigna mungo Linn.). J Res to plant viruses. In: Hadidi A, Khetarpal RK, Ko- (BAU) 19(1):73Ð79 ganezawa H (eds) Plant virus diseases control. Ameri- Suarez RF, Gonzalez NL (1983) Evaluation of losses can Phytopathological Society, Saint Paul, pp 1Ð13 in soybean varieties caused by cowpea mosaic virus Xia JQ, Wang QH, Yan DY, Zuh HC, Zheng JF (1986) On (CpMV). Cienciers de la Agricultura 17:25Ð29 the mosaic disease of asparagus lettuce II. The distri- Tu JC (1989) Effect of different strains of soybean mo- bution, loss, hosts and transmission of asparagus let- saic virus on growth, maturity, yield, seed mottling tuce mosaic virus. Acta Phytopathol Sin 16(1):37Ð40 and seed transmission in several soybean cultivars. J Xu Z, Zhang Z (1986) Effect of infection by three major Phytopathol 126:231Ð236 peanut viruses on the growth and yields of peanut. USDA (1965) Losses in agriculture. Agric. Res. Serv., Agric Sci China 4:51Ð56 Agric. Handbook No. 291. Washington DC, 120 p Zink FW, Grogan RG, Welch JE (1956) The effect Vishwadhar, Gupta SN (1990) Effect of bean common of the percentage of seed transmission upon subse- mosaic virus in yield and yield attributes in French quent spread of lettuce mosaic virus. Phytopathology bean. Indian J Pulses Res 3:147Ð150 46:662Ð664 Virus Transmission 4

Abstract The secondary spread of the seed-transmitted virus diseases both in the field and glass houses takes place through different vectors. Under field conditions, the infected seedlings raised from virus-infected seeds act as primary foci of infection and further spread takes place through insect vectors. Transmission through mite and insects is a natural and main method of virus spread, and the major vectors of viruses are from the phylum Arthropoda (94%) and other important vectors (about 6%) are nematodes (Phylum: Nematoda). Nearly 35 seed-transmitted viruses are from Potyvirus group, which have aphid vectors. The next highest seed- transmitted viruses (28) are found in Nepovirus group, in which nematodes are the vectors. Even beetle-transmitted seed-borne viruses are found in Fabavirus group (3) and Sobemovirus group (5). Soil-borne fungal vectors have also transmitted 4 and 2 viruses in Furovirus and Necrovirus groups. While dealing with the insect vector transmission, the virusÐvector relationship of some of the seed-transmitted viruses was also discussed. Doubtful reports of seed transmission of certain viruses by whiteflies and mites are to be confirmed by further studies. Information on role of pollen in seed transmission is discussed. Potexvirus and Tobamovirus groups, which are externally seed-borne in certain solanaceous crops, were also presented.

the next generation infected seeds. More than 4.1 Vector Transmission 231 seed-transmitted viruses are known to be vertically transmitted. The secondary spread of All seed-transmitted viruses are vertically seed the seed-transmitted virus diseases both in the transmitted and the movement of the virus is field and glasshouse takes place through different temporal from generation to generation down vectors as reviewed. the pedigree line. In the present context, the Under field conditions, the diseased plant vertical transmission means the movement of raised from infected seed serves as focus of a virus from a male and/or female parent via infection for further secondary spread either pollen and/or ovule leading to the formation of mechanically or by the vectors, and hence

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 4, 75 © Springer India 2013 76 4 Virus Transmission seed-transmitted virus diseases are compound was carried up to 60 km or even more in a interest diseases. Further, horizontal spread of nonstop flight. Certain vectors like the the virus to the contemporary plants in the and mites also spread viruses to nearby plants field from the initial foci of infection takes by crawling or walking. No reports are available place mechanically or through pollen or vectors on the transmission of any seed-transmitted virus resulting in heavy infections. The word ‘vector’ either by mealybug or by leaf and plant hop- is derived from the Latin word ‘vectus’ as past pers. Although the mite transmission of Wheat participle of vehere meaning ‘to carry’, and streak mosaic virus is reported to be seed trans- in a biological sense, vector is an organism mitted in corn (Hill et al. 1974), it requires carrying pathogenic agents. Barring a few confirmation. viruses, majority of the seed-transmitted plant viruses spread through arthropod vectors like aphids, whiteflies, beetles, mites and thrips. Only 4.1.1 Aphids about three decades ago, certain nematodes and fungi have joined the vector group for some seed- Aphids constitute the most important group of transmitted viruses. The insect vectors transmit virus vectors and transmit more plant virus dis- plant viruses by four major transmission modes eases than any other group of vectors. About 50% and a number of virus and insect have of seed-transmitted viruses are aphid transmitted been found to control virusÐvector association. and belong to Caulimovirus, Carlavirus, Cucu- Majority of the seed-borne plant viruses are movirus, Alfamovirus, and Potyvirus groups. It transmitted by aphids in nonpersistent manner. is a nonpersistent relationship in which both the Most vectors are piercingÐsucking insects that acquisition and inoculation probes of 15Ð60 s transmit plant viruses in either the circulative each are optimal for transmission. Both winged virus (CV) or noncirculative virus (NCV). NCVs and wingless single aphids can transmit the virus. are carried on the lining cuticle of vector stylets. High percent of transmission is achieved by the Circulative viruses (CVs) cross the vector’s gut, preliminary fasting period with optimal aphid move internally to the salivary glands (SG) and numbers; however, the inoculative capacity of the cross the SG membranes to be ejected upon aphid decreases as the period between acquisition feeding. Transmissibility of NCVs depends and inoculation of the virus increases. Aphids on motifs of coat protein and for almost always probe in the anticlinal grooves and Caulimoviruses also on helper proteins of adjacent epidermal cells, which they locate (encoded by the virus). NCV proteins were via receptors-mechano pegs present on the labial found to associate with vector’s cuticle proteins. tip. Aphids always secrete saliva at the start of Transmissibility of CVs depends on proteins the probe as well as during stylet penetration comprising the virus capsid (the coat protein forming a sheath. Stylets move fairly rapidly and the read-through protein) and on symbionin within this sheath but subsequently extend be- (produced by vectors symbionins). Passage of yond the sheath for ingestion of food material CV through the gut has been also associated from host cells. During the process of feeding, the with vector’s proteins. Raccah and Fereres viruses present in the sap usually adhere to their (2009) have furnished more details on vector mouth parts and are introduced subsequently into transmission. another plant when the viruliferous aphids feed, The arthropod vectors have a diverse assem- which is termed as mechanical contamination hy- blage of mouth parts with great differences be- pothesis. Forbes (1977) has extensively reviewed tween the various groups in life cycle, behaviour the feeding mechanism of aphids. In general, the and activity. The viruliferous winged adult aphids nonpersistent seed-transmitted viruses have low and thrips are blown by wind to distant places level of vector specificity because they are trans- where they initiate infection. For example, John- mitted by several aphid species, such as AMV son (1967) recorded that a nonpersistent virus by 14 aphid species (Crill et al. 1970), CMV 4.1 Vector Transmission 77 by 60 aphid species (Kennedy et al. 1962)and confirmed. Cowpea mottle virus in cowpea has SMV by 31 aphid species (Irwin and Goodman beetle vector, namely, Ootheca mutabilis,which 1981). acquired virus in 10 min, transmitted within 1 h Domier et al. (2007) have studied eight SMV and remained viruliferous for 5 days (Shoyinka strains in soybean and showed that there was et al. 1978). direct correlation with transmission through The beetle vectors are confined to the families aphid vector, and seed coat mottling caused by of Chrysomelidae, Coccinellidae, Curculionidae SMV virus isolate. The poor seed transmission and Meloidae. They have biting type of mouth showed even poor transmission by the soybean parts and transmit viruses mechanically by car- aphid, Aphis glycines. The loss of aphid and rying them on the mouth parts. These vectors seed transmissibility by repeated mechanical acquire the virus during acquisition feeding pe- transmission suggests that constant selection riods ranging from a few minutes to 24 h and pressure is needed to maintain the regions of the transmit them immediately. The length of time SMV genome controlling the two phenotypes a beetle retains and transmits the virus primar- from genetic drift and loss of function. ily depends on the type of virus, its host and environmental conditions. Both Walters (1969) and Selman (1973) suggested that virus retention 4.1.2 Beetles time by the vector is characteristic of a particular virus. The viruses transmitted for 1 or 2 days Another active vector of seed-transmitted viruses fall into one group, while that of longer periods is beetle and five members from Comovirus, three into the other group. The Mexican bean beetle from Carmovirus, two from Tymovirus, two from retains different viruses and transmits only for Sobemovirus and one each from Bromovirus and short periods of time, whereas the Fabavirus groups are beetle transmitted (Fulton retains the same viruses for extended periods. et al. 1980, 1987). There is a high degree of Beetles can retain viruses up to a period of 8 specificity between the beetle vectors and the days and can be detected in haemolymph, but viruses they transmit. Squash mosaic and South- apparently do not multiply in their beetle vectors. ern bean mosaic viruses are seed transmitted VirusÐvector relationship appears to be similar to in a number of cucurbitaceous and leguminous semi-persistent type. hosts, respectively. As low as 0.1% seed trans- As early as 1949, Markham and Smith pointed mission has been recorded in different virusÐhost out that beetles regurgitate during feeding which combinations, namely, Bean pod mottle virus in contain virus over several days. The viruses are soybean which is transmitted by bean leaf beetle also detected in the faecal matter of beetles which (Cerotoma trifurcata) (Lin and Hill 1983;Giesler have fed on infected plants. Some viruses like et al. 2002; Krell et al. 2003), while as high as Broad bean stain and Broad bean true mosaic 93% seed transmission in C. melo with Squash (BBTMV) are transmitted by weevils, Apion vo- mosaic virus was recorded (Rader et al. 1947). rax (Cockbain et al. 1975; Jones 1978). BBTMV There are certain controversial reports of seed is transmitted through seed of faba bean up to transmission of viruses having beetles as vectors. 17% (Cockbain et al. 1976) and 2.7Ð10% by Seed transmission (1Ð5%) of beetle-transmitted Broad bean stain virus in the same host (Gibbs Cowpea mosaic virus was reported by Gilmer et and Smith 1970). al. (1973), but Thottappilly and Rossel (1987) could not confirm the seed transmission. Urdbean leaf crinkle virus in Vigna mungo is also seed 4.1.3 Thrips transmitted up to 83% (Pushpalatha et al. 1999) and reported to be transmitted by leaf-feeding The transmission of seed-transmitted viruses by beetle Henosepilachna dodecastigma (Beniwal thrips vectors has been reported for Ilarvirus and Bharathan 1980), and this vector has to be and Tomato spotted wilt virus groups. Tobacco 78 4 Virus Transmission streak virus (TSV), seed transmitted in hosts like not observe the seed transmission of CMMV in Datura stramonium, Glycine max and Phaseo- soybean and groundnut. Even in extensive seed lus vulgaris, was transmitted by Frankliniella transmission tests conducted by Rossel and Thot- sp.andThrips tabaci (Costa and Lima Neto tappilly (1993) with two isolates of Cowpea mild 1976; Sdoodee and Teakle 1987). During 2010, mottle virus in 25 soybean accession numbers, Vemana and Jain have detected TSV both in seed transmission was not noticed both in grow groundnut pod shell and seed coat (testa) and out tests and by ELISA. no virus was detected either in cotyledons or embryos obtained from infected seeds, indicating absence of true seed transmission in groundnut 4.1.5 Mites and also in other leguminous hosts. Similarly, Tomato spotted wilt virus (TSWV) was also seed The Eriophyid mite vector is elongated and transmitted in Senecio sp., but not in peanut. Ex- tiny, about 0.2 mm long, and virusÐvector tensive serological tests carried out at ICRISAT, relationship is of noncirculative type. For the Hyderabad (India), have clearly indicated the first time, Khetarpal (1989) and Khetarpal and non-seed-transmitted nature of GBNV in mature Maury (1989) have reported the transmission dried peanuts (Reddy et al. 1983;PrasadaRao of the Pea seed-borne mosaic virus in pea by et al. 2009). the mite, Tetranychus urticae under glasshouse conditions and requires confirmation. Another seed-transmitted virus, Wheat streak mosaic 4.1.4 Whiteflies virus, in wheat is transmitted by mite vector Aceria tosichella (Jones et al. 2005), and this The whiteflies are small, piercing and sucking virus is reported to be seed transmitted in wheat insects belonging to the family Aleyrodidae in the as low as 0.22% (Lanoiselet et al. 2008). order Homoptera. Seed transmission of whitefly- transmitted virus diseases has been reported in Begomovirus and Carlavirus groups for which 4.1.6 Nematodes Bemisia tabaci is the vector and further seed transmission researches are required with dif- The soil-inhabiting nematodes are the vectors ferent whitefly-transmitted viruses. For example, responsible for transmission of members of Keur (1933, 1934) has reported Abutilon mosaic Tobravirus and Nepovirus groups which are begomovirus to be seed transmitted and subse- also seed transmitted (Table 1.2), and this has quent studies have not confirmed the seed trans- been reviewed exhaustively (Taylor 1980;Brown mission. Abutilon mosaic virus belonging to Be- et al. 1995). Four nematode genera, namely, gomovirus group is transmitted by whitefly vector Xiphinema, , Paratrichodorus and in a semi-persistent manner, and the Cowpea mild Trichodorus, are known vectors which measure mottle virus (CMMV) of Carlavirus group in 2Ð12 mm long and are migratory ectoparasites a nonpersistent manner. Muniyappa and Reddy feeding mainly on tips. Regarding their (1983); Jeyanandarajah and Brunt (1993)have habitat, X. diversicaudatum is generally associ- reported that CMMV was transmitted even by a ated with moist and shady sites in the vicinity of single whitefly and the adult whitefly acquired water or medium soils with high moisture levels the CMMV in an access period of 10 min and (Fritzsche et al. 1972). L. elongatus is found in transmitted in a 2 min inoculation period in serial light medium soils and L. attenuatus in light 5 min transfers, the virus was retained for a sandy soils (Harrison 1977). T. pachydermatus maximum period of 20 min. However, according usually occurs in light rather than heavy soils to Briddon (2003), Begomoviruses belonging to (Van Hoof 1962). Both adult and larval stages family Geminiviridae has 102 viruses, which are can transmit the viruses with equal efficiency. not seed transmitted. Horn et al. (1991) could L. elongatus retains the virus for 8Ð9 weeks and 4.1 Vector Transmission 79 the Xiphinema sp. for many months, and there and Furovirus groups. Peanut clump virus is no evidence that viruses multiply inside the (Pecluvirus) occurring to a limited extent in sandy nematode vector. soils of Punjab, Rajasthan and Andhra Pradesh Based on the particle morphology of the states in India is transmitted both by Polymyxa nematode-transmitted viruses, Cadman (1963) graminis and also through seed (Thouvenel and andHarrison(1964) categorised them into Fauquet 1981a; Reddy et al. 1983; Delfosse et al. Nepovirus and Tobravirus groups having 1999, 2002; Dieryck et al. 2009). The life cycle polyhedral and tubular morphology, respectively. of the fungus vector in its graminaceous hosts has The spread of these viruses is very slow since been studied by Ratna et al. (1991). Similarly, Pea the vector nematodes move over short distances stem necrosis virus, transmitted by Olpidium sp., of approximately 50 cm in a year. Transport of is seed transmitted (Brunt et al. 1996). Soil-borne vector nematodes over longer distances in soil is wheat mosaic virus (SBWMV) transmitted by probably less feasible since they are susceptible Polymyxa graminis is seed transmitted in rye and to desiccation, but movement of moist soil on wheat crops (Delfosse et al. 1999). Another soil agricultural implements, plant and the feet and seed-transmitted virus, Tomato bushy stunt of animals and birds may play some role in virus of Tombusvirus group, has been suspected nematode vector distribution. to have a fungus vector, but attempts to implicate a specific vector have not been successful. Potato virus X, transmitted by the fungus Synchytrium 4.1.7 Bumblebees endobioticum, is seed transmitted in potato to an extent of 0.6Ð2.3% and requires confirmation Antignus et al. (2007) have confirmed that (Darozhkin and Chykava 1974). Different aspects bumblebees (Bombus terrestris) transmit the of fungus transmission of plant viruses have Tomato apical stunt viroid from infected to been reviewed by Teakle (1983). Campbell et al. healthy tomato plants in the form of secondary (1996) have tested the hypothesis of vector- spread. Okada et al. (2000) have demonstrated assisted seed transmission (VAST) while working that TMV was transmitted to tomato plants with seed-transmitted Melon necrotic spot carmo grown in glasshouses by the bumblebees. The virus (MNSV) infection in melons (Cucumis virus transmission by bumblebees may result melo) which is transmitted by Olpidium from the wounding of flowers during visits of the radicale. Seed-transmitted virus rarely infected insects as was reported for Pepino mosaic virus melon seedlings unless the vector was present, or from the introduction of infected pollen to the confirming VAST as a novel means of seed stigma, from which the viroid could enter the transmission. embryo after the fusion of the sperm cells with the ovules. Lacasa et al. (2003) have implicated the involvement of bumblebees on Pepino mosaic 4.1.9 Mealybug virus spread in tomato crop. In Japan, Tomato chlorotic dwarf viroid is also transmitted from Among the mealybug-transmitted viruses is Co- infected tomato plants to neighbouring healthy coa swollen shoot virus for which the vector is plants through bumblebees (Bombus ignites) dur- Pseudococcus njalensis and also transmitted by ing pollination activities (Matsuura et al. 2010). seed to the extent of 34Ð54% (Quainoo et al. 2008). This virus is located in testa, cotyledon and embryo and virus is pollen-borne. Another 4.1.8 Fungi seed-transmitted virus with mealybug Planococ- cus citri vector is Piper yellow mottle virus of Certain fungi like Polymyxa sp., Olpidium sp. and black pepper, and rate of seed transmission in Synchytrium sp. are also implicated as vectors of black pepper seeds is up to 30% (Bhat et al. seed-transmitted viruses belonging to Necrovirus 2005). 80 4 Virus Transmission

vulgaris) and other Compositae members, viruses 4.2 Nonvector Transmission like Arabis mosaic, Tomato black ring and Straw- berry latent ring spot are seed transmitted and 4.2.1 Mechanical Spread the infected seed is carried through wind. Seeds of S. vulgaris also carry LMV (Costa and Duf- The limited persistence of certain viruses fus 1958). Wind-mediated spread of Rice yellow and viroids outside living cells restricts the mottle virus (RYMV) in irrigated rice crops was opportunities for their transmission by direct reported by Sarra et al. (2004). contact. However, majority of the viruses which are spreading by mechanical contact have no known vectors. Viruses like TMV, PVX, BSMV, 4.2.3 Water PepMV and PStV spread by handling, by pruning or by contact with infected plants, debris or Viruses present in water can infect plant through contaminated implements. With BSMV being the root system and cause the appearance of dis- seed transmitted to the tune of 90%, mechanical ease symptoms. They can also be released from spread is an efficient source for its introduction in infected plants into drainage and then spread to uninfected barley growing areas. No other weed other plants (Koenig 1986). In a Hungarian study, host acts as a significant reservoir of the virus the presence of 26 plant viruses in 47 environ- except wild oats. The secondary spread of this mental water samples was detected (Horvath et virus inside the field takes place mechanically by al. 1999). leaf contact of infected plant with healthy plants. Yakovleva (1965) reported that Cucumber Even Tobacco mosaic virus (TMV), Tomato green mottle mosaic virus (CGMMV) is being mosaic virus Pepino mosaic virus (ToMV) and seed transmitted in Cucumis sativus to an extent (PepMV) strains in most of the solanaceous crops of 44% and spreads with surface water used for like tobacco, tomato and chillies are spreading watering. In the Netherlands, in areas where through contact, contaminated tools, workers’ CGMMV prevails, it has been demonstrated hands and clothing. Wind currents and walking that the virus is present in high concentrations of animals and workers in the field promote in the surface water contaminated from waste increasing spread. Besides this, TMV infection heaps at dumping sites. Under glasshouse or persists in the soil even 20 months after removal field conditions, the virus may be released from of the diseased plants and the virus also survives dying roots and end up in drainage water. It has in dried debris. TMV spreads through irrigated also been observed that a high percentage of the water or soil as a contaminant and poses a healthy plants are infected due to the sprinkling of problem in glass houses. Mechanical spread of contaminated water (Van Dorst 1988). In India, Rice yellow mottle virus by cows, donkeys and CGMMV was detected in Jamuna river waters grass rats in irrigated rice crops was reported by flowing near cucurbit cultivated areas (Vani and Sarra and Peters (2003). In almost all the viroid Varma 1988). Similarly, CMV and TMV were diseases, the secondary spread takes place via also detected in some rivers of Southern Italy workers’ infested hands and tools (Singh et al. (Piazzolla et al. 1986). During 2007, Boben et al. 1998a, b; Ramachandran et al. 1996). have done detection and quantification of Tomato mosaic virus in irrigation waters in Slovenia by using RTÐPCR technique. 4.2.2 Wind

Dispersal of the infected seed over long distance 4.2.4 Obligate Symbiosis through wind has been observed among seed- transmitted viruses. In wild hosts such as Betula Obligate symbiosis is a mutual molecular spp. and in weeds such as groundsel (Senecio beneficial mechanism of sharing of genetic References 81 material of two different viral strains at the cellular system. Majority of the plant viruses are References independent of their genetic material replication Antignus Y, Lachman O, Pearlsman M (2007) The spread in the in vivo system, but very few virus of Tomato apical stunt viroid (TAS Vd) in green house strains depend on their replication mutually, for tomato crops is associated with seed transmission and example, Pea enation mosaic virus (PEMV) is bumble bee activity. Plant Dis 91:47Ð50 caused by a complex of two viruses, PEMV-1 Beniwal SPS, Bharathan N (1980) Beetle transmission of Urdbean leaf crinkle virus. Indian Phytopathol is a Luteoviridae member and PEMV-2 is an 33(4):600Ð601 Umbravirus. Two sizes of isometric particles are Bhat AI, Devasahayam S, Hareesh PS, Preethi N, Thomas found, that of PEMV-1 (28 nm diameter) and that T (2005) Planococcus citri (Risso)Ðan additional Badnavirus of PEMV-2 (25 nm diameter) having icosahedral mealybug vector of infecting black pepper (Piper nigrum L.) in India. Entomon 30:85Ð90 and quasi-icosahedral symmetry, respectively. Boben J, Kramberger P, Petrovic N, Cankar K, Peterka Both types of particles are composed of coat M, Strancar A, Ravnikar M (2007) Detection and protein encoded by PEMV-1. PEMV-1 can infect quantification of Tomato mosaic virus in irrigation waters. Eur J Plant Pathol 118:59Ð71 protoplasts but does not spread in plants unless Briddon R (2003) Tomato pseudo-curly top virus. AAB PEMV-2 is present. Unlike other members of descriptions of plant viruses no. 395. Available at the Luteoviridae, Pea enation mosaic virus is http://dpvweb.net transmissible mechanically, as well as by aphids, Brown DJF, Robertson WM, Trudgill DL (1995) Trans- mission of viruses by plant nematodes. Annu Rev and spreads to mesophyll tissues; these two Phytopathol 33:223Ð249 properties are conferred by PEMV-2. Virions Brunt AA, Crabtree K, Dallwitz MJ, Gibbs AJ, Watson of some strains of PEMV-1 plus PEMV-2 also L (eds) (1996) Viruses of plants. Description and lists contain a satellite RNA. Aphid transmission from VIDE data base. CAB International, Wallingford, p 1484 of PEMV-1 plus PEMV-2 is conferred. Aphid Cadman CH (1963) Biology of soil-borne viruses. Annu transmissibility can be lost after multiple Rev Phytopathol 1:143Ð172 passages of mechanical transmission. Differences Campbell RN, WipfÐScheibel C, Lecoq H (1996) Vector Ð have been found in the electrophoretic profiles associated seed transmission of Melon necrotic spot virus in melon. Phytopathology 86:1294Ð1298 of the particles of aphid-transmissible and Cockbain AJ, Bowen R, Etheridge P (1975) Attempts to non-transmissible isolates, but the cause of control the spread of BBSV/EAMB. Rep Rothmsted these differences has not been fully resolved Exp Stn 1974:235Ð236 (Hull 2002). Cockbain AJ, Bowen R, Vorra-urai S (1976) Seed transmission of broad bean stain virus and Echtes Ackerbohnenmosaik-virus in field bean (Vicia faba). Ann Appl Biol 84:321Ð332 4.3 Conclusions Costa AS, da Lima Neto VC (1976) Transmissao de virus da necrose branca de fumo por Freankliniella sp. IX congress Soc. Bras Fitopatol Cryptic viruses which induce little or no disease Costa AS, Duffus JE (1958) Observations on lettuce symptoms are not transmitted mechanically or mosaic in California. Plant Dis Rep 42:583Ð586 by any other vector, but are transmitted through Crill P, Hagedorn FJ, Hanson EW (1970) Alfalfa mosaic the seed; 100% of progeny is infected when the disease and its virus incitant. University of Wis- consin Research Bulletin. No:280, 39 pp both parents are carriers of the virus. Future Darozhkin MA, Chykava SIG (1974) Da pytannya studies are very much essential to identify the peredachy X-virusa nasennem bul ‘by. (Transmission factors involved in seed infection or the lack of virus X by potato seeds). Vestsi Akademii Navuk of it: the prevalence of seed transmissible virus BSSR. Biyalagichnykh Navuk 5:80Ð85 Delfosse P, Reddy AS, Legreve A, Devi PS, Devi KT, isolates, potential source of virus, occurrence and Maraite H, Reddy DVR (1999) Indian peanut clump behaviour of vectors of such viruses before and virus (IPCV) infection on wheat and barley: symp- after virus acquisition and their interactions with toms, yield loss and transmission through seed. Plant the environment and persistence of virus in seed Pathol 48:278Ð282 Delfosse P, Reddy AS, Thirumala Devi K, Legreve A, and pollen in nature and development of effective Risopoulas J, Doucet D, Shoba Devi P, Maraite H, novel control measures. Reddy DVR (2002) Dynamics of polymyxa graminis 82 4 Virus Transmission

and Indian Peanut clump virus (IPCV) infection on K, Harris KF (eds) Plant diseases and vectors: ecology various monocotyledonous crops and groundnut dur- and epidemiology. Academic, New York, pp 182Ð215 ing the rainy season. Plant Pathol 51:546Ð560 Jeyanandarajah P, Brunt AA (1993) The natural occur- Dieryck B, Otto G, Doucet D, Legreve A, Delfosse P, rence, transmission, properties and possible affinities Bragard C (2009) Seed, soil and vegetative trans- of cowpea mild mottle virus. J Phytopathol 137: mission contribute to the spread of pecluviruses in 148Ð156 Western Africa and the Indian subÐcontinent. Virus Johnson CG (1967) International dispersal of in- Res 141:184Ð189 sects and insect-borne viruses. Neth J Plant Pathol Domier LL, Steinlage TA, Hobbs HA, Wang Y, Her- 73((suppl)):21Ð43 rera Rodriguez G, Haudenshield J, McCoppin NK, Jones AT (1978) Incidence, field spread, seed transmis- Hartman GL (2007) Similarities in seed and aphid sion and effects of broad bean stain virus and Echtes transmission among soybean virus isolates. Plant Dis Ackerbohnenmosaik-virus in Vicia Faba in eastern 91:546Ð550 Scotland. Ann Appl Biol 88:137Ð144 Forbes AR (1977) The mouth parts and feeding mecha- Jones RAC, Coutts BA, Mackie AE, Dwyer GI (2005) nism of aphids. In: Harris KF, Maramorosch K (eds) Seed transmission of wheat streak mosaic virus shown Aphids as virus vectors. Academic, London, pp 83Ð unequivocally in wheat. Plant Dis 89:1048Ð1050 104, 559 pp Kennedy JS, Day MF, Eastop VP (1962) A conspectus Fritzsche R, Karl E, Lehmann W, Proesler G (1972) of aphids as vectors of plant viruses. Commonwealth Tierische Vektoren Pflanzen Pathogener viren. Veb Inst. Entomol, London Gustav. Fischer Verlag, Jena, p 521 Keur JY (1933) Seed transmissions of virus causing var- Fulton JP, Scott HA, Gamez R (1980) Beetles. In: Harris iegation of Abutilon. Phytopathology 23:20 KF, Maramorosch K (eds) Vectors of plant pathogens. Keur JY (1934) Studies on the occurrence and transmis- Academic, New York, pp 115Ð132, 467 sion of virus diseases in the genus Abutilon. Bull Fulton JP, Gergerich RC, Scott HA (1987) Beetle trans- Torrey Bot Club 61:53Ð70 mission of plant viruses. Annu Rev Phytopathol Khetarpal RK (1989) Contribution A L’etude des relations 25:111Ð123 pea seed-borne mosaic virus Ð Pois Universite De Paris Gibbs AJ, Smith HG (1970) Broad bean stain virus. Sud, Centre D’orsay, Paris CMI/AAB descriptions of plant viruses, no.29. Khetarpal RK, Maury Y (1989) Transmission of pea seed Kew/Surry borne mosaic virus on peas by the mite Tetranychus Giesler LJ, Ghabrial SA, Hunt TE, Hill JH (2002) Bean urticae Koch. in the glasshouse. In: Proceedings of the pod mottle virus Ð a threat to US soybean production. IVth international plant virus epidemiology workshop, Plant Dis 86:1280Ð1289 Montpellier, France, p 313, 3Ð8 Sept 1989 Gilmer RM, Whitney WK, Williams RJ (1973) Epi- Koenig R (1986) Plant viruses in rivers and lakes. Adv demiology and control of cowpea mosaic in Western Virus Res 31:321Ð333 Nigeria. In: Proceedings of the 1st IITA grain legume Krell RK, Pedigo LP, Hill JH, Rice ME (2003) Potential improvement workshop, p 269 primary inoculum sources of Bean pod mottle virus in Harrison BD (1964) Specific nematode vector for sero- Iowa. Plant Dis 87:1416Ð1422 logically distinctive forms of raspberry ringspot and Lacasa A, Guerrero MM, Hita I, Martinez MA, Jorda tomato black ring viruses. Virology 23:544Ð550 C, Bielza P, Contreras J, Alcazar A, Cano A (2003) Harrison BD (1977) Ecology and control of viruses Implication of bumble bees (Bombus spp.) on Pepino with soil-inhabiting vectors. Annu Rev Phytopathol mosaic virus (PepMV) spread on tomato crops. Plagas 15:331Ð360 29:393Ð403 Hill JH, Martinson CA, Russell WA (1974) Seed transmis- Lanoiselet VM, Hind-Lanoiselet TL, Murray GM (2008) sion of maize dwarf mosaic and wheat streak mosaic Studies on the seed transmission of Wheat streak viruses in maize and response to inbred lines. Crop Sci mosaic virus. Australas Plant Pathol 37:584Ð588 14:232Ð235 Lin MT, Hill JH (1983) Bean pod mottle virus: occurrence Horn NM, Saleh N, Baladi Y (1991) Cowpea mild mottle in Nebrasca and seed transmission in soybeans. Plant virus could not be detected by ELISA in soybean Dis 67:230Ð233 and groundnut seeds in Indonesia. Neth J Plant Pathol Markham R, Smith KM (1949) Studies on the virus of 91:125Ð127 turnip yellow mosaic. Parasitology 39:330Ð342 Horvath J, Pocsai E, Kazinczi G (1999) V: Zbornik pre- Matsuura S, Matsushita Y, Kozuka R, Shimizu S, Tsuda S davanj in referatov. 4. In: Macek J (ed) Slovensko (2010) Transmission of Tomato chlorotic dwarf viroid posvetovanje o varstvu rastlin, Portoroz, 3Ð4 Mar by Bumblebees (Bombus ignites) in tomato plants. Eur 1999. Drustvo za varstvo rastlin Slovenije, Ljubljana, J Plant Pathol 126:111Ð115 pp 353Ð356 Muniyappa V, Reddy DVR (1983) Transmission of cow- Hull R (2002) Mathews plant virology, 4th edn. Aca- pea mild mottle virus by Bemisia tabaci in a non- demic, London persistent manner. Plant Dis 67:391Ð393 Irwin ME, Goodman RM (1981) Ecology and control of Okada K, Kusakari SI, Kawaratani M, Negoro JI, Okhi ST, soybean mosaic virus in soybeans. In: Maramorosch Osaki T (2000) Tobacco mosaic virus is transmissible References 83

from tomato to tomato by pollinating bumblebees. J Selman BJ (1973) Beetles-phytophagus coleoptera. In: Gen Plant Pathol 66:71Ð74 Gibbs AJ (ed) Virus and invertebrates. North-Holland Piazzolla P, Castellano MA, De Stradis A (1986) Presence Publishing Co., London, pp 157Ð177 of plant viruses in some viruses of Southern Italy. J Shoyinka SA, Bozarth RF, Reese J, Rossel HW (1978) Phytopathol 116:244Ð246, OmV and TMV Cowpea mottle virus: a seed borne virus with dis- Prasada Rao RDVJ, Jyothirmai Madhavi K, Reddy AS, tinctive properties infecting cowpeas in Nigeria. Phy- Varaprasad KS, Nigam SN, Sharma KK, Lavakumar P, topathology 68:693Ð699 Waliyar F (2009) Non transmission of Tobacco streak Singh RP, Singh M, King RR (1998a) Use of citric acid virus isolate occurring in India through seeds of some for neutralizing polymerase chain reaction inhibition crop and weed hosts. Indian J Plant Prot 37:92Ð96 by chlorogenic acid in potato extracts. J Virol Methods Pushpalatha KC, Prakash HS, Albrechtsen SE, Setty HS, 74:231Ð235 Mathur SB (1999) Transmission of Urdbean leaf crin- Singh RP, Singh M, McDonald JG (1998b) Screening by kle virus through urdbean seeds. Seed Res 27:112Ð115 a 3-primer PCR of North American PVYN isolates Quainoo AK, Wetten AC, Allainguillaume J (2008) Trans- for European type members of the tuber necrosis mission of Cocoa swollen shoot virus by seeds. J Virol inducing PVYNTN subgroup. Can J Plant Pathol 20: Methods 150:45Ð49 227Ð233 Raccah B, Fereres A (2009) Plant virus transmission Taylor CE (1980) Nematodes. In: Harris KF, by insects. In: Raccah B (ed) Encyclopedia of life Maramorosch K (eds) Vectors of plant pathogens. sciences (ELS). Wiley, Chichester Academic, New York, pp 375Ð416, pp 467 Rader WE, Fitzpatvick HF, Hildebrand EM (1947) A Teakle DS (1983) Zoosporic fungi and viruses: dou- seed borne virus of muskmelon. Phytopathology 37: ble trouble. In: Buczacki ST (ed) Zoosporic plant 809Ð816 pathogens. Academic, New York, pp 231Ð248 Ramachandran P, Ahlawat YS, Varma A (1996) Viroids: Thottappilly G, Rossel HW (1987) Seed transmission of the potential plant pathogens. In: Second internat. cowpea (yellow) mosaic virus unlikely in cowpea. Crop Science Congress, New Delhi, p 433. 18. 093, Trop Grain Legume Bull 34:27Ð28 17Ð24 Nov Thouvenel JC, Fauquet C (1981a) Further properties of Ratna AS, Rao AS, Reddy AS, Nolt BL, Reddy DVR, Peanut clump virus and studies on its natural transmis- Vijayalaxmi M, McDonald D (1991) Studies on trans- sion. Ann Appl Biol 97:99Ð107 mission of Indian peanut clump and virus disease by Thouvenel JC, Fauquet C (1981b) Peanut clump virus. No. Polymyxa graminis. Ann Appl Biol 111:353Ð358 235 In: Descriptions of plant viruses common Mycol. Reddy DVR, Amin PW, McDonald D, Ghanekar AM Lust. Assoc. Appl. Biol. Kew/Surrey, 4 pp (1983) Epidemiology and control of groundnut bud Van Dorst HJM (1988) Surface water as source in the necrosis and other diseases of legume crops in India spread of cucumber green mottle mosaic virus. Neth caused by tomato spotted wilt virus. In: Plumb RT, J Agric Sci 36:291Ð300 Thresh JM (eds) Plant virus epidemiology. Blackwell Van Hoof HA (1962) Trichodorus pachydermus and I. Scientific Publications, Oxford, pp 93Ð102 teres vectors of early browning virus of peas. Tijdschr Rossel HW, Thottappilly G (1993) Seed transmission of Pl Ziekt 68:391Ð396 viruses in soybean (Glycine max) in relation to sanita- Vani S, Varma A (1988) Properties of cucumber green tion and international transfer of improved germplasm. mottle mosaic virus isolated from water of Jamuna Seed Sci Technol 21:25Ð30 river. Indian Phytopathol 41:266Ð267 Sarra S, Peters D (2003) Rice yellow mottle virus is trans- Vemana K, Jain RK (2010) New experimental hosts of mitted by cows, donkeys and grass rats in irrigated rice tobacco streak virus and absence of true seed trans- crops. Plant Dis 87:804Ð808 mission in leguminous hosts. Indian J Virol 21(2): Sarra S, Oevering P, Guindo S, Peters D (2004) Wind 117Ð127 mediated spread of rice yellow mottle virus (RYMV) Walters HJ (1969) Beetle transmission of plant viruses. in irrigated rice crops. Plant Pathol 53:148Ð153 Adv Virus Res 15:339Ð363 Sdoodee R, Teakle DS (1987) Transmission of tobacco Yakovleva N (1965) Borba s zelenoi mazaikoi Ogurtsov. streak virus by Thrips tabaci: a new method of plant (Control of green mosaic of cucumber). Zashch Rast virus transmission. Plant Pathol 36:377Ð380 Vredit Bolez 10:50Ð51 Mechanism of Seed Transmission 5

Abstract The specific mechanism by which some plant viruses are transmitted through seed, while others are excluded, is known very little. Genetic tests designed to identify the viral genes which control seed transmission may aid in the discovery of this mechanism. Cytoplasmic connections between the infected mother plant, flower and the developing seeds influence the seed infection. The higher the embryonic seed infection, the greater will be the size and number of cytoplasmic connections. Since legumes develop cytoplasmic connections more frequently than cereals, the percentage of virus transmission through seeds in legumes is higher than in cereals. The information on the distribution of virus in the seed is also pre- sented. Survival of the virus in seed varies with virus strain, the cultivar and storage conditions. Some aspects of genetics of seed transmission are also presented. Factors like environmental aspects, stage of infection, host cultivar and virus strain/isolate play major role in seed transmission in different virusÐhost combinations. The information on reasons for failure of seed transmission is also presented.

made up of more than one carpel and may contain 5.1 Embryology one or more locules, each of which contains one and Development of Seed or more ovules. Thus, a large number of ovules Structures are formed in the ovary which after fertilisation develop into seeds. The developing ovule gener- A typical angiosperm flower consists of four ally is attached to the placenta by funiculus. The types of floral organs, sepals, petals, stamens and outer layers of the ovule are the outer integument pistil(s), and only the last two are directly con- which is joined with funiculus, and within this is cerned in seed production. Stamens form pollen the inner integument. These integuments do not grains which produce male gametes. The pistil completely enclose the inner parts; at one end is consists of ovary which contains the ovules, style a small opening called micropyle. and stigma. A simple ovary consists of one carpel The central part of the ovule is the nucellus with one locule or cavity; a compound ovary is with a central single large megaspore mother

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 5, 85 © Springer India 2013 86 5 Mechanism of Seed Transmission cell (2n) which gives rise to an oval or elongate form the endosperm. The seed coat results from embryo sac. During the development of the em- integuments around the ovule. bryo sac, the megaspore nucleus divides three Many embryo-borne viruses are transmitted times by mitosis to form eight genetically iden- through pollen. The pollen grains when released tical nuclei. Three of the nuclei become located from infected plants, may carry the virus either at the micropylar end of the sac and three at the on exine or intine. When such pollen grains ger- opposite end. The two polar nuclei move towards minate on the stigma, pollen tubes grow through the central area of the polar embryo sac and fuse, the style and reach the embryo sac, into which forming the secondary nucleus. The three cells at they release two male gametes. The infected the micropylar end constitute the egg cell and the gamete unites with the egg cell, and the resulting two synergids. The three cells at the opposite end embryo may be infected. However, union of other of the embryo sac are the antipodals. The ovule gamete with polar nuclei may lead to infected and the organised embryo sac may be oriented in endosperm. different ways, depending on the positions of the Cytoplasmic connections between the infected hilum and micropyle. mother plant, flower and developing seeds may The pollen grains are transferred to stigma by influence seed infection. The higher the embry- wind currents or through insects or by rain water. onic seed infection, the greater will be the size The transfer of pollen either to the stigma of or number of cytoplasmic connections. Since the same flower or another flower in the same legumes develop cytoplasmic connections more plant is known as ‘self-pollination’ and that to frequently than cereals, the percentage of virus the stigma of flowers on other plants is termed seed transmission in legumes is higher than in ‘cross-pollination’. Mature pollen consists of a cereals. Ovule infection occurs with a number spore wall, a tube cell nucleus and a generative of viruses which may move into different parts nucleus. The pollen germinates on the stigma, of the seed including the embryo sac. Similarly, and the pollen tube grows through the style and cytoplasmic connections between the developing ovary, reaches the tip of an ovule. Fertilisation is pollen grains and the infected mother plant result of three types depending on how the pollen tube in virus-infected pollen grains. The viruses may enters into the ovule. enter the embryo sac from the nucellus until cy- The pollen tube enters through the micropyle toplasmic connections exist. When cytoplasmic which is the most common form of fertilisation connections are broken, the membrane of the egg and is known as porogamy. The second type is cell is formed which prevents the movement of called chalazogamy, wherein the pollen tube en- the virus. The capability of viruses to infect either ters the ovule at the chalazal end as in Casuarina. the ovule or pollen grains systematically prior to The third type is mesogamy where the pollen fertilisation assists in seed transmission. tube enters through the funiculus or through the Seed transmission is achieved either by direct integuments as in Cucurbita. The tip of the pollen invasion of the embryo via the ovule or by in- tube passes through the nucellus and enters the direct invasion of the embryo, mediated by in- embryo sac where it bursts discharging the two fected gametes. For some viruses in certain hosts male germ cells called sperms. (e.g. BSMV in barley), both processes operate Out of the two sperm cells, one fuses with the simultaneously, although the relative contribu- egg and forms a zygote. The second fuses with tion of the two processes will vary depending the polar nuclei and results in primary triploid upon a large number of factors (Mandahar 1981). endosperm nucleus. The process of fertilisation For direct embryo invasion, there is currently no where both the sperm cells on fusion form a explanation for how the virus is able to cross zygote and an endosperm is known as double the boundary between the parental and progeny fertilisation. The fertilised egg or zygote devel- generations in the ovule, and no routes have been ops into an embryo, and the primary endosperm identified that lead to the establishment of the nucleus divides a number of times to form many virus in the tissues of the developing embryo. nuclei. These nuclei are separated by cell walls to Furthermore, genetic and cell biological studies 5.2 Distribution of Virus in the Seed 87 have never been combined to obtain an overall invasion after fertilisation. For many virusÐhost understanding of the principles involved in seed interactions, both modes of embryo infection may transmission. result in maximal seed transmission (Johansen et al. 1994). The seed-transmitted viruses of the direct 5.2 Distribution of Virus in the virus invasion after fertilisation are found in Seed different seed tissues and may be confined to the embryo or endosperm tissues. In such cases, The location of the virus in seed determines the infection originates at the female gamete transmissibility of virus through seed. The virus production. Some examples of internally seed- is considered to be externally seed transmitted transmitted viruses are BCMV, PSbMV, TRSV, when it is outside the functional seed and in- SBMV, LMV, BSMV, PMV, PEBV, CMV, Urd ternally seed transmitted when it is within the leaf crinkle virus and CpAMV. Each virus tissue of the seed, respectively. When externally has been detected internally in cotyledons and seed transmitted, the virus is confined to the embryos of their respective hosts (Ekpo and testa as a contaminant. In the case of seeds from Saettler 1974;Ladipo1977; Adams and Kuhn fleshy fruits like tomato, cucumber, watermelon 1977; Uyemoto and Grogan 1977; Hoch and and apple, viruses such as TMV, ToMV, PVX, Provvidenti 1978; Datta Gupta and Summanwar CGMMV and Tomato bushy stunt, respectively, 1980; Beniwal et al. 1980; Beniwal and Chaubey adhere to the seed coat. During germination, 1984; Bharatan et al. 1984; Patil and Gupta the virus infection takes place through the tiny 1992; Varma et al. 1992;Yangetal.1997;Wang abrasions caused by small soil particles. It is sug- et al. 1997; De Assis Filho and Sherwood 2000; gested that the contact points between the testa Roberts et al. 2003; Ali and Kobayashi 2010). and suspensor as a route of entry. They further Some viruses like BSMV and TMV infect the suggested that the virus may be able to pass endosperm (Carroll 1972). Hoch and Provvidenti through the cell wall between the testa and sus- (1978) provided electron micrographic evidence pensor by an as yet unidentified mechanism that for localisation of BCMV particles and virus in- does not require plasmodesmata, or it may be able clusion bodies in mature embryos of dormant and to induce formation of new plasmodesmata, thus germinating bean seeds. They have also recorded allowing direct invasion of the embryo. There are virus in thin sections of embryo and endosperm a large number of research articles which sup- of infected soybean seeds (Tu 1975). The em- port the view of transmission of Tomato mosaic bryo invasion with Turnip yellow mosaic virus virus, a strain of TMV which is carried as ex- (TYMV) in Arabidopsis thaliana was reported by ternally seed transmitted primarily in tomato and De Assis Filho and Sherwood (2000). The distri- capsicum in number of countries and has been bution of viruses within the seed could be studied confirmed by extensive serological tests. Ear- by bioassay/serology and electron microscopy. lier, Huttings and Rast (1995) have reported that Large number of viruses are located in embryo tomato mosaic virus in tomato seed is localised and hence high percentage of seed transmission. on seed coat and sometimes in the endosperm. Some of the embryo-borne viruses are PSbMV During Pradhanang et al. (2005) have expressed in peas (Wang and Maule 1992, 1994; Roberts the doubt of Tomato mosaic virus transmission et al. 2003), BCMV in beans (Hoch and Provvi- through tomato seeds, and negative results were denti 1978), SMV in soybean (Bowers and Good- also obtained in DAS ELISA and grow out tests man 1979; Porto and Hagedorn 1975)andCMV (Pradhanang 2005). in lupin (Lupinus angustifolius) (O’keefe et al. Now it is clearly understood that there are 2007) and Pepper (Ali and Kobayashi 2010). two ways to infect the developing embryo: by Thus, viral access to embryo is gained either either indirect invasion through infection of the indirectly by infection of reproductive tissues gametes before fertilisation or by direct embryo before embryogenesis, that is, ovule, megaspore 88 5 Mechanism of Seed Transmission mother cell and pollen mother cells, or directly by transmission of two pathotypes of TSV, which invasion of embryo during stages of embryogen- differ in physicoÐchemical properties and RNA esis (Carroll 1981). The ultimate levels of seed secondary structure. Their results indicated that transmission in some virusÐhost combinations infectivity of TSV Mel 40 and Mel F isolates may in fact result from a combination of both appears to be dependent on homologous RNAs. routes of infection. BSMV contains three RNA species: RNA ’, RNA “ and RNA ”. Major seed determinants are located in the 50 untranslated region (UTR) and 5.3 Virus Longevity in Seeds a 369 nt repeat present in the ”aand”b genes. Mutations in these determinants significantly Stability of the virus in the seed depends on influence replication and translation of the virus. its type, location and the host. In a majority of It was shown that seed transmission is completely cases, the viruses infecting embryo survive for blocked when the repeat sequence is present. This longer periods, sometimes even as long as the sequence is suggested to enhance the replication seeds (Erkan 1998). For example, BCMV in bean and movement of BSMV, while its mutation seed remained viable for 30 years, sowbane mo- affects both the transmission and symptom saic virus in Chenopodium murale for 14 years, expression in barley plant. PNRSV in Prunus pensylvanica for 6 years and It is worth mentioning that the 12 K gene Squash mosaic (SqMV) in Cucurbita pepo and of tobraviruses, HC-Pro of potyviruses and the TRSV in soybean for over 5 years. Many po- ”b gene of hordeiviruses share cysteine-rich tyviruses like SMV and CpAMV survive for 2Ð domains that regulate the transmission process 3 years in their respective legume host seeds. by affecting the replication and movement of TMV in tomato seed has been known to be active the virus in the reproductive tissues of respective for 9 years. In true potato seed, the Potato spindle hosts and thus influences the infection of the tuber viroid survives for 21 years (Singh et al. embryo (Johansen et al. 1994). 1991). Longevity of important seed-transmitted viruses in crops is furnished in Table 5.1. 5.5 Factors Influencing Rate of Seed Transmission 5.4 Genetics of Seed Transmission Some of the seed-transmitted viruses become inactive within a short span of time, while oth- Various researchers have expressed different ers remain active as long as the seeds remain opinions regarding the role of genetics in viable. The active period of the virus generally transmission of viruses through seed. For depends on the seed longevity and location in the example, Keller et al. (1998) have reported seed. It appears to be characteristic of the virusÐ that Potyvirus genome-linked protein (VPg) host combination (Table 5.1). Some nematode- determines the Pea seed-borne mosaic virus transmitted viruses persist in the seed of certain pathotype. On the other hand, Johansen et al. weed plants and increase the chances of virus (1996) found that CP and HC-Pro coding regions perpetuation under field conditions. Some other of the Pea seed-borne mosaic virus genome were viruses show high seed transmission since they required for efficient transmission through seed are effectively translocated into the developing of Pisum sativum. The ”b protein of Barley stripe seeds in a passive way along with carbohydrates. mosaic virus has been shown to the involved But in other cases, certain inhibitors may im- in seed transmission (Edwards and Steffenson pede translocation. Considerable variation in seed 1996). transmission is influenced by one or more factors Walter et al. (1995) have studied the viral such as host variety, virus strain, the time of in- genetic basis of symptom severity and seed fection of the host and the prevailing temperature 5.5 Factors Influencing Rate of Seed Transmission 89

Table 5.1 Longevity of some important seed-transmitted viruses in different crops Viability Virus Crop (years) Reference Alfalfa mosaic Medicago sativa 3 Frosheiser (1970) M. sativa 5 Frosheiser (1974) M. sativa 7 Babovic (1976) Barley stripe mosaic Hordeum vulgare 3 McNeal et al. (1961) H. vulgare 19 McNeal et al. (1976) Triticum aestivum 6:5 Scott (1961) Bean common mosaic Phaseolus vulgaris 30 Pierce and Hungerford (1929) P. vulgaris 3 Nelson (1932) P. vulgaris 38 Walters (Walters 1962a, b) P. vulgaris 6 Polak and Chod (1967) P. angularis 4 Tsuchizaki et al. (1970) Bean southern mosaic P. vulgaris 0:6 Zaumeyer and Harter (1943) Bean western mosaic P. vulgaris 3 Scotland and Burke (1961) Broad bean stain Vicia faba 1 Cockbain et al. (1973) Vicia faba 4 Vorra-urai and Cockbain (1977) Cowpea aphid-borne mosaic Vigna unguiculata 2 Khatri and Chohan (1972) Cucumber green mottle mosaic Cucumis sativus 0:5 Van Koot and Van Dorst (1959) Cucumis sativus 3 Yakovleva (1965) Cucumber mosaic P. vulgaris 2:25 Bos and Maat (1974) P. vulgaris 0:5 Meiners et al. (1977) C. sativus 1 Meiners et al. (1977) Stellaria media 1:75 Tomlinson and Walker (1973) Dodder latent mosaic Cuscuta campestris 1 Bennett (1944) Eggplant mosaic Solanum melongena 0:6 Mayee (1977) Hop mosaic Humulus lupulus 2 Blattny and Osvald (1954) Lychnis ring spot Lychnis divaricata 3:3 Bennett (1959) Silene noctiflora 2:15 Bennett (1959) Muskmelon mosaic Cucumis melo 3 Rader et al. (1947) C. melo 5 Middleton and Bohn (1953) Prune dwarf Prunus sp. 3:5 Gilmer (1964) Prunus necrotic ring spot P. pensylvanica 6 Fulton (1964) Raspberry ring spot Capsella bursa-pastoris 6 Lister and Murant (1967) Stellaria media 6 Lister and Murant (1967) Sowbane mosaic Chenopodium murale 6:5 Bennett and Costa (1961) C. murale 14 Bennett (1969) Soybean mosaic Glycine max 2 Kendrick and Gardner (1924), Sinclair and Backman (1993) G. max 1 Provvidenti et al. (1982) Squash mosaic Cucurbita pepo 3 Middleton (1944) C. pepo 5 Middleton and Bohn (1953) Tobacco mosaic Lycopersicon esculentum 3 Alexander (1960) L. esculentum 9 Broadbent (1965) Tobacco ring spot Petunia violacea 0:6 Henderson (1931) Glycine max 5 Laviolette and Athow (1971) G. max 0:75 Athow and Bancroft (1959) Nicotiana sp. 5:5 Valleau (1939) Tomato black ring C. bursa-pastoris 6 Lister and Murant (1967) S. media 6 Lister and Murant (1967) Source: Agarwal and Sinclair (1987) 90 5 Mechanism of Seed Transmission conditions (Pathipanawat et al. 1997; Singh and PMV differed in frequency of seed transmission Mathur 2004). in Starr peanut M1 D 0.3%; M2 D 0; M3 D 8.5 Maule and Wang (1996) suggested four factors and N D 0 (Adams and Kuhn 1977). The impact that possibly regulate the seed transmission. of virus strains on seed transmission has also 1. Host genes control the ability of the virus to been noticed with Bean yellow mosaic virus invade the gametes or the meristematic tissues (Anderson 1957), BSMV (Hamilton 1965), as in the case of cryptic viruses (Kassanis et al. AMV (Frosheiser 1974), BCMV (Morales and 1978). Castano 1987), CMV (Davis and Hampton 1986; 2. Host genes control the survival of the gametes Sandhu and Kang 2007), SMV (Tu 1989;Domier and the embryo in the presence of the virus. et al. 2007)andSubterranean clover mottle virus 3. Host factors regulate the multiplication and (Wroth and Jones 1992), Broad bean stain virus movement of the virus. and Pea seed borne mosaic viruses (Hampton 4. Host factors govern the maturation of the em- et al. 1981; Kumari and Makkouk 1995; Johansen bryo that affects the longevity of the virus. et al. 1996) and TSV in soybean (Ghanekar and Schwenk 1974).

5.5.1 Number of Infection Sources 5.5.3 Mixed Infections The effect of number of sources of infection has attracted particular attention where infection The rate of seed transmission of a virus may originates partially in infected stocks. In England, also be influenced by infection with other viruses. lettuce crop grown in five commercial seed lots Seed transmission of SMV in seeds from soy- with 2.2Ð5.3% infection by LMV had 25Ð96% bean plants infected with either SMV alone or infection with mosaic at the end of the growing in combination with Bean pod mottle virus was season. But six batches of seed with LMV infec- 6 and 11%, respectively (Ross 1963). Similarly, tion up to 0.1% have yielded crops with infected Southern bean mosaic virus (SBMV) was 12% plants up to 0.5% (Tomlinson 1962). in cowpea but increased to 20% in the pres- ence of Cowpea chlorotic mottle virus (CCMV) (Kuhn and Dawson 1973). Seed transmission of 5.5.2 Virus Strain/Isolate Turnip yellow mosaic virus (TYMV) in Ara- bidopsis thaliana doubly infected with TYMV The amount of seed transmission varies greatly and TMV was 70% compared to infection of with the virus strain/pathotype. At Corvallis TYMV alone, which was only 31% (De As- (USA)., two isolates of PSbMV (P-1 and P-4) sis Filho and Sherwood 2000). Mechanism of were transmitted through seed at 24 and 0.3%, increases in transmission of a virus in mixed respectively, in Pisum sativum 549 (Johansen infections is not clearly understood. et al. 1996). Earlier, Ghanekar and Schwenk (1974) noticed 2.6Ð30.6% seed transmission for soybean isolates of Tobacco streak virus, 5.5.4 Host Species whereas a tobacco isolate of the same virus did not show any transmission. Isolates belonging The degree of seed transmission of a virus varies to serological group I of SqMV were seed with the host species which was noticed as early transmitted in pumpkin, squash, cantaloupe, as 1935 by Hewitt, while working with BCMV. honey dew and watermelon, while isolates Bock and Kuhn (1975) reported seed transmis- belonging to group II of SqMV were seed sion of PMV in peanut, but not through cowpea transmitted only in pumpkin and squash (Nelson or soybean. Raspberry ring spot virus (RRV) and Knuhtsen 1973a). Similarly, four isolates of was seed transmitted at 2Ð3% in Capsella bursa- 5.5 Factors Influencing Rate of Seed Transmission 91 pastoris but at more than 50% in Beta vulgaris. and Varma 1986). The examples in different virus Tomato black ring virus (TBRV) was transmitted and cultivars/breeding lines cited here clearly at 5% in Rubus idaeus but reached up to 100% in indicate that the variation was primarily due to Cerastium vulgatum, Fumaria officinalis, Myoso- different environmental conditions, virus strain, tis arvensis and Polygonum persicaria (Lister and hostÐplant age and host plant constitution physi- Murant 1967). ology. Seed transmission rates of Broad bean stain High degree of seed transmission in different virus (BBSV) varied between 0.2 and 32% in host cultivars of cryptic viruses has also been 19 lentil genotypes (Makkouk and Kumari 1990; recorded. The incidence of Beet cryptic virus M Kumari et al. 1996). Even, Al-khalaf et al. (2002) was between 42 and 90% in seedlings of 8 culti- reported the variation in seed transmission of vars of Beta vulgaris (Kassanis et al. 1977; White BBSV in lentil based on genotype variability and and Woods 1978), Vicia cryptic virus incidence seed size. was from 50 to 75% in 9 cultivars of Vicia faba Considerable differences exist in the magni- (Kenten et al. 1978), Radish yellow edge virus tude of seed transmission in different virusÐhostÐ was detected in 80Ð100% of seedlings of 6 cul- variety combinations. LMV was very commonly tivars of Raphanus sativus (Natsuaki et al. 1979), seed transmitted in some but not through the seed Carnation cryptic virus seed transmission ranged of certain lettuce varieties (Couch 1955). Simi- from 85 to 100% in 13 cultivars of Mediterranean larly, seed transmission of CMV was dependent hybrids of carnation but only 9% in the garden in cowpea on the host variety (Anderson 1957). carnation cultivar Chabaud (Lisa et al. 1981)and At Hyderabad (India), Reddy et al. (1998a, b) Ryegrass cryptic virus incidence varied from 0 have studied seed transmission of Indian peanut to 82%y in 21 species and cultivars of Lolium clump virus in 22 peanut genotypes of botanical (Plumb and Lennon 1981a, b). type of Spanish, Virginia, Valentia and runner, A similar variation in seed transmission which varied from 3.5 to 17% depending on the among different cultivars was reported for other genotype. A similar results were observed by viruses such as SMV (Kendrick and Gardner Eslick and Afanasiev (1955) and Singh et al. 1924;Ross1961; Kennedy and Cooper 1967), (1960) in some barley varieties infected with LMV (Fegla et al. 1983), BCMV (Agarwal et al. BSMV. The PSbMV seed transmission varied 1979), CpAMV (Ladipo 1977; Ata et al. 1982), from 0 to 87.5% in 148 pea varieties (Stevenson PSbMV (Musil et al. 1983), Brome mosaic virus and Hagedorn 1973; Musil et al. 1983) and 1.9Ð (BMV) (Von Wechmar et al. 1984), CMV (Davis 32.7% in 19 pea varieties with the same virus and Hampton 1986), AMV (Frosheiser 1974), infection (Gallo and Jurik 1995). While working White clover cryptic virus (Boccardo et al. 1985; at France, Khetarpal et al. (1993) recorded varied Natsuaki et al. 1986), Alfalfa cryptic virus M degrees of seed transmission of PSbMV in dif- (Boccardo et al. 1983; Natsuaki et al. 1984), ferent cultivars of pea. With the same virusÐhost BgMV (Varma et al. 1992), TYMV (De Assis combination, Wang et al. (1993) noticed no seed Filho and Sherwood 2000) and CMV (Gillaspie transmission in pea cultivars like Maro, Princess et al. 1998a, b). and Progreta, while it was 74% in the cultivar The effect of host plant on seed transmission Vedette. In India, Mali et al. (1987) recorded a lot of virus is mostly related with its susceptibility of variation in seed transmission of CpAMV and or resistance to specific virus/strain. Therefore, CMV in 14 and 10 promising varieties of cowpea, greater disease severity of the mother plant results respectively. Seed transmission of Cowpea band- in highly susceptible varieties than less suscepti- ing mosaic and Cowpea chlorotic spot virus in ble/resistant varieties. This close relationship has 15 cultivars ranged from 0% in P-910 and R-352 been explained on the basis of mechanisms of lines to 19% in Pusa Phalguni and 0% in R-339 disease resistance. to 18% in Iron New line, respectively (Sharma 92 5 Mechanism of Seed Transmission

5.5.5 Stage of Infection Highly determinant plants which flower over a relatively short period might give no seed trans- Voluminous literature is available on the degree mission from the inoculations after flowering of seed transmission in relation to the infection begins (Eslick and Afanasiev 1955). However, stage of the plant. Infection of plants before indeterminate plants like peanut and late flow- flowering leads to embryonic infection resulting ering may produce virus transmitting seeds if in maximum seed transmission (Schippers 1963). inoculation occurs early enough. Plants infected For example, in Columbia, the seed transmission after flowering generally do not produce infected of BCMV in the bean cultivar Similac was 40 , seed, and transmission is successful only before 9% and nil and 41.8, 2.8 and 10.1% in cultivar cytoplasmic separation of the developing embryo Pubbele Witte, when inoculated at 10, 20 and from maternal tissue (Bos 1977). 30 days after sowing, respectively (Morales and Generally, early virus infection leads to high Castano 1987). Similarly, 18Ð19%y seed trans- seed-transmitted virus in number of virusÐhost mission of SMV in soybean was recorded when combinations. Studies carried out by Chitra et al. inoculated at 3Ð4 week and only 3Ð4%y when in- (1999) have shown that ToMV and TMV can oculated at 9Ð10 week after planting (Bowers and enter in seeds of tomato and bell pepper, irre- Goodman 1979; Irwin et al. 2000). In the same spective of growth stage at the time of inocula- crop, TRSV was transmitted up to 100% through tion. Inoculation even at flowering and fruit-set seeds from plants infected before flowering, but stage can lead to establishment of virus in seeds. the percentage decreased when infected prior However, the concentration of virus in seeds will to or immediately after flowering (Athow and be higher, if the plants are infected at an early Bancroft 1959). While working with TRSV in growth stage. Since ToMV and TMV occur in the Phaseolus aureus, Shivanathan (1977) reported seed coat, even late infection of the host leads to that for effective seed transmission, the mother establishment of virus particles in the seed. plant must become infected at least 10 days prior to pollination. Similar results were observed with LMV/lettuce crops, but plants infected after 5.5.6 Environmental Factors flowering did not transmit the virus through seed (Couch 1955). In India, seed transmission of The seed transmission also markedly depends on ULCV infection in urad bean seed (Vigna mungo) the season and temperatures that prevail during was 68% when 10-day-old plants were inocu- plant growth. High seed transmission of CpBMV lated. Subsequently, the rate of seed transmission and CpCSV was noticed in seeds of rainy season declined to 46, 22 and 10% in 20-, 30- and cowpea crop (Sharma and Varma 1986). But 40-day-old inoculated plants, respectively. The in Georgia, Peanut stripe virus (PStV) infection virus failed to become seed transmitted when reached 37% and 18% in summer and winter 50-day-old plants were inoculated (Dubey and harvest, respectively. While in Spanish cultivar Sharma 1985). Similar trend was noticed with while it was 19 and 11% in a runner type during Cowpea banding mosaic (CpBMV) and Cowpea the two seasons (Demski and Warwick 1986). chlorotic spot viruses (CpCSV) when inoculated Barley cultivar seed was more prone to BSMV to 10-day-old cowpea plants under field con- in spring than in autumn (Slack et al. 1975). ditions, with 18Ð25% and 15Ð20% seed trans- Thus, difference in seed transmission in sowings mission, respectively. However, plants inoculated at various seasons was due to factors like occur- after 35 days of sowing had 6Ð8% and 5Ð6% rence of susceptible crop growth stage at a time seed transmission, respectively, for CpBMV and when appropriate aphid vector activity was at a CpCSV (Sharma and Varma 1986). peak. The rate of infection with BSMV in barley in- Seed transmission of BCMV in bean seeds at ı creased when plants were inoculated 10 days be- 20 C varied between 16 and 25%, while it was ı fore heading than from earlier or later infections. negative at 16.5Ð18.5 C(Crowley1957a, b). On 5.6 Reasons for Failure of Seed Transmission 93 the other hand, the Southern bean mosaic virus has been studied by Wang and Maule (1994). infected 95% of the embryos of the immature Pea seed-borne mosaic virus (PSbMV), a seed- seed in bean plants grown at 16Ð20ıC and only transmitted virus in pea and other legumes, 55% when grown at 28Ð30ıC(Crowley1959). invades pea embryos early in development. This Maximum (88%) seed transmission of PSbMV process has been controlled by maternal genes was recorded at 28ıC in peas only after 5 weeks and, in a cultivar that shows no seed transmission, of plant growth but not at 21 nor 32ıC (Khetarpal is prevented through the action of multiple and Maury 1990). Singh et al. (1960) have noted host genes segregating as quantitative trait loci. greater seed transmission of BSMV from bar- These genes control the ability of PSbMV to ley plants grown at 20Ð24ıC than at higher or spread into and/or multiply in the nonvascular lower temperatures. In Scotland, Stellaria media, testa tissues, thereby preventing the virus from an important weed host of Raspberry ring spot crossing the boundary between the maternal and virus, is slightly more readily seed transmitted progeny tissues. Immuno-cytochemical and in at 14ıC than at 18Ð22ıC, whereas Strawberry situ hybridisation studies suggested that the virus latent ring spot virus is less frequently trans- uses the embryonic suspensor as the route for the mitted at 14Ð18ıCthanat22ıC (Hanada and direct invasion of the embryo. The programmed Harrison 1977). While working with LMV in degeneration of the suspensor during embryo lettuce, Ryder (1973) observed high rate of seed development may provide a transient window transmission from mother plants at lower day for embryo invasion by the virus and could temperatures than those at higher temperature. explain the inverse relationship between the age However, the results of environmental factors on of the mother plant for virus infection and the seed transmission are often contradictory (Ben- extent of virus seed transmission. It appears nett 1969; Shepherd 1972). that two mechanisms, namely, embryonic and non-embryonic, operate for the virus in seed transmission, based on whatever the virus is 5.6 Reasons for Failure of Seed unable to enter the embryo or get eliminated Transmission after entry. Bennett (1969), Neergaard (1977, 1979a, b), Bos (1977) and Carroll (1981)have The presence of virus in a seed (either in seed- reviewed this particular aspect in detail. coat, cotyledons, embryo) does not always lead to seedling infection. This property distinguishes a seed-transmitted virus that is carried by the seed 5.6.1 Inability to Infect Embryos but does not infect the seedling produced from the seed (Neergaard 1979). Maximum seed transmission occurs when the Most viruses are not seed transmitted. The embryo is infected. This is apparently the reasons for the same are still obscure. However, common mode of virus transmission, and Bennett certain inferences can be made based on the (1969) terms this as embryonic transmission. available experimental data. The seed-transmitted During the process of embryo initiation by viruses are found in different seed tissues and the union of nuclei in an infected embryo sac, may be located in the embryo, endosperm tissues the embryo begins to develop in a virus-free (embryonic) or, in some cases, only in or on seed medium. There are no protoplasmic connections coats (non-embryonic). It is generally accepted between the cells of the embryo and cells of that for true seed transmission, the virus must the adjacent tissue. The embryo absorbs food enter and survive in the embryo. The ability materials from virus-infected areas without to invade embryos and get transmitted through becoming infected. Only a few viruses are seed is determined not solely by either the virus able to pass through cell walls, and in the or the host. Genetic and structural analysis of absence of penetrating protoplasmic strands, seed-transmitted and non-seed-transmitted Pea usually the cell wall acts as a formidable barrier seed-borne mosaic virus in certain pea lines to virus passage (Neergaard 1977). Lack of 94 5 Mechanism of Seed Transmission direct vascular/cellular contact through the virus was introduced into ovules, it failed to infect plasmodesmata with the mother plant inhibits but succeeded in susceptible ones (Neergaard seed transmission (Bennett 1969; Carroll 1972). 1977). Absence of such virus pathways explains why embryonic infection exclusively requires mother plant infection before the production of 5.6.2 Inability of Virus Survival gametes. in the Embryos Many researchers have suggested plasmodes- mata as the probable avenue of virus movement It is now clearly known that a virus-inactivating in infected plants based on their observations of system operates in the infected seed when it virus and virus-like particles in plasmodesmata is drying during maturity (Duggar 1930;Cheo (Esau et al. 1967;Davidson1969; de Zoeten and 1955). Southern bean mosaic virus infection de- Gaard 1969; Kitajima and Lauritis 1969; Roberts creases abruptly or disappears as the seed matures and Harrison 1970; Kim and Fulton 1971;Wein- even after heavy infection in the immature seed. traub et al. 1976). The virus can move from cell In West Africa, Konate et al. (2001) reported to cell in the form of complete particles or viral that in 17 rice genotypes, Rice yellow mottle nucleic acid. virus was detected in all seed parts including It has been hypothesised by Caldwell (1934) glumolla, endosperm and embryo at 65Ð100%. that marked differences between the growth rate Nevertheless no seed-transmitted infection was of embryonic and endosperm tissues within de- found as the virus decreased throughout the pro- veloping ovules normally lead to rupture of the cess of seed formation, suggesting inactivation protoplasmic bridges between these tissues and of virus due to seed maturation and desicca- the nucellus. This view has been based on the tion. Even Abo et al. (2004)havealsoproved assumption that protoplasmic continuity is nec- the nontransmissibility of RYMV through rice essary to bring about embryonic infection by seeds. Allarangaye et al. (2006) have reported translocating the virus from the surrounding ma- nontransmission of RYMV through seeds of wild ternal tissues to the developing ovules. Another rice species. RYMV was infectious in freshly possibility is that, generally, most viruses do not harvested seed extracts; however, most infectivity multiply in meristematic tissues. Embryo being was lost in dried rice seeds, possibly due to a site of very intense metabolic activity may be virus inactivation following dehydration of the viewed as a particular type of meristematic tissue seeds (Konate et al. 2001). A similar phenomenon responsible for the failure of the virus invasion. was observed in pea and cowpea infected with It is likely that natural cytokinins present in the Pea streak and Cowpea chlorotic mottle viruses, embryo may make the virus inactive. Caldwell respectively (Ford 1966;Gay1969). Although (1962) suggested another reason for failure of TSWV was not seed transmitted in peanut, virus seed transmission. Many plant viruses lack the has been recorded from the testa of immature ability to invade meristematic tissue since path- and freshly harvested mature seeds. However, it ways for virus synthesis cannot compete suc- could only be detected serologically in the testa of cessfully with cellular systems for high-energy dried seeds. Freshly harvested mature seeds with phosphorylated compounds. testa-containing infective virus failed to transmit Resistance of gametes to virus infection has TSWV when tested by grow out tests (Reddy also been implicated as one of the factors. Evi- et al. 1983a). dence of such resistance was reported by Medina Seed-transmitted viruses possess a unique and Grogan (1961) in the bean cultivars Red property which enables them to invade even Mexican and Idaho Refugee, which were resis- reproductive tissues. This property may be related tant to BCMV. No seed transmission was estab- to the glycoprotein portion of the coat protein. lished when pollinated from infected susceptible The carbohydrate residues in the viral coat cultivars. In resistant embryos, even though the may function as recognition sites to attach the References 95 virus to gametophyte cell surfaces. However, the Alexander LJ (1960) Inactivation of tobacco mosaic virus association of the carbohydrate with virus coat from tomato seed. Phytopathology 50:627 protein has not been demonstrated conclusively Ali A, Kobayashi M (2010) Seed transmission of cu- cumber mosaic virus in Pepper. J Virol Methods 163: (Partridge et al. 1974). 234Ð237 The abortion of microspores due to virus in- Al-Khalaf M, Makkouk KM, Kasem AH (2002) Seed fection may have a bearing on lack of seed trans- transmission of broad bean stain virus in lentil with mission (Valleau 1939). Caldwell (1962) sug- respect to genotype variability and seed size. Arab J Plant Protect 20:106Ð110 gested virus-induced meiotic irregularities to be Allarangaye MD, Traore O, Traore EVS, Millogo RJ, concerned with lack of seed transmission. Konate G (2006) Evidence of non-transmission of rice yellow mottle virus through seeds of wild host species. J Plant Pathol 88:309Ð315 Anderson CW (1957) Seed transmission of three viruses 5.7 Conclusions in cowpea. Phytopathology 47:515 Ata AEA, Allen DJ, Thottappilly G, Rossel HW (1982) Perusal of the Table 1.2 indicates that consider- Variation in the rate of seed transmission of cow- pea aphid borne mosaic virus in cowpea. Trop Grain able number of plant viruses of different virus Legume Bull 25:2Ð7 groups are seed transmitted in large number of Athow KL, Bancroft JB (1959) Development and trans- crop plants. The percentage of virus transmission mission of tobacco ring-spot virus in soybean. Phy- in different hosts varies due to virus strain, topathology 49:697Ð701 Babovic MV (1976) The transmission rate of alfalfa mo- host variety, environment factors, etc. There saic virus by lucerne seed. Acta Biol Yugosl 13:83 are certain controversial results regarding seed Beniwal SPS, Chaubey SN (1984) Internal seed borne transmission in certain virusÐhost combinations. nature of Urdbean leaf crinkle virus in urdbean seed. For example, Jain et al. (2006) reported the Seed Res 12(2):8Ð10 Beniwal SPS, Chaubey SN, Bharatan N (1980) Presence transmission of Tobacco streak virus through of urdbean leaf crinkle virus in seeds of mungbean seed of cucumber (Cucumis sativus) and gherkins germplasm. Indian Phytopathol 33:360Ð361 (Cucumis anguria) to the extent of 3Ð68%. Bennett CW (1944) Latent virus of dodder and its effect on sugarbeet and other plants. 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Roberts IM, Wang D, Thomas CL, Maule AJ (2003) Pea Tsuchizaki T, Yora K, Asuyama H (1970) Seed trans- seed-borne mosaic virus seed transmission exploits mission of viruses in cowpea and Azuki bean plants. novel symplastic pathways to infect the pea embryo II. Relations between seed transmission and gamete and is, in part, dependent upon chance. Protoplasma infection. Ann Phytopathol Soc Japan 36:237Ð242 222:31Ð43 Tu JC (1975) Localization of infections soybean mosaic Ross AF (1961) Systemic acquired resistance induced virus in mottled soybean seeds. Microbiology 14: by localised virus infections in plants. Virology 14: 151Ð156 340Ð358 Tu JC (1989) Effect of different strains of soybean mo- Ross JP (1963) Interaction of the soybean mosaic and bean saic virus on growth, maturity, yield, seed mottling pod mottle viruses infecting soybeans. Phytopathology and seed transmission in several soybean cultivars. 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Abstract The methods for detecting seed-transmitted virus infection should be rapid, reliable and sensitive besides being simple to achieve results. This has been a challenge since the advent of the discipline of plant virology over 100 years ago, and a great variety of methods have been developed since that time. Based on biological, serological and molecular tests, the virus and viroid diseases are diagnosed. In biological tests, a close and careful observation on the seed morphology in certain cases gives a tentative indi- cation of the presence of virus (es). Grow-out test also helps in seed-borne virus diagnosis. Among serological tests Ouchterlony, ELISA and its variants, dot-immunobinding assay, TBIA, and Immunosorbent electron microscopy are widely used for virus detection. Among antibody-based detections, and Polymerase chain reaction (PCR) and its variants, (real- time PCR, RT-PCR, IC-PCR, IC-RT-PCR, multiplex PCR), are extensively practice. Protocols have been implemented in the field using portable real- time PCR machines for same-day, on-site results; cDNA probes which are labelled with radioactive markers or nonradioactive markers are used for diagnosis of seed-borne virus and viroid diseases. Array technology has revolutionised the world of viral diagnosis because of its efficiency in screening a large volume of infected seed samples in a single array plate or reaction.

and of little use if one requires quick information. 6.1 Introduction Hence, methods for detecting seed-transmitted virus infection should be rapid, reliable and sen- The movement of seed material from one country sitive besides being simple to undertake and give to another poses a serious problem, as it intro- clear-cut results. As some of the devastating virus duces new diseases to that area. The presence of viruses in true seed is often established by raising diseases are seed-transmitted, quick and thorough seedlings from suspected seed lots and observing evaluation of the seed lot is essential at the symptoms on them. This test is time-consuming quarantine stations.

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 6, 101 © Springer India 2013 102 6 Detection of Plant Viruses in Seeds

6.1.1 Seed Health Testing over 75% of the world’s vegetable seed supply (Sheppard 1999; Sheppard and Wesseling 1997). It is well known that about 90% of all the food ISHI published many protocols, organised by crops grown are propagated by seed. Seeds are crop. For horticultural crops, the ISHI-Veg, the both vehicles and victims of disease. The signif- Seed Health Testing Methods Reference Manual icance of transmission of plant diseases through (van Ettekoven 2002) and this one included 21 seeds was realised long ago. It is a well-known different protocols with diverse status ranking. fact that infected or contaminated seed is a pri- mary source of inoculums for a large number of 6.1.1.1 ISHI-Veg Status destructive diseases of important food, fodder and 1. An ISHI-validated reference method is a fibre crops (Neergaard 1977b). Besides affecting method that has been through the ISTA/ISHI the crop yields, the seed-borne pathogens affect comparative testing process and is being the nutritive quality and value of the seed, leading published as ISTA working sheet. to trade barriers. In some cases infected seeds 2. An ISHI reference method is a method that has are the only source of initial inoculums in the been through ISHI comparative testing and field. has been reviewed by the ISTA/ISHI reviewers One of the most relevant aspects about con- for publication as ISTA working sheet. trolled health quality seed is referred to the de- 3. An ISHI-accepted method is a method that tection methods used against different pathogens. is commonly used by the seed industry for International Seed Testing Association (ISTA) determining seed health. The method has been took the leading role in studying seed-borne dis- published in a journal, as a working sheet, eases. In 1924, people were interested in seed’s and/or is publicly available for comparative gene purity and germination aspects. Later then, testing and commonly in use by the seed thanks to the efforts made by Dr. L.C. Doyer industry. The method is in comparative publishing the first manual for the determina- testing or aspects of the test are being tion of seed-borne diseases and being the first tested for inclusion or deletion from the director of the PDC (Plant Disease Committee), method. the importance of seed-transmitted diseases was 4. An ISHI-reviewed method is a method turned into first priority, but detection methods that is publicly available or published, were applied according to the criterion of each documented and prioritised by the ITG, but laboratory, so their results were not compara- not subjected to a comparative test. The tive. So, in 1957, PDC established a compara- method usually is accepted by other agencies tive health testing programme and standardised or groups. applied methods. The complexity for comparison After the second ISTAÐPDC symposium, a of these methods made a considerable step back Joint ISTA/ISHI Guidelines for Comparative on the publication of working sheets (protocols). Testing of Methods for Detection of Seed ISTA-approved methods became rules. By 1999, Transmitted Pathogens was edited. Through this only 64 protocols were being compared, and combined effort of many laboratories, organised only 14 of them were accepted as rules. As the under this alignments, the ISTA developed the solutions proposed by ISTA did not satisfy the Manual of Seed Health Testing Methods that has needs of international seed industry, this industry, two sections: (1) Ð validated test methods and in 1994, organised the International Seed Health (2) Ð peer-reviewed methods. Initiative for Vegetables (ISHI-Veg) chartered by The transition process from working protocol the vegetable seed industries in the Netherlands to ruled method is long and laborious, so working and France. The initiative was soon joined by the protocols can be considered as adequate methods seed companies in the United States, Israel and for seed analysis. Nowadays, it is common Japan. This group represents the production of that technological advances may become 6.1 Introduction 103 obsolete and research institutions, universities From the earlier Chapter 2, it is clearly and also individual scientists/researchers have known that the economic losses caused by published protocols for the easy access to viruses are very high and hampers agricultural the research scientists (Duncan and Torrance yields. In India, several government and non- 1992; Foster and Taylor 1998; Dijkstra and government organisations import germplasm for de Jager 1998;Hull2004; Nayudu 2008; crop diversification and crop improvement. The Ahlawat 2010). government of each country should restrict the At most of the germplasm centres, the entry of the viruses through seed which were not cultivars of some crops like peas, peanuts, recorded in their respective country. Germplasm beans and potatoes are generally contaminated of different crops are being exchanged between with seed-transmitted viruses (Hampton and countries for their breeding programmes and Braverman 1979; Hampton et al. 1982;Bharathan also for distribution to the farmers directly for et al. 1984; Alconero and Hoch 1989). For agriculture with the view of introducing new crop example, from survey of USDA Phaseolus or variety which is high yielding. In this regard germplasm, Klein et al. (1988) found BCMV National Bureau of Plant Genetic Resources in accessions of 10 Phaseolus species. This (NBPGR) is the nodal agency in India for is a serious problem in Phaseolus accessions quarantine processing of imported germplasm. since approximately 60% of the germplasm Similarly, Directorate of Plant Protection, was contaminated. Even in Canada, more Quarantine and Storage, Ministry of Food and than 14,000 pea germplasm lines maintained Agriculture, New Delhi (India), is also involved at the gene bank of the Crop Improvement in intercepting the new pests and diseases Research Centre, University of Saskatchewan carried in germplasm received from different found contaminated with PSbMV were discarded countries. Inevitably the large-scale introduction (Kartha and Gamborg 1978). In the United States, of germplasm involves a risk of accidentally seed-transmitted CMV was introduced through introducing seed-transmitted pathogens. In international exchanges of bean germplasm, particular diseases that are often symptomless despite the fact that the plants grown in bean- such as those caused by viruses pose a great producing areas of the Pacific Northwest were risk. In order to avoid this risk, sensitive and virus-free (Davis et al. 1981). Hence, it is reliable testing procedures are required to detect imperative that all the incoming seed and the viruses in seeds to ensure that the introduced plant material from various countries should seed material is free from viruses of quarantine be subjected to strict quarantine regulations importance. Large number of virus diseases that before releasing for use in breeding work or were intercepted at NBPGR stations, which were for cultivation. Any negligence in examination not known in India, were detected (Khetarpal of imported seeds and planting stocks may lead et al. 2001;PrasadaRaoetal.2004;Chalam to introduction of several unknown viruses into et al. 2005). Harvest from only virus-free plants the country. Intensive researches have been made are being released to the intenders. in different parts of the world to evolve simple diagnostic techniques. The Danish Government Institute of Seed Pathology established in 1967 6.1.2 Low Seed Transmission/ at Copenhagen by the Danish International Symptomless Carriers Development Agency (DANIDA) has been doing laudable service in the field of seed pathology In some crops, very low rate of seed transmission by training plant pathologists from developing has caused enormous crop losses due to countries and assisting in programmes on secondary spread. To quote a few examples, education in the field of seed health certification LMV in lettuce seed even at a reasonably low and quarantine methods (Albrechtsen 2006). level of infection caused a high disease incidence 104 6 Detection of Plant Viruses in Seeds due to secondary spread through aphids. If be present at a low percentage and also the hosts seed transmission exceeds 0.1%, control of this may be symptomless carriers. The tests will be virus will not be satisfactory (Broadbent et al. quite helpful at quarantine centres as well as field 1951; Zink et al. 1956). The number of infected trials. Appropriate control procedures can only be lettuce seedlings that emerged per 0.4 ha at 0.1% applied effectively if the virus is correctly identi- rate would be 200/0.4 ha or 20,000 infected fied and distribution in an area or crop is known. seedlings/40 ha (Greathead 1966). Similarly, low In advanced countries, identification of seed transmission (0.3%) of PMV in peanut was seed-transmitted viruses is well attended by the found to be the primary source for secondary virologists. However, due to meagre facilities spread (Demski et al. 1983). in a majority of the underdeveloped countries, Maize/sweet corn is the most extensively there is a great risk of introducing the virus (es) grown crop; low levels of seed transmission into the countries. Diagnosis is mostly based of viruses have been documented in several on the reactions produced on indicator hosts reports. Maize dwarf mosaic virus (MDMV) where only greenhouse facility is available. By was transmitted at a low level, with only one serological tests, one can easily identify the seed transmission in 22,189 seedlings in one virus in a short time involving meagre space. study (Mikel et al. 1984), one seed transmission Tremendous improvements have been made in 11,448 seedlings in another study (Hill et al. in developing new techniques in recent years. 1974), and two transmissions in 29,735 seedlings However, one should not exclusively depend in a third study (Williams et al. 1968). Similarly, only on serological tests, since in certain cases Zhu et al. (1982) reported 14 seed transmissions even with high antibody titres, false-positive of MDMV-B, now known as Sugarcane mosaic serological reaction may be obtained between virus-MDB, in 22,925 seedlings, and Shepherd different viruses possessing the same antigenic and Holdeman (1965) found somewhat higher sites of virus particle. For instance the viruses transmission of MDMV, with 17 transmissions like BCMV, Bean yellow mosaic, CpAMV, SMV from 9,485 seeds, but 14 of these came from one and WMV belonging to the Potyvirus genus lot of 3,163 seeds. Jensen et al. (1996) found are serologically interrelated (Martyn 1968a; 21 seed transmissions of Maize chlorotic mottle Matthews 1970). Under such circumstances, one virus in over 42,000 seeds tested. During 2001, should guess the identity of the causal agent, but only 3 plants of sweet corn were infected with confirmatory tests including recently developed High plains virus out of 38,473 seedlings, which molecular assays and electron microscopy tests is again seed transmitted at low frequency. are essential for precise diagnosis. The success of Some of the viruses and viroid diseases serology, however, depends on the availability of will not be causing characteristic and clear antisera against a wide range of plant viruses, and symptoms. The example is of the cryptic viruses one can purchase the antisera from the American in different crop plants (Boccardo et al. 1983, Type Culture Collection centre at Rockville, 1987). Another important example is of Avocado Maryland, and also as gratis samples from sunblotch viroid, which has high percentage of persons who have produced the antisera. Logical seed transmission. The root stock and scions and systematic 10-step approach suggested by are symptomless and remain undetected (Stace- Bos (1976) is of immense use in diagnosing virus Smith and Hamilton 1988). In olive plants, latent diseases. infection is noticed with Olive latent virus-1 Intensive researches in these directions have (Necrovirus genus) and Cherry leaf roll virus led to the development of a number of biological, (Nepovirus genus), which are seed transmitted physical, serological and molecular methods for (Saponari et al. 2002). detecting the presence of virus infections. The There is, therefore, an urgent need for de- value of plant health certificates issued for seed veloping sensitive, reliable and quick tests for is entirely dependent on the testing procedure detection of seed-transmitted viruses which may applied. 6.2 Biological Methods 105

There are few examples wherein removal of 6.2 Biological Methods small shrivelled and discoloured seeds minimised the risk of introduction of seed-transmitted 6.2.1 Visual Examination viruses in the case of three important leguminous food crops (Jeyanandarajah 1992) and mottled A close and careful observations on the seed mor- and non-mottled seeds in the case of soybean due phology in certain cases gives tentative indication to Soybean mosaic virus (Khetarpal et al. 1992; of the presence of virus(es). Seed morpholog- Parakh et al. 1994, 2005; Chalam et al. 2004). ical abnormalities like shrunken shape, shriv- It is also known that the external morphology elled seed coats, discolouration, size, weight, of seed should not be considered alone as a major cracking, and necrotic spots or bands are the criterion for identification, since in some virus common morphological criteria used for detect- hosts, it is misleading. For example, Porto and ing the presence of viruses in seeds (Fig. 6.1). Hagedorn (1975) considered mottling of soybean The extensive studies carried out on seed coat seeds to be due to hereditary or environmental morphology and virus transmission by Hobbs factors and not due to viral infection. Even Kon- et al. (2003) revealed that the seed coat mot- ing et al. (2003) reported that Soybean mosaic tling in soybean caused by Bean pod mottle virus (SMV) and also Phomopsis spp. (fungus) virus (BPMV) and Soybean mosaic virus has cause seed coat mottling, and this seed coat resulted into infected plants when grown. Even mottling was inconsistent across cultivars and the seeds collected from certain crop plants in- years. Pacumbaba (1995), Bazwa and Pacum- fected with viruses are small, poorly filled with baba (1996) and Andayani et al. (2011)have disfigured seed coats and are lighter in weight. also reported no correlation between mottling of For example, in seeds of cowpea and barley soybean seeds and SMV transmission. Similarly, infected with Cowpea mosaic virus and Barley seed coat cracking in pea seeds, was not due to false stripe virus, respectively, maximum virus Pea seed-borne mosaic virus (PSbMV) infection transmission was associated with small shrivelled (Stevenson and Hagedorn 1970). Even Chalam seeds (Phatak and Summanwar 1967). Similarly, et al. (2004), while working with SMV in soy- small-sized peanut seeds showed maximum seed bean, have observed that many a time, mottled transmission of PMV (Paguio and Kuhn 1974). soybean seeds are found to be free from SMV and The available information on the morphological healthy looking seeds were found to be infected seed abnormalities due to different viruses are when tested by ELISA. Thus, seed abnormali- furnished in Table 6.1. ties are not necessarily being implicated as seed

Fig. 6.1 Morphological abnormalities due to seed-borne viruses 106 6 Detection of Plant Viruses in Seeds

Table 6.1 Morphological seed abnormalities correlated with infection of some seed-transmitted viruses Plant species Virus Seed abnormality Reference Arachis hypogaea Peanut mottle Small size, discoloured Kuhn (1965) and Paguio and Kuhn seed coat (1974) Cicer arietinum Pea seed-borne mosaic Darkening of the seed Coutts et al. (2008) coat Cucumis melo Squash mosaic Poorly filled, deformed Middleton (1944) Cucurbita pepo Squash mosaic Poorly filled, deformed Middleton (1944) Glycine max Bean pod mottle Seed coat mottling Hobbs et al. (2003) Glycine max Soybean mosaic Discoloured seed coat Koshimizu and Iizuka (1963), Ross (1963), Phatak (1974), Almeida (1981), Taraku et al. (1987), Hobbs et al. (2003), Koning et al. (2003), and Domier et al. (2007) Glycine max Soybean stunt Discoloured seed coat Koshimizu and Iizuka (1963), Ross (1963), Van Niekerk and Lombard (1967), Kennedy and Cooper (1967), Phatak (1974), and Laguna et al. (1987) Hordeum vulgare Barley stripe mosaic Small size, shrivelled, Inouye (1962) and Phatak and lightweight Summanwar (1967) Lactuca sativa Lettuce mosaic Lightweight Ryder and Johnson (1974) Lens culinaris Pea seed-borne mosaic Necrotic rings Coutts et al. (2008) Lupinus luteus Bean yellow mosaic Large or sharp edged Blaszczak (1963) (narrow-leaf lupin strain) Lupinus luteus Cucumber mosaic Large size Troll (1957) Lycopersicon esculentum Tobacco mosaic Necrotic, blackened Broadbent (1965) Nicotiana tabacum Ring spot Small, lightweight Marcelli (1955) Phaseolus aureus (syn. Mung bean mosaic Wrinkled, greenish grey Phatak (1974) Vigna radiata var. radiata) Pisum sativum Pea early browning Wrinkled, green grey Bos and Van der Want (1962)and seed coat Stevenson and Hagedorn (1969) Pisum sativum Pea seed-borne mosaic Seed coat splitting, Hamptom and Mink (1975), mottling, irregular Kumar et al. (1991), and Coutts depressions, et al. (2008) discoloration Sorghum vulgare Sugarcane mosaic Shrunken, reduced size Edmunds and Niblett (1973) Triticum aestivum Brome mosaic Small size, lightweight Von Wechmar et al. (1984a) Vicia faba Broad bean stain Shrunken seed coat El-Dougdoug et al. (1999) Vicia faba Bean yellow mosaic Discoloured and Kaiser (1973) and El-Dougdoug Crinkled seed coat et al. (1999) Vicia faba Pea seed-borne mosaic Necrotic rings and line Coutts et al. (2008) markings on seed coat malformation, reduced size and splitting. Vigna unguiculata Cowpea aphid-borne Shrunken, shrivelled Phatak and Summanwar (1967) Vigna unguiculata Blackeye cowpea mosaic Mottling of testa Jeyanandarajah (1992) infection albeit is an indication. Definitive tests visual judgement alone does not yield absolute are essential to relate seed abnormalities with disease-free seed, since certain plants that carry infection. Seed collection from a healthy crop by the virus exhibit no symptoms of infection. 6.2 Biological Methods 107

Fig. 6.2 Detection of BCMVÐBlCM by growing-on test. Two-leaf stage seedlings showing mosaic and banding symptoms (Source: Udayashankar et al. 2010; H.S. Prakash)

6.2.2 Grow-Out Test and was able to detect TRSV infection by grow- out test as well as by sap inoculation. From It involves germinating the infected seeds in ster- Karnataka (India), Puttaraju et al. (1999)have ile soil under glasshouse conditions either in Petri conducted grow-out test for detection of BCMV dishes or in moist paper towels in controlled in French bean seed collections. Similarly from conditions of light and temperature. Emerging NBPGR, New Delhi, Dinesh Chand et al. (2004) leaves of the seedlings are observed for character- detected Blackgram mottle virus in blackgram istic visible symptoms develop according to the by grow-out test. Effectiveness of this test was virusÐhost combinations. The symptoms could be also observed by Pena and Trujillo (2006)and mild mottle or mosaic on cotyledons or primary Udayashankar et al. (2010) while working with leaves. Period of incubation varies from crop to cowpea and French bean seeds against different crop and virus to virus. Germinating and growing seed-transmitted viruses (Fig. 6.2). seed samples in glasshouse for virus detection is Chowdhury and Nath (1983) developed a tech- also known as sand bench germination test. For nique in which 48-h young seedlings of black- instance, LMV in lettuce was detected by this test gram were inoculated with Blackgram mottle after 16Ð21 days when seedlings exhibit clear- virus. The virus inoculum was prepared in 0.2 M cut mosaic symptoms (Rohloff 1967). Similarly, phosphate buffer and young healthy seedlings Hampton (1972), Naim and Hampton (1979)and that were 48 and 72 h are placed in the virus Hampton et al. (1981) have detected pea fiz- extract with Carborundum and shaken for 40Ð50 zle top (Pea seed-borne mosaic virus)bygrow- times. Seedlings were washed gently by dipping ing seeds under glasshouse conditions. Alfalfa in water and transplanted in pots. After 21Ð mosaic virus was detected in 7-day-old alfalfa 28 days, the symptoms will be noticed if the virus seedlings and also even in the cotyledonary stage transmission has taken place. (Tosic and Pesic 1975). Phatak (1974) observed The seedling inspection method of detecting typical symptoms of BSMV in 1-week-old barley virus-infected seed lots is unsatisfactory for some seedlings. He also developed a ‘blotter test’ in virusÐhost combinations under certain environ- Petri dishes for nepoviruses in Petunia violacea mental conditions since seedlings from infected 108 6 Detection of Plant Viruses in Seeds seeds fail to exhibit symptoms (McKinney 1954; culturing on artificial medium. Besides provid- Afanasiev 1956; Hampton et al. 1957;Mac ing an improved ‘growing-on’ system, culturing Withey et al. 1957; Lister 1960; Jeyanandarajah embryos on synthetic media allows the virus to 1992). For example, Lister (1960) observed latent develop a certain detectable concentrations not infections in one or more hosts with three seed- only for the expression of symptoms of newly transmitted viruses and the growth of seedlings formed leaves but also for detection by other was similar in both healthy and diseased lots. methods. For the first time, Crowley (1957)em- Subsequently, several viruses producing latent or ployed this technique to study the nature of seed semi-latent infections in their hosts were found transmission. Examples wherein this technique transmitted by seed which escaped detection was successfully employed for seed-transmitted (Frosheiser 1964; Hampton 1962, 1963, 1967). viruses detection are Runner Bean mosaic virus Even in red clover (Trifolium pratense) infected in runner bean (Mishra et al. 1967), BCMV in with Clover yellow mosaic or White clover urd bean (Agarwal et al. 1979), mosaic virus mosaic viruses, detection of virus by assaying in berseem (Fugro 1980) and LMV in lettuce on host plants was not possible between July seeds (Ramachandran and Mishra 1987). More and September (Hampton and Hanson 1968). At information on embryo culture is provided in a Montana (USA), under glasshouse conditions, review by Mishra and Ramachandran (1986). many plants did not show distinct symptoms, even though serological tests confirmed their infection. Moreover, in some crops physiological 6.2.3 Indicator Hosts spotting of the leaves tend to be confused with symptoms associated with virus infection In order to increase the reliability of growing- (Hamilton 1965). Hampton et al. (1957) found on tests and to eliminate symptomless infections light intensity as a critical factor for the symptom especially for quarantine work, infectivity tests expression of Barley stripe mosaic virus and are appropriate. In this test, the indicator plants that when plants are grown under 10,000 fc, the after mechanical inoculation produce character- diseased plants exhibit clear-cut symptoms. In Sri istic either systemic or local lesion symptoms, Lanka, Jeyanandarajah (1992) could not detect which help in identification of the virus(es) and Blackeye cowpea mosaic, Cucumber mosaic sometimes even the latent or mixed infections. and Bean common mosaic viruses by grow-out The local lesion hosts are faster to react although tests in cowpea, winged bean and mung bean they may not be as sensitive as systemic hosts hosts. Under such conditions, large number of (Green 1991); Albrechtsen (2006) have provided seedlings need to be tested to derive precise extensive information on indicator hosts. recommendations on seed transmission. The most commonly used technique is the The major demerits of grow-out test are as direct seed test, which includes examination of follows: dry seed and an infectivity assay. In this test, 1. Large space in the form of controlled room or suspected abnormal seeds are soaked in an aque- a greenhouse/glasshouse is required. ous medium and then triturated in a mortar with 2. Long time for standardisation to record repro- pestle or in a mixer or a Wiley mill. The slurries ducible symptoms. produced are applied to the abrasive dusted in- 3. Positive result may not indicate involvement dicator plants and their leaves rinsed with water of virus(es). immediately after inoculation. The inoculated as 4. Negative result may not mean the absence of well as the subsequently developed leaves are ob- virus, since the less virulent strains and latent served for symptom development. The symptoms infections do not produce external symptoms. like local lesions (hypersensitive reaction) or sys- Embryo culture technique is quite useful for temic reactions such as vein clearing, various quick detection of viruses in seed lots and this types of mosaics, line pattern, chlorosis, necrosis technique involves excision of embryo and its and stunting are recorded on the inoculated test 6.3 Physical Methods 109 plants. Single-seed or composite-seed samples tions in accordance with the seed material as well may be processed by the infectivity test. as the virus strain. Any tentative diagnosis from Sometimes viruses like Tomato mosaic virus the reaction of host plants requires confirmation in tomato can be detected by inoculating the by the serological or other authentic tests. seed washings to indicator hosts like N. gluti- Problems associated with virus indexing based nosa, N. tabacum var. Xanthi nc. and P. v u l- on the symptomatology are many and variable. garis var. Scotia (Phatak 1974); hypersensitive Some such problems are (1) symptoms induced cultivars of tobacco, bean and cucumber cotyle- by a specific virus may be mimicked by another dons for TMV (Shmyglya et al. 1984); Nicotiana virus; (2) symptoms vary with plant species, crop tabacum ‘Xanthi NN’, a local lesion host for variety age and physiological condition of the test detection of tobamoviruses carried on tomato plant; (3) indexing based on symptomatology is seeds (Grimault et al. 2012); bean for BCMV time-consuming and expensive with respect to (Quantz 1957) Chenopodium amaranticolor, C. manpower and plant growth facilities with large quinoa, C. murale, Phaseolus vulgaris and Vigna number of test samples. sinensis for majority of the nepoviruses (Lister and Murant 1967); tobacco for Tobacco rattle virus (TRV) (Sol and Seinhorst 1961); pea for 6.2.4 Biological Properties Pea early browning virus (Van Hoof 1962); C. amaranticolor and pea cv. ‘Perfection’ for PS- Up to 1975, virologists used to depend on bio- bMV (Hampton et al. 1976); Bountiful and Top logical properties like thermal inactivation point, Crop beans for AMV (Frosheiser 1970, 1974); dilution end point and ageing in vitro for virus C. quinoa for LMV (Rohloff 1962; Pelet 1965; identification besides host range, vector transmis- Marrou et al. 1967;Phatak1974; Kimble et al. sion and particle morphology. With technologi- 1975); and tomato and Solanum berthaultii for cal progress in virology, the biological proper- PSTVd (Yang and Hooker 1977; Singh 1984; ties gradually lost their weightage. For instance, Singh et al. 1991b). These indicators are now Francki (1980) summarised the ranges of the routinely used in different countries. Hampton above properties in 20 taxonomic virus groups. et al. (1978) have listed diagnostic hosts for the His graphical illustrations clearly show that al- identification of mechanically transmitted legume though these data may differ for distinct groups, viruses which are now being used in most of the there is widespread overlap between many of the laboratories. Boswell and Gibbs (1986)haveas- groups. Therefore, these properties should not be sembled virus identification data exchange, com- exclusively considered for virus characterisation, prising the list of infected species and description identification and classification. of virus-associated symptoms. To accommodate more samples and minimise space, detached leaf technique which involves in- 6.3 Physical Methods cubation of detached leaves on moist filter paper in Petri dishes at 30Ð32ıC under artificial illumi- 6.3.1 Inclusion Bodies nation of ca.1000 lx for 3Ð4 days is also used. Quantz (1962a, b) employed this technique for The morphology and structure of inclusion the detection of BCMV by using the bean cultivar bodies are highly characteristic of the infecting ‘Top Crop’. Detached leaves of N. glutinosa and viruses in spite of the host variability which N. tabacum var. Xanthi nc have been used for greatly aid in diagnosis. The plant virus the detection of Tomato mosaic virus in tomato subcommittee of the International Committee seed (Phatak 1974). For some viruses, where the on Taxonomy of Viruses (ICTV) listed ‘types titre values are very low in dry seed coats, the of inclusion bodies’ as one of the criteria to be inoculum has to be prepared by removing the used in plant virus classification (Harrison et al. seed coat (Quantz 1962a, b; Gilmer and Wilks 1971; Matthews 1982). The inclusions contain 1967). Both these techniques require modifica- either virus particles and/or other products of 110 6 Detection of Plant Viruses in Seeds

Table 6.2 Inclusions in different virus groups of seed-transmitted viruses Group Diagnostic at Virus genus Inclusions characteristic group level Alfamovirus Hexagonally packed layers as whorl in cytoplasm vacuole CC Bromovirus Granular and crystalline in cytoplasm and nuclei C Carlavirus Some banded, most paracrystalline arrays with cell components CC Closterovirus Cross-banded masses in phloem CC Cucumovirus Crystals in vacuole but usually no inclusions CC Comovirus Crystalline arrays of virus particles C Hordeivirus Particles in cytoplasm and nuclei C Potexvirus Stranded and fibrous masses of LIC C Potyvirus Cylindrical, pinwheels CC Sobemovirus Crystalline aggregates, hexagonal CC Tobamovirus Crystalline, monolayer CC Tombusvirus Multi-vesicular body CC Tymovirus Flask-shaped double membrane CC viral genome with occasionally modified cell Phloxine staining proved extremely simple and constituents. Most of these inclusions are located rapid for fresh tissues (Christie and Edwardson in the cytoplasm of mesophyll, epidermal and 1977, 1986). hair cells and sometimes in vacuoles, nuclei and Viruses within groups are now known to in- phloem cells depending on the viruses. The use duce the same or closely similar type of inclu- of these inclusions for the rapid identification of sions (Fenner 1976a, b; Martelli and Russo 1977; seed-transmitted virus diseases plays an integral Christie and Edwardson 1977, 1986; Matthews role at research and plant quarantine stations 1982; Edwardson and Christie 1986). In gen- where adequate facilities are not available for eral, the virus identification should not be de- tentative virus identification. pendent exclusively on inclusion morphology, The inclusions can easily be observed by light since the viruses of different groups also in- microscopy in epidermal strips on the underside duce similar type of inclusion bodies, and hence, of the leaves, but easily from main veins, petioles virus identification needs consideration of other or young herbaceous stems because of larger size characteristics. and regular shape than those of laminar leaf parts Both light microscopy and electron mi- and in leaf hairs. Several selective stains such croscopy are being used in studies on inclusion as trypan blue, azure A, Luxol brilliant green, bodies induced by seed-transmitted viruses. Light phloxine and calcomine orange have been used microscopy with staining of test material helps in for distinguishing inclusions from normal cell differentiating the virus-induced inclusions from components, even though they may have similar each other and from cell organelles. Since the morphology and locations. resolving power of light microscope is low, one With trypan blue (0.5% in 0.9% NaCl) can study only the location and morphology staining, the nuclei turn blue quickly and of the inclusions in the tissues. However, it inclusions stain strongly. For some inclusion is possible to study the internal structure and bodies, aqueous phloxine (1%) is better and exact shape of the inclusion bodies and also stains bright red. The azure A stain imparts the smaller inclusions which are not seen under a red to magenta colour to inclusions high in light microscope by using electron microscope ribonucleoprotein and does not stain other type at higher magnifications. However, the usage of proteins. The Luxol brilliant greenÐcalcomine of electron microscope for routine detections is (OÐG) combination stain imparts green colour to costly. The characteristic inclusions found from inclusions containing different types of protein. each virus group are listed in Table 6.2. 6.4 Serological Techniques 111

Based on inclusion body morphology and viruses. Depending on the particle morphology location, it is possible to identify several seed- and size of the virus in test samples, a tentative transmitted viruses among various virus groups. virus grouping can be done. Especially, viruses For instance, when a diagnostician detects with elongated particles are easy to assign as the banded body inclusions, he/she will know that size range of most groups of viruses is suffi- he/she is dealing with a carlavirus or a clos- ciently distinct from others (Harrison et al. 1971; terovirus or a potexvirus or a potyvirus. Similarly, Hamiltan et al. 1981; Matthews 1982; Francki in nepovirus infections the formation of inclusion et al. 1991). The particle morphology of isometric bodies at early stage of infection, usually viruses (25Ð30 nm in diameter) is also useful in adjacent to the nucleus containing elements of preliminary separation, that is, round and smooth endoplasmic reticulum, membranous vesicles (cucumoviruses and bromoviruses); round and and ribosomes are noted in ultrathin sections. knobby (tombusviruses and tymoviruses); ovoid Another characteristic feature of the nepovirus or imperfectly spherical (ilarviruses); and angular infection is the occurrence of virus particles, (nepoviruses, and fabaviruses). The usually in single files with membrane-walled virus extraction procedure varies with host and tubules, present in the cytoplasm of infected routine tests require modification when infected cells (Murant 1981a). One of the diagnostic seeds are processed containing inhibitors (Pena characters of Potyviridae family recognised by and Trujillo 2006). However, limited information ICTV is the formation of cytoplasmic inclusions is available on the application of this test. Gold called pinwheels, laminated aggregates, scrolls, et al. (1954) observed virus particles of BSMV etc. Seed-transmitted viruses of Potyvirus genus in embryo and endosperm of individual seeds of like Bean common mosaic, Blackeye cowpea barley and wheat. From Poland, Garbaczewska mosaic and Water melon mosaic virus-1 induce et al. (1997) have detected Soil-borne wheat mo- cytoplasmic scroll inclusions, while Bean yellow saic virus particles and inclusion bodies in the tis- mosaic and Lettuce mosaic viruses induce sue of 3-day-old rye seedlings. Similarly, Walkey laminated aggregates. Both scrolls and laminated and Webb (1970) observed virus particles of aggregates are induced by PSbMV, SMV and Cherry leaf roll virus and tubular inclusion bodies Turnip mosaic virus (Edwardson 1974). in homogenates of mature seeds of N. rustica. It is also possible to identify virus strains Phatak (1974) found virus particles of BSMV, based on the shape of inclusion bodies, that is, BCMV and SMV in plumule extracts of barley, hexagonal crystals have been found with TMV bean and soybeans, respectively, but failed to find vulgare and Tomato mosaic strains; rounded- LMV in lettuce seeds. In recent years, electron plate inclusions with Sunn-hemp mosaic virus microscopy is also used successfully in the quar- strain, Cucumber green mottle mosaic virus, antine division of NBPGR, New Delhi, (India), orchid strain and Holmes rib grass strain; and while processing the imported germplasm of a angled-layer aggregates with aucuba strain number of crops from different countries and and U5 strain (Warmke and Edwardson 1966; number of seed-transmitted viruses are also in- Warmke 1968; Milicic et al. 1968; Milicic and tercepted (Chalam et al. 2009b). The high cost Stefanac 1971). of electron microscope makes limitation to most Precise comparisons of the information de- of the workers. However, if available it requires rived from both light and electron microscopic less time for diagnosis when compared to the techniques should be related with other virus- germination test. induced inclusions.

6.4 Serological Techniques 6.3.2 Electron Microscopy Serological techniques are indispensable for the An electron microscope is useful in conjunction detection and identification of plant viruses. with grow-out tests for several seed-transmitted Many of these techniques have been developed 112 6 Detection of Plant Viruses in Seeds for the detection of seed-transmitted viruses results from the linking of antigen molecules by and certain tests are highly specific and most antibody, forming an aggregate and precipitates. promising in the regular diagnosis. The details of different serological techniques are covered in certain annual reviews and laboratory manuals 6.4.1 Monoclonal and Polyclonal (Van Slogteren and Van Slogteren 1957; Antibodies Ouchterlony 1968;Ball1974; Torrance and Jones 1981; Padma and Chenulu 1985a, b;Clark Polyclonal antibodies represent the antibodies et al. 1986; Hampton et al. 1990; Bashir and from multiple clones or §-lymphocytes and, Hassan 1998;Bos1999; Webster et al. 2004; therefore, bind to a number of different epitopes. Akinjogunla et al. 2008; Rao and Singh 2008; Polyclonal antisera have served us well over the Koenig et al. 2010). years. For the past 30 years, ELISA involving The serological tests involve physico- polyclonal antibodies are still the widely used chemical interactions between antisera produced method for practical plant virus detection. Nalini to a plant virus in a warm-blooded animal et al. (2006) and Puttaraju et al. (2004b)have and plant viruses (antigen). The antigen may produced polyclonal antibody against Bean be a protein or polysaccharide and capable of common mosaic virus (BCMV) in French and inducing an immune response when introduced Blackeye cowpea mosaic virus in cowpea and into the warm-blooded animals. Most of the tested their application in seed health. Many plant virus proteins are effective antigens and commercial plant virus kits are designed based on the antibodies to the viruses are produced that principle. The quantity, quality and affinity of via their immunological protective system. antibodies specific for the antigenic determinants Antisera are produced by injecting purified virus of an antigen often vary from animal to animal preparations into rabbit, goat, chicken, mouse and even from different bleedings of the same or other suitable animals. Several injections are animal. The hybridoma technology introduced administered intravenously or intramuscularly by Kohler and Milstein (1975) has provided or a combination of both over a period of 4Ð a revolutionary advances in the method of 5 weeks. The virus preparations are emulsified antibody production that eliminates many of the with equal volumes of Freund’s incomplete problems associated with polyclonal antisera. adjuvant before injection. Test bleeds to check Hybridoma technology has the potential for the level of antibody are made 3 weeks after producing an unlimited quantity of monospecific the immunisation. Several large bleeds can be antibodies (monoclonal antibodies). Monoclonal made without sacrificing the immunised animal antibodies (mAbs) potentially eliminate the when the antibody level is satisfactory. Such variation inherent in whole animal systems of blood samples are allowed to coagulate for an serum production, should have maximal usable overnight and kept in a refrigerator; then the sensitivity and are currently marketed for routine serum may be decanted and clarified by low- virus testing of seed stocks. The monoclonal speed centrifugation. Small aliquots of serum are antibodies have become extremely useful for stored in 1 vol of glycerol/1 vol of antiserum or detecting specific virus strains or viruses that are with 0.02% sodium azide or may be lyophilised. present in low concentrations in infected seed Serological reactions rely on the unique nature material. However, due to high cost of producing of the way in which the antigen and antibody monoclonal antibodies, they are not as widely molecules fit together. The combining sites on used as the polyclonal antibodies. antibody molecules are complementary to differ- Hybridomas are somatic cell hybrids made ent antigenic determinants on the surfaces of the by the fusion of mouse spleen B-lymphocytes homologous antigen. When antibody and antigen to mouse myeloma cells. The resulting hybrids are mixed in suitable combinations, a visible (hybridomas) acquire the properties of antibody- precipitate results. Such a precipitate probably producing ability of the spleen lymphocyte and 6.4 Serological Techniques 113 the ability to grow in culture as well and produce diseases of vegetatively propagated crops such as tumours of the myeloma cells. Antibodies pro- potato, fruit trees, cassava and bulb crops and of duced by a single hybridoma clone are identical seed-transmitted viruses (Halk et al. 1984;Wang and are specific for a single antigenic determi- et al. 1984; Huguenot et al. 1996; Bashir and nant. They are, in essence, a chemically defined Hampton 1996; Ouizbouben and Fortass 1997; immunological reagent (Halk and De Boer 1985). Shang et al. 2011). Monoclonal antibodies offer several advan- Monoclonal antibodies to SMV, MDMV and tages over conventional polyclonal antiserum: (a) LMV were also incorporated into a competi- An unlimited quantity of antibody can be pro- tive radioimmunoassay (RIA) utilising antigen- duced from a small quantity of antigen. (b) Pure coated plastic beads and tritium-labelled anti- antibodies specific for a single antigenic determi- body. The sensitivity of these assays for the three nant can be obtained, even when impure antigen viruses was 10Ð50 ng/ml with purified virus (Hill or antigen mixtures are used as the immunogen. et al. 1984). The mAbs produced against PVY (c) Hybridomas can be preserved by freezing in and PVX performed well in dot-ELISA, reacting liquid nitrogen, thereby assuring a continuous with similar virus and strain specific as in the supply of antibody over time. (d) Highly specific direct or indirect forms of ELISA. Sensitivity mAbs may reveal serological relationships be- of NCM-ELISA was similar to that obtained tween viruses and antigens that were previously in the direct or indirect ELISA (Lizarraga and unrecognised with polyclonal sera. (e) The use Fernandez-Northcote 1989). of monoclonal antibodies eliminates the qualita- Monoclonal antibodies can serve as versatile tive and quantitative variability in specific anti- and powerful immunochemical tools in problems body content in different batches of polyclonal involving viruses and their interactions with serum (Halk and De Boer 1985). (f) It is possible host or insect vectors. These antibodies in for avoidance of background reaction and non- essence are chemically defined reagents capable specific detection of host proteins. of discriminating minute differences on the Monoclonal antibody technology has a few surface properties of biologically important disadvantages as well. The production and char- macromolecules. Diagnostic applications are the acterisation of a collection of mAbs for strain use of many mAbs for seed-transmitted virus and epitope specificity can take over a year. detection. Encouraging results have already been By comparison, the preparation of mAbs is an produced with some viruses, which indicate expensive, complicated and labour-intensive pro- that mAbs may eventually replace polyclonal cedure. Some mAbs do not form precipitin bands serum in large-scale diagnostic progress (Halk in double-gel diffusion tests and some classes and De Boer 1985; Seddas et al. 2000). The or subclasses of immunoglobulins do not bind mAbs also serve as analytical, functional and protein A. Some mAbs may be too specific for structural probes to help in elucidating the virus diagnosis, recognising only a rare isolate biochemical and physiological interaction of or a small group of isolates of a particular virus gene products and viruses within their host (Oxford 1982). In such cases, a mixture of several plants. Monoclonal antibodies will become more mAbs may be needed to detect all strains of a widely available in the future and permit a virus. While testing mAbs for specificity, tests high degree of sensitivity in serological assays. should include as broad a range of isolates as (Van Regenmortel 1984, 1986; Martin 1987). possible (Martin 1985). Monoclonal and polyclonal antisera for many Rishi and Dhawan (1994) have reviewed the viruses are available commercially (Agdia, production and applications of mAbs in plant USA, A.C. Diagnostics Inc., USA; BIOREBA, virology. mAbs have been produced to more than Switzerland; Loewe diagnostics, Germany, and 40 plant viruses in different taxonomic groups. Neogen Europe Ltd., UK) and in individual The greater use of mAbs is to diagnose virus laboratories. The mAbs are to be used to identify 114 6 Detection of Plant Viruses in Seeds the strainal variation or isolates with single amino smaller quantities of virus compared to acid differences in the viral coat proteins (Chen microprecipitin or immunodiffusion tests et al. 1997) and epitope profiling. (Koenig et al. 1979). It can be carried out Before the development of advanced with lower concentrations of reactants than techniques like ELISA, PCR, microarrays and are required for precipitin tests. The results other tests, virologists have tried flocculation are expected within 15 min to 1 h. The tests, which were visible to the naked eye, in method can detect one infected seed per 100 liquid media by using test tubes. Later slide seeds or 1 g/ml of virus (Carroll 1979). This agglutination and latex agglutination tests were test has been routinely employed for large- employed in potato certification schemes (Hardie scale testing of potatoes, both in certification 1970; Seaby and Caughey 1977). schemes and in screening disease resistance The commonly used serological tests are (Khan and Slack 1978, 1980). It was used to grouped as follows: detect BSMV in germinated barley seedlings 1. Flocculation tests in liquid media (Phatak 1974; Lundsgaard 1976)andSMV 2. Immunodiffusion tests in soybean seeds (Phatak 1974). 3. Labelled antibody techniques 4. Immuno-electron microscopy 6.4.2 Immunodiffusion Tests 6.4.1.1 Flocculation Tests in Liquid Media In immunodiffusion tests, the antibodyÐantigen The combination between antigen and specific reactions are carried out in gel instead of liquid. antibody can be demonstrated in a variety of The reactants are allowed to diffuse and precipi- ways. The most direct method is to observe the tation bands form wherever the reactants of suit- formation of an aggregate or a precipitate. In able concentrations meet. These tests separate the this the size of reacting antigen greatly influences mixtures of antigens and antibodies by their sizes, the number of interacting antibody molecules diffusion coefficient and concentrations. Thus, required to produce an aggregation visible to the they are extremely useful for virus detection and naked eye. Accordingly, two types of tests are identification from seed extract. recognised, namely, (a) Precipitation test and (b) There are two types of immunodiffusion tests: agglutination test. (1) single or simple immunodiffusion, in which (a) Precipitation Tests one of the reactants diffuse into the gel, and (2) Precipitation is caused by the formation of a double immunodiffusion, where both the reac- lattice of an antigen and antibody molecules tants diffuse into a gel. Diffusion can occur in that grow in complex size and become in- one or two dimensions depending on whether the soluble. This precipitate can be observed in reaction takes place in tubes or plates. tubes (tube test) or in drops on flat surfaces In immunodiffusion tests, isometric viruses (microprecipitin test). This test was success- will readily diffuse through the gels and without fully applied in the early diagnostic period; pretreatment, but the larger rod-shaped viruses PSbMV is detected in pea seeds by this have to be degraded into smaller units before method (Kumar et al. 1991). they diffuse to give a reaction. They have to be (b) Agglutination Test broken down either by treatment with chemicals In this test the size of the reacting antigenic such as pyridine pyrrolidine, ethanolamine and complexes is large and absorbed to larger guanine HCl or by detergents such as sodium do- particles such as red blood cells or latex decyl sulphate (SDS), sodium dibutyl napthalene or bentonite. These tests include slide sulphonate (Leonil SA) and sodium-N-methyl- agglutination and latex agglutination. Even N-oleoyl taurate (Igepon T-73) or by physical the latex agglutination test is rapid, specific treatments such as freezing and thawing and and sensitive. It can detect 100- to 1,000-fold ultrasonic vibrations. 6.4 Serological Techniques 115

6.4.2.1 Single Diffusion in Tubes The double diffusion method is generally quite In this method, one reactant usually antiserum specific and reasonably sensitive. It can detect is incorporated in the gel, while the other, the virus concentrations of 10Ð25 g/ml and can virus (antigen), is allowed to diffuse into it. The assay a single seed or parts of a seed. This method position of the leading edge of the precipitin band has been extensively used for the detection of in the tube is proportional to the square root of several seed-transmitted viruses, namely, BSMV time (Oudin 1952). This technique has restricted in barley embryos (Hamilton 1964; Slack and use in the detection of seed-transmitted viruses. Shepherd 1975; Carroll et al. 1979a), Blackeye However, this method was employed for testing cowpea mosaic virus in cowpea and SMV in soy- BSMV in barley seed by Slack and Shepherd bean hypocotyls (Lima and Purcifull 1980), TMV (1975) and BMV in wheat by Von Wechmar et al. in tomato seeds (Phatak 1974), Brome mosaic (1984a). virus (BMV) in wheat seeds (Von Wechmar et al. 1984a) and CMV in French bean (Padma and 6.4.2.2 Radial Immunodiffusion Test Chenulu 1985b). This technique is based on the principle of single Occasionally in gel diffusion method, non- diffusion in two dimensions. It is performed in specific precipitates develop when the antigen Petri dishes, with the addition of antibody or was prepared directly from the seed (Phatak antigen to the liquid gel before it sets. A well 1974;Shrestha1984). Similar problem was faced is then cut in the gel, the antibody or antigen is in the embryo detection of Broad bean true added and a halo or ring of precipitation is formed mosaic and Echtes Ackerbohnen mosaic viruses around the well if the reaction is positive. in broad bean seed (Cockbain et al. 1976). This This method is also sensitive and rapid. It can problem can be overcome by clarifying the seed detect as little as 1 g/ml of degraded virus. extract in low-speed centrifugation. This test has been used for the detection of seed- Depending on the type of seed material and transmitted Brome mosaic virus (BMV) in wheat the virus, double immunodiffusion test was mod- seeds (Von Wechmar et al. 1984) and BSMV in ified by many researchers. Carroll et al. (1979a) barley (Slack and Shepherd 1975; Carroll 1979) successfully modified this technique with filter and in mass indexing programme for the viruses paper discs serving as sero-reactant depots for the detected in potato seed stock (Shepard and Secor detection of BSMV in barley. The SDS antisera 1969; Shepherd 1972). The drawback of this test were used successfully by the Montana State is that it requires large amount of reactants. Seed Testing Laboratory at Montana State Uni- versity, Bozeman (USA), for the Montana Seed 6.4.2.3 Gel Double Immunodiffusion Growers Association and by plant virology labo- (Ouchterlony) ratory, and this test has been largely responsible Before ELISA invention, this test was the most for significant reduction of BSMV in barley at widely used technique in plant virology research Montana. and in quarantine inspections. Thin agar or Hamilton (1965) also modified this technique agarose gel layers are prepared on glass slides for rapid detection of BSMV even in a single or in Petri dishes, and suitably arranged wells embryo of barley. In this test, the infected seeds are cut to place the reactants. The antibody and are soaked overnight at room temperature which antigen (infected seed extract) are added to wells results in their swelling considerably causing the and are allowed to diffuse towards each other. The embryos partially separated from the endosperm. reactants meet forming a white precipitin band Complete separation is achieved by ‘rolling’ a when the optimal proportions of antigens and layer of seeds on a piece of aluminium foil with antibodies diffuse. The number of bands, size, a metal rolling pin. The seeds are compressed shape and position of the precipitin bands are by the force of rolling pins resulting in effective characteristic to the particular antigenÐantibody release of the embryos, which are then separated system. by washing. 116 6 Detection of Plant Viruses in Seeds

In this test, quadrant type of Petri dishes is the biological detectability of the viruses nor the employed. A filter paper disc (Whatman No.1, seed viability (Green 1991; Albrechtsen 2006). 6.5 mm dia.) is placed in each of 100 holes, 1 mm deep and of 9 mm diameter in a Lucite ‘embryo crusher’, and individual embryos are 6.4.3 Labelled Antibody Techniques transferred to each disc. The discs are sprayed with phosphate-buffered saline and a top plate The sensitivity of detection of antigenÐantibody with 100 pegs of 2 mm length and pressed for reactions can be increased by labelling antibody crushing embryos. The top plate is removed and that can readily be noticed or increasing sensi- the discs supporting the crushed embryos are tivity or by both. The use of labelled antibodies transferred directly to serological test plates. can help in detecting the virus in minute quan- The immunodiffusion system is incubated for tities (Fateanu 1978). Antibodies are generally 12Ð24 h and the number of embryos showing labelled with ferritin, fluorescent dyes or radioac- the immunoprecipitates indicates the seed- tive iodine or enzymes to locate the viruses in transmitted infection. This test is extremely tissue sections by light, fluorescent microscopes, useful in large-scale screening of commercial autoradiography and electron microscopy (Ball seed lots of barley and possibly other cereal 1974; Andres et al. 1978; Johnson et al. 1978). crops too. These techniques are being applied for virus detection in seeds. The various labelled antibody 6.4.2.4 Direct Immunostaining techniques are: Assay (DISA) 1. Fluorescent antibody technique (immunofluo- The DISA was developed by Takeuchi et al. rescence) (1999) as a technique for detecting tobamoviruses 2. Radioimmunoassay (RIA) on pepper seeds. In this technique, the seeds are 3. Enzyme-linked immunosorbent assay ELISA used as the solid phase in an immunoassay by 4. Dot-immunobinding assay (DIBA) which infected seeds become stained. In the experimentation of DISA, pepper 6.4.3.1 Fluorescent Antibody seeds were soaked in a polyclonal antiserum Techniques solution for 1 h at room temperature, washed Immunofluorescence techniques are the most in phosphate-buffered saline with addition of widely used techniques for studying virus Tween-20 (PBST), incubated with anti-rabbit location and distribution within the tissues of IgGÐAP conjugate for 1 h at room temperature host plants (Coons et al. 1942; Nagaraj and and washed with PBST and distilled water, before Black 1961; Tsuchizaki et al. 1978; Thornley being incubated with nitro blue tetrazolium and Mumford 1979) as well as in insect vectors (NBT)/5-bromo-4-chloro-3-indolyl phosphate (Sinha and Black 1962; Reddy and Black 1972). (BCIP). After 30Ð60 min, infected seeds then It is being presently employed for detecting and stained dark purple, while healthy seeds did locating viruses in seed. not. To test whether the detected virus was This technique is perhaps the most specific infective, stained seeds were homogenised and versatile among all the histochemical meth- and assayed on a hypersensitive tobacco ods, since it provides a means of observing an cultivar, Nicotiana tabacum cv ‘Xanthi nc’ and antigenÐantibody reaction by chemically linking Nicotiana tabacum cv ‘White Burley’ on which a fluorescent dye such as fluorescein isothio- tobamoviruses (TMV, ToMV and PMMoV) cyanate (FITC) or rhodamine B to specific anti- produced differential symptoms in the form of body molecules. Such labelled antibodies retain necrotic local lesions or systemic mosaic (Green the ability to react specifically with their respec- et al. 1987; Green and Kim 1991; Albrechtsen tive antigens and when viewed under a fluores- 2006). The DISA treatment influenced neither cent microscope, the reaction site is relatively 6.4 Serological Techniques 117 more common. There are two ways of perform- acids, namely, brick-red fluorescence for RNA ing the immunofluorescence techniques, that is, and yellowish-green fluorescence for DNA (Hirai direct and indirect methods. and Wildman 1963). (a) Direct Method: In direct method, antigens Jagadish Chandra and Summanwar (1971) are mixed with FITC-labelled specific employed A.O. stain for the detection of CPMV antibodies. The reaction gives a brilliant in cowpea seeds of cv. P. 378. The seeds are yellow-green fluorescence when examined soaked for 2 days. On the third day, the sections under a microscope with an ultraviolet are taken from germinating embryos and stained light source. Blackgram mottle virus was with A.O. The stained tissues are viewed under detected by using this method in embryo a fluorescent microscope. Infected seed sections and germinated seeds of blackgram (Krishna showed aggregation of red fluorescence material. Reddy and Varma 1994). This technique is also found successful for the (b) Indirect Method: In indirect method, the anti- detection of Wheat mosaic virus (Mayee and gens are first allowed to react with unlabelled Ganguly 1974). antibodies and then with FITC-labelled sheep antiserum prepared against gamma globu- 6.4.3.2 Radioimmunoassay (RIA) lin obtained from animal species (rabbit) in Antibodies labelled with radioisotopes have been which the virus-specific antiserum was pro- employed in the detection of seed-transmitted duced. viruses. These techniques are very sensitive and Phatak (1974) used the indirect method well suited to detect and quantify the virus infec- of immunofluorescence for the detection tion in seeds. However, the application of these of SMV in germinated seeds of soybean. techniques requires strict safety precautions and Free-hand sections of seed and squashes of highly trained personnel; the conjugate isotopes plumule and small shoots are fixed for 5Ð have a short shelf life and expensive equipment is 10 min in acetone, dehydrated with neutral required to assess the results. phosphate-buffered saline and followed The techniques mostly applied for plant virus by treatment with SMV antiserum and detection are as follows: subsequently with the conjugate for 30 min (a) Solid-Phase Radioimmunoassay (SPRIA): at 37ıC. Sections of virus-infected material There are two types of SPRIA. In a under a fluorescent microscope appeared competitive type of assay, unlabelled antigen with more bluish-green fluorescence than is first incubated in an antibody-coated healthy ones. Similarly, SqMV is detected polystyrene centrifuge tubes, and 125I- in infected embryos, seedling protoplasts labelled antigen is added afterwards. The from cotyledons and microtome sections of radioisotope-labelled antigen combines dry embryos or seedlings by staining with specifically with the antibody to give a fluorescein isothiocyanate Ð labelled and test with sensitivity in nanogram quantities. distributed in clusters of cells in epidermal, An indirect assay can be performed in palisade and spongy mesophyll tissues. which known incremental quantities of The virus is detected only in sections unlabelled antigen is used. This test is simple, of cotyledons from 6-day-old seedlings economical and sensitive and requires very (Alvarez and Campbell 1978). small quantity of reactants (Ball 1973). This test was successfully employed to detect Fluorescent Staining of Nucleic Acids SMV in soybean seeds by Hill (1981). Vital stains (fluorochromes) such as acridine or- Even Diaco et al. (1985) used to detect ange (A.O.) are gaining importance as compared SMV in seed by using two mAbs that to the stains used for light microscope because recognised different epitopes on SMV. The of their differential staining colours for nucleic assays used mAbs S2 as the capture antibody 118 6 Detection of Plant Viruses in Seeds

and mAbs S1 as either the radiolabelled or This technique consists of sodium enzyme-labelled detecting antibody and had dodecyl sulphate (SDS) polyacrylamide gel a sensitivity of detection of ng/ml of SMV. electrophoresis (SDS-PAGE) of the virus During 1982, a method based on SPRIA is infected seed extract, electrophoretic transfer developed which detects all strains of SMV of protein bands to the activated paper by the with equal sensitivity using antibody-coated electro-blot technique, subsequent probing polystyrene beads (Bryant et al. 1982, 1983). of the viral coat protein bands by specific It consists of coating polystyrene beads (solid antiserum which was prepared against intact phase) with antibodies and addition of anti- virus and detection of immune complex with gens into the tubes containing beads. The 125I-labelled protein A for autoradiographic antigens (infected seed extract) bind to the and scintillation counter detection of virusÐ solid-phase antibodies. Radioactive virus an- antibody immune complexes (O’Donnell tibodies are then added to the antigens. The et al. 1982; Shukla et al. 1983, 1991). amount of radioactivity in the tube is directly proportional to the amount of virus antigen. 6.4.3.3 Enzyme-Linked If the virus is absent, there will be no binding Immunosorbent Assays sites for the radioactive antibodies and hence (ELISAs) Techniques are removed during beads washing. The ad- Enzyme immunoassays were introduced in med- vantage of this method is its ability to detect ical diagnostics during the early 1970s and have virus antigen in the presence of extraneous distinct advantages over radioimmunoassays. As seed material. a result, the method known as enzyme-linked (b) Radioimmunosorbent Assay (RISA):Aimmunosorbent assay (ELISA) was adopted to simple and highly sensitive method RISA plant viruses by Voller et al. (1976)andClark was described by Ghabrial and Shepherd and Adams (1977). Use of an enzyme as marker (1980). It is a microplate method based on which is linked to the virus-specific antibody the principle of double-antibody sandwich augments the detection of a specific reaction in (DAS) ELISA and follows essentially the several hundredfold relative to the visibility of an protocol of the enzyme-linked immunosor- immunoprecipitate. bent assay (ELISA) with the exception that The basic principle of ELISA is that the 125I-labelled gamma globulin is substituted virus in test sample is selectively trapped and for the globulin enzyme conjugate. The immobilised by specific antibody adsorbed 125I-labelled gamma globulin is dissociated on polystyrene or polyvinyl microtitre plates. by acidification from the double-antibody Enzyme-labelled antibodies are then complexed sandwich, and the released radioactivity is with the trapped virus and detected by either proportional to virus concentration. colorimetric or spectrophotometric method by This test is a valuable tool for viruses in adding a suitable substrate. Removal of non- which the ELISA values are too low to be specific substances is carried out by washing dependable. Another advantage is that it also after each step of the procedure, leaving only detects strains of viruses that differ serolog- specific immobilised reactors. ically. This technique is effectively used for Greater use of ELISA in plant virology has detection of LMV in very low proportions of been in the large-scale testing and certification lettuce seed lots (Ghabrial et al. 1982). of seeds and vegetatively propagated plant (c) Electro-blot Radioimmunoassay (EBRIA): materials of crops (Morrison 1999). Visually, This technique combines the principles of it is often possible to distinguish the healthy serology with an analytical technique and sample reactions from the infected ones. Now is capable of detecting viruses occurring in ELISA has been increasingly used for detecting extremely low concentration in plants or in virus in seed due to the following major seed. advantages: 6.4 Serological Techniques 119

(f) Scope for specificity in differentiating enzyme-labelled antibodies, which requires serotypes considerably less time for completion of the (b) Shorter time for its operation test (Bar-Joseph et al. 1979; Flegg and Clark (a) Very much sensitive in detecting low concen- 1979; Hamdi and Rizkallah 1997;Chalam trations of virus up to 1Ð10 ng/ml et al. 2009a). (c) Direct application to large- or small-scale Besides the above methods, depending on testing the virusÐhost combination and convenience, (d) Requires small amounts of antiserum Yolken and Leister (1982), Stobbs and Baker (e) Used for plant or seed extracts as well as (1985) and Van Vuurde and Maat (1985) purified virus as antigen have also modified the standard DAS-ELISA (h) Amenability to obtain quantitative measure- technique. ments Although the method is convenient and effec- (i) Involves low cost and long shelf life of the tive, it has some disadvantages: reagents 1. It necessitates the preparation of specific anti- (j) Enables economical and efficient use of anti- body conjugates for each virus under test. bodies 2. It is highly strain specific and not suitable (g) Suitable for both intact and fragmented virion for virus detection in disease surveys or for of different sizes as well as morphology probing a single antigen with several different Numerous variations of ELISA have been de- antisera. veloped, but basically there are two main cat- egories, namely, ‘direct’ and ‘indirect’ ELISA Indirect ELISA techniques that are widely used in plant virology. In indirect ELISA test, immobilised antigen is the target for unconjugated specific antibody. Direct ELISA Trapped antibody is detected by enzyme-labelled In this test, r-globulins prepared for specific virus protein A conjugate or anti-FC-specific or anti- are used in coating and the same r-globulins IgG conjugates. Thus, in indirect ELISA, de- conjugated with an enzyme are employed for tecting antibodies are not labelled with enzyme. detection. Certain details of some direct ELISA There are several variations among the indirect procedures in common use are as follows: ELISA procedures which are as follows: (a) Double-Antibody Sandwich-ELISA (DAS- (a) Indirect DAS-ELISA: Bar-Joseph and ELISA): This method was first described by Saloman (1980), Koenig (1981), Rybicki and Clark and Adams (1977). As the antigen Von Wechmar (1981) and Van Regenmortel is sandwiched between two r-globulins, the and Burckard (1980) described modified type test is called as double-antibody sandwich- of direct ELISA. This method is sometimes ELISA. In this method, the wells of the called as second antibody system. In this test, microtitre plate are first coated with antivirus antigen-specific antiserum is prepared in two globulin and the virus in the test sample is animal species. Gamma globulins from one then trapped by the adsorbed antibody. The species of antiserum is used to coat the plate presence of virus is revealed by an enzyme- and trap antigen from the same. Antiserum labelled antivirus conjugate. This method or r-globulin from the other species is is highly strain specific and requires the reacted with the immobilised antigen and preparation of a different enzyme-conjugated the resulting immune complex is detected Ig for each virus to be tested. with an enzyme conjugate specific for the Several modifications have been made to r-globulin of the second animal species. This the standard DAS-ELISA to suit a particular allows the full binding property of specific r- virusÐhost combination or to reduce the assay globulin to be used, giving greater sensitivity time. One such method is simultaneous incu- and overcoming the extreme specificity of bation of virus-containing samples with the DAS-ELISA. 120 6 Detection of Plant Viruses in Seeds

This ELISA is particularly suitable for FC-specific enzyme-conjugated antibodies. virus detection in disease surveys, testing the This method is advantageous over the DAS presence of viruses in seed and determining method because of the use of crude antiserum serological relationships when specific conju- instead of IgG and a single enzyme conjugate gates cannot be prepared. It is relatively more with all detection systems (Hobbs et al.1987). economical to perform than the DAS form. Cooper et al. (1986) have detected the cherry (b) Indirect F(ab’)2ELISA:PurifiedIgGisused leaf roll and Prune dwarf viruses in cherry for coating the plate in common forms of (Prunus avium) seeds by using PAS-ELISA. ELISA but Barbara and Clark (1982)used This technique is also effectively used in F(ab’)2 portion by cleaving the FC region of detecting cowpea aphid-borne mosaic virus the IgG. This technique relies on trapping in cowpea germplasm at IITA, Nigeria virus with adsorbed F(ab’)2 fragment and (Ojuederie et al. 2009). subsequent probing with an unlabelled virus- (f) Clq ELISA: A different indirect method which specific antibody. This immune complex is uses Clq (a component of complement ob- detected with enzyme-labelled antiglobulin tained from bovine serum) was developed antibody or Staphylococcus protein A (Bar- by Torrance (1980a, b). The plates are first bara and Clark 1982). This combines the coated with Clq. Since the Clq component advantages of an indirect assay with that of of complement binds immune complexes, the DAS-ELISA. virus and its specific antiserum are incubated (c) Indirect Direct Antigen Coating-ELISA together in the wells. The resulting virus IgG (DAC-ELISA) or Plate-Trapped Antigen- complexes are trapped by the Clq molecules ELISA (PTA-ELISA): This method is similar and detected by an enzyme-labelled antiglob- to the indirect DAS-ELISA except that the ulin conjugate. wells are initially coated with antigen. The reaction of antigen attached on solid phase is Choice of Enzyme Labels and Substrates then carried out as in indirect DAS-ELISA. The enzymes most widely used in plant virology This method has the advantage of simplicity are alkaline phosphatase (ALP) and horseradish and convenience of application specially as peroxidase (HRP) (Clark 1981; Clark and Bar- a general diagnostic procedure where a virus Joseph 1984). Both of these enzymes have prob- isolate may have to be tested for reaction with lems associated with their use. These include several antisera (Mowat and Dawson 1987; high cost, limited availability, the potential car- Behl et al. 1995). cinogenic activity of some of the substrates and (d) Protein A Sandwich-ELISA (PAS-ELISA or the hazardous nature of hydrolyzed products of PA-ELISA): Edwards and Cooper (1985)have the substrates. Later urease (Evans et al. 1983) described a novel form of indirect ELISA, and “-lactamase, generally known as penicilli- which utilises protein A in two applications to nase (PNC) (Joshi et al. 1978;Yolkenetal. sandwich antibodyÐantigenÐantibody layers. 1984; Sudarshana and Reddy 1989), have been The first applied layer of protein A prepares introduced in ELISA. Urease has the prospect of the plate for coating the antibody layer. The becoming routine because it catalyses the release second layer of protein A is conjugated to of ammonia from the low-cost substrate urea. the enzyme and detects the second antibody The product of this reaction can be conveniently layer. detected by pH indicator such as bromocresol (e) Protein A Coating-ELISA (PAC-ELISA):In purple which changes colour from yellow to this method, protein A is only used for initial purple. plate coating and subsequently antigen is Alkaline phosphatase and penicillinase added followed by antisera or r-globulins. exhibit the linear reaction kinetics with The resultant protein antigenÐantibody their substrates p-nitrophenyl phosphate and complexes are detected by anti-rabbit penicillin, respectively. The reaction kinetics 6.4 Serological Techniques 121 of horseradish peroxidase (HRP) is not linear. Substrates should ideally be cheap and non- Its cost is reasonably low and easily conjugated toxic and must provide a sensitive quantitative to immunoglobulins, but the main disadvantage measure of the bound enzyme. They should be is that many chromogenic substrates used for easily prepared, stable and easily soluble for peroxidase are suspected mutagens. Further, the enzyme. hydrolysed products are hazardous. Penicillinase has several features that make it a desirable Improvements in ELISA enzyme label for ELISA tests. It has relatively Various modifications and improvements have low molecular weight (24 KDa), a high turnover been introduced that give the test a higher degree rate, is not too expensive and more readily of sensitivity and better versatility (Koenig and available in developing countries than other Paul 1982). New developments in the immunoen- enzymes. The visual result reading from this zymatic diagnosis of plant viruses are (a) biotinÐ test is easier than that of the alkaline phosphatase avidin techniques and (b) use of fluorescent sub- system. Additionally, hydrolysed products in strates. PNC assays are not known to be hazardous (a) BiotinÐavidin System: Biospecific bridges (Sudarshana and Reddy 1989). PNC has also involving biotinÐavidin or lectin polysaccha- been used for labelling biotin and used in rides seem promising alternative strategies in conjunction with streptavidin for improving the ELISA. This system is based on the very high sensitivity of the ELISA (Yolken et al. 1984). affinity of avidin for biotin. In the modified The choice of substrates depends on the ELISA test, the virus is trapped between enzymes used to make conjugate. For alkaline the coated antibodies and biotinylated phosphatase, the substrate most commonly used antibodies; avidin molecules are used to is p-nitrophenyl phosphate. The fluorogenic trap non-specific biotin enzyme conjugate. substrate, 4-methylumbelliferyl phosphate, has Though this procedure involves additional been reported to be more sensitive than the steps, the sensitivity is significantly increased p-nitrophenyl phosphate when ALP was used over standard ELISA (Kendall et al. 1983; (Yolken and Stopa 1979; Torrance and Jones Zrien et al. 1986). This new procedure 1982). With p-nitrophenyl phosphate, the reac- appears to be very promising for detecting tion is stopped by adding excess alkali, and with viruses in seed (Maury and Khetarpal 1989). 4-methylumbelliferyl phosphate, the reaction is (b) Use of Fluorescent Substrates: The desire to stopped by adding K2HPO4.KOH (Martin 1985). enhance sensitivity of ELISA for detection For HRP, the substrates most commonly used of antigens led to the development of the are o-phenylenediamine (OPD) or 2,2 azino-di(3- enzyme-linked fluorescent assay (ELFA). ethylbenzthiazoline-6-sulphonic acid) (ABTS) or Recent immunological evidences have 3,30,5,500-tetramethylbenzidine (TMB). OPD is suggested that utilisation of fluorescent photosensitive; both OPD and ABTS are muta- substrates such as 4-methylumbelliferyl genic and should be handled accordingly. TMB phosphate (MUP) or 4-methylumbelliferyl is neither photosensitive nor mutagenic. With all 1-“-D-galactopyranoside (MUG) in ELISA- these substrates, the reaction is stopped by adding based assays enhances detection for several H2SO4 (Clark 1981). diverse antigens (Shalev et al. 1980;Neurath For penicillinase, bromothymol blue (BTB), and Strick 1981; Konijn et al. 1982; Torrance starch iodine complex (SIC) and mixed pH indi- and Jones 1982). cator of bromocresol purple (BCP C BTB (2:1) The use of fluorescent substrates does not are the substrates used. BTB substrate colour modify or complicate the normal ELISA test. changes from blue to greenish yellow to deep yel- Only the usual substrates are modified. Anti- low, depending on the amount of penicilloic acid bodies conjugated with alkaline phosphatase produced and measured at 620 nm (Sudarshana or galactosidase and a fluorogenic substrate and Reddy 1989). is used. It has been demonstrated that 122 6 Detection of Plant Viruses in Seeds

ELFA, using polyclonal antibodies to SMV, can be detected when diluted up to 2,000 w/v can provide sensitive levels of detection in case of SMV in soybean (Bossennec and (Dolores-Talens et al. 1989), equivalent to Maury 1978), 3,600 w/v for PMV in peanut biotinÐavidin ELISA (Diaco et al. 1985)or (Bharathan et al. 1984)orwhenmixed radioimmunosorbent assay (RISA) (Ghabrial with 1,400 and 200 healthy seeds in case and Shepherd 1980), and is 10- to 25-fold of LMV in lettuce and BCMV in bean, more sensitive than DAS-ELISA (Hill and respectively (Jafarpour et al. 1979). The Durand 1986; Benner et al. 1990). Enzyme- relative concentration of virus in different linked fluorescent assay (ELFA) can detect embryos has been analysed such as BSMV 1 ng/ml of purified virus and has been in barley (Lister et al. 1981), LMV in applied for the detection of SMV in soybean lettuce (Ghabrial et al. 1982), PMV in seed using polyclonal antibodies (Hill and peanut (Bharathan et al. 1984), PSbMV Durand 1986) and monoclonal antibodies in pea (Maury et al. 1987a, b) BCMV in (Benner et al. 1990)andalsoforLMVin cowpea (Udayashankar et al. 2010)and lettuce seeds (Dolores-Talens et al. 1989). SqMV in cucurbits (Nolan and Campbell Benner et al. (1990) have also successfully 1984). In barley infected with BSMV, a large used mAbs of S1 or S2 labelled to biotin variation of virus concentration has been and detected SMV in soybean by using found in different infected embryos from enzyme-linked fluorescent assay (ELFA). the same seed lot (Miller et al. 1986;Huth The indirect ELISA using mAbs is found 1988). Amount of PMV detected in axis and most sensitive in detecting 2.5 ng/ml of cotyledon extracts from the same peanut seed virus and used to screen seed from the were found to be the same (Bharathan et al. PStV-infected peanut cultivars. The mAbs 1984), contrary to another isolate of PMV in the indirect ELISA readily detected PStV which was not detected in cotyledons (Adams antigen in peanut cotyledonary tissue and and Kuhn 1977). ELISA also confirms that PStV-infected seed part diluted in 32 healthy the concentration of SMV and BgMV was seed parts (Sherwood et al. 1987; Culver and not the same for cotyledons and axis of Sherwood 1988;Culveretal.1989). single embryo, and the virus could even be (c) Mixed Antisera: To reduce the time and cost, restricted to either of these two parts (Varma attempts were made by number of workers by et al. 1992). mixing the different antisera for the detection (b) Correlation Between Embryo Positive in of more than one virus at a time. Some of ELISA and Seed Transmission:Thistest the successful hostÐvirus combinations of further helps in determining whether the detection of viruses by mixed antisera are number of infected embryos found positive cowpea and soybean viruses (Joshi and in ELISA is the same as the infected plants Albrechtsen 1992), nepoviruses (Etienne in a progeny. A good correlation has been et al. 1991) and potato viruses (Bantarri and found for SMV in soybean seed (Maury et al. Franc 1982; Grimm and Daniel 1984). 1983), PMV in peanut seed (Bharathan et al. 1984), PSbMV in pea seed (Maury et al. Application of ELISA for Detection 1987b) and LMV in lettuce seed (Falk and of Seed-Transmitted Viruses Purcifull 1983). With increased advantages of ELISA, it has been On the contrary, for SqMV in cucurbit successfully employed for detecting several seed- seed, the number of ELISA-positive embryos transmitted viruses (Table 6.3). Further, it has largely exceeded the number of virus- been useful in revealing and detecting virus sit- infected plants in the ‘grow out’ test (Nolan uation in different seed parts, tolerance limits and and Campbell 1984). In this case, when detection of latent infections. dissected embryos were cut into two halves (a) Virus Detection in Embryos: ELISA is so transversely, the distal half having been sensitive that even a single infected embryo tested by ELISA and the germinative half 6.4 Serological Techniques 123

Table 6.3 Application of ELISA techniques in the detection of seed-transmitted plant viruses Virus Crop Reference Alfalfa mosaic Alfalfa Halk et al. (1982), Lange et al. (1983), and Pesic and Hiruki (1986) Annual medics Jones and Pathipanwat (1989) Broad bean Chalam et al. (2009a, b) Clover Bariana et al. (1994) Apple mosaic Almond Barba (1986) Barley stripe mosaic Wheat Lister et al. (1981), Qiu et al. (1982), and Khetarpal et al. (2006b) Barley Qiu et al. (1982), Lange et al. (1983), Mukhayyish and Makkouk (1983), Miller et al. (1986), and Huth (1988) Bean common mosaic French bean Jafarpour et al. (1979), Lister (1978), Wang et al. (1982), Mukhayyish and Makkouk (1983), Lange and Heide (1986), Dusi et al. (1988), Klein et al. (1992), Khetarpal et al. (1994), Saiz et al. (1994), Puttaraju et al. (1999), Njau and Lyimo (2000), Nalini et al. (2004, 2006), and Chalam et al. (2007) Blackgram/green gram Dinesh chand et al. (2004) Cowpea Hao et al. (2003), Chalam et al. (2007), and Udayashankar et al. (2010) French bean Puttaraju et al. (1999) Cluster bean Gillaspie et al. (1998b) Mung bean Jeyanandarajah (1992), Choi et al. (2006) Bean common mosaic necrosis Bean Njau and Lyimo (2000) Bean pod mottle Soybean Krell et al. (2003) and Pedersen et al. (2007) Bean yellow mosaic Broad bean Eppler and Kheder (1988), Raizada et al. (1991), El-Dougdoug et al. (1999), and Chalam et al. (2007) Lentil Makkouk et al. (1992) Lupin Robertson and Coyne (2009) Black eye cowpea mosaic Cowpea Jeyanandarajah (1992); Puttaraju et al. (2002, 2003; Puttaraju et al. 2004b) Blackgram mottle Mung bean Saleh et al. (1986), Dinesh Chand et al. (2004) Blackgram Varma et al. (1992) Blackgram mild mottle Blackgram Varma et al. (1992) Blueberry leaf mottle Blueberry Childress and Ramsdell (1986) Broad bean stain Lentil Makkouk and Azzam (1986) Vetch Makkouk et al. (1986) Broad bean Anon (1984), Makkouk et al. (1987), Eppler and Kheder (1988), El-Dougdoug et al. (1999), Khetarpal et al. (2001), Chalam et al. (2007), and Chalam et al. (2009b) Broad bean true mosaic Faba bean Anon (1984) Brome mosaic Wheat Von Wechmar et al. (1984a) Cherry leaf roll Cherry Cooper et al. (1984) French bean Chalam et al. (2005) Soybean Chalam et al. (2007) Walnut Kolber et al. (1982) Cowpea aphid-borne Cowpea Yilmaz and Ozaslan (1989), Hampton et al. (1992), Bashir and Hampton (1993), Ndiaye et al. (1993), Konate and Neya (1996), Khetarpal et al. (2001), Chalam et al. (2007), and Ojuederie et al. (2009) Cowpea mild mottle Soybean Iwaki (1986), Horn et al. (1991), and Hampton et al. (1992) (continued) 124 6 Detection of Plant Viruses in Seeds

Table 6.3 (continued) Virus Crop Reference Cowpea severe mosaic Cowpea Hampton et al. (1992) and Bashir and Hampton (1993) Cucumber mosaic Barley Von Wechmar et al. (1983) Cowpea Hampton et al. (1992), Bashir and Hampton (1993), Gillaspie et al. (1998a), Abdullahi et al. (2001), and Ojuederie et al. (2009) French bean Davis and Hampton (1986) and Hampton and Francki (1992) Lupin Njeru et al. (1997) and O’keefe et al. (2007) Peanut Reddy et al. (1984), Cai et al. (1986), and Demski and Warwick (1986) Cucumber Ertunc (1992) Spinach Yang et al. (1997) Subterranean clover Jones and Mckirdy (1990) Lupin Wylie et al. (1993), Njeru et al. (1997), and O’Keefe et al. (2007) Cucumber green mosaic mottle Cucumber Kawai et al. (1985)andShangetal.(2011) Hibiscus latent ring spot Kenaf Rubies-Autonell and Turina (1997) High plains virus Sweet corn Froster et al. (2001) Indian peanut clump Peanut Reddy et al. (1988), Reddy et al. (1998a, b) Wheat Reddy et al. (1998a, b) and Delfosse et al. (1999) Iris yellow spot Onion Crowe and Pappu (2005) Lettuce mosaic Lettuce Jafarpour et al. (1979), Ghabrial et al. (1982), Falk and Purcifull (1983), Van Vuurde and Maat (1983, 1985), Falk and Guzman (1984), Gusenleitner (1985), and Dolores-Talens et al. (1989) Maize chlorotic mottle Maize Jensen et al. (1991) Maize dwarf mosaic Maize Hill et al. (1974), Mikel et al. (1984), Khetarpal et al. (2006a, b) Melon necrotic ring spot Melon Avegelis and Barba (1986) Melon rugose mosaic Melon Mahgoub et al. (1997) Onion yellow dwarf Garlic/shallot Delecolle et al. (1985) Pea early browning Lettuce Van Vuurde and Maat (1985) Pea Van Vuurde and Maat (1985) Pea seed-borne mosaic Broad bean Chalam et al. (2009a, b) Lentil Varma et al. (1991) Pea Hamilton and Nichols (1978), Maury et al. (1987b), Kheder and Eppler (1988), Khetarpal and Maury (1990), Haack (1990), Varma et al. (1991), Phan et al. (1997), Khetarpal et al. (2001), Parakh et al. (2006), and Coutts et al. (2009) Peanut clump virus Peanut Dieryck et al. (2009) Peanut mottle Peanut Bharathan et al. (1984), Hobbs et al. (1987), Gillaspie et al. (2000), Puttaraju et al. (2001), Prasada Rao et al. (2004), Khetarpal et al. (2006a, b), and Chalam et al. (2007) Peanut mild mottle Peanut Cai et al. (1986) Peanut stripe Peanut Demski and Warwick (1986) Culver and Sherwood (1988), Warwick and Demski (1988), Matsumoto et al. (1991), Xu et al. (1991), Prasada Rao et al. (2004), and Khetarpal et al. (2006a, b) Peanut stunt Peanut Cai et al. (1986) Pepino mosaic Tomato Cordoba Ð Selles et al. (2007) (continued) 6.4 Serological Techniques 125

Table 6.3 (continued) Virus Crop Reference Pepper mild mottle Pepper Svoboda et al. (2006) Plum pox Prunus spp. Thomidis and Karajiannis (2003) Prune dwarf Sweet cherry Mink (1984), Mink and Aichele (1984), Cooper et al. (1986), and Kelley and Cameron (1986) Prunus necrotic ring spot Almond Mink (1984), Mink and Aichele (1984), Barba (1986), andKelleyandCameron(1986) Sweet cherry Mink (1984), Mink and Aichele (1984), Barba (1986), andKelleyandCameron(1986) Prunus spp. Mink (1984), Mink and Aichele (1984), Barba (1986), andKelleyandCameron(1986) Ryegrass seed-borne Lolium sp. Chester et al. (1983) Soybean mosaic Soybean Lister (1978), Chen et al. (1982), La et al. (1983), Iwai et al. (1985), Diaco et al. (1985), Hill and Durand (1986), Taraku et al. (1987), Maury et al. (1983, 1985, 1987), Benner et al. (1990), Khetarpal et al. (1992, 2001), Chalam et al. (2004), Golnaraghi et al. (2004), Parakh et al. (2005, 2008), Pedersen et al. (2007), and Andayani et al. (2011) Southern bean mosaic Cowpea Hampton et al. (1992) and Bashir and Hampton (1993) Squash mosaic Cucumber Nolan and Campbell (1984) Melon Lange et al. (1983), Avegelis and Katis (1989b), and Franken et al. (1990) Strawberry latent ring spot Quinoa Hicks et al. (1986) Parsley Bellardi and Bertaccini (1991) Parsnip Hicks et al. (1986) Sugarcane mosaic Maize Li et al. (2007) Tobacco mosaic Tomato Cicek and Yorganci (1991), Chitra et al. (1999, 2002), and Sevik and Tohumcu (2011) Capsicum Chitra et al. (1999, 2002) Tobacco ring spot Soybean Lister (1978) and Golnaraghi et al. (2004) Tobacco streak Beans Walter et al. (1992) Tomato black ring French bean Chalam et al. (2005, 2007) Tomato mosaic Tomato Chitra et al. (1999, 2002)andIsmaeiletal.(2011) Tomato ring spot Soybean Golnaraghi et al. (2004), Chalam et al. (2007) Turnip yellow mosaic Arabidopsis de Assis Filho and Sherwood (2000) Oil seed rape Spak et al. (1993) Winter turnip rape Spak et al. (1993) Urdbeanleafcrinkle Urdbean Beniwaletal.(1984) Wheat streak mosaic Wheat Jones et al. (2005)

by the grow-out test, none of the ELISA- lots could be related for the frequent false negative embryos produced a virus-infected positives by ELISA. The decline with time plant. This absence of false negatives would level of embryonic transmission of SqMV by suggest that the virus is more homogenously the cucurbit seed lots could be related for the distributed in the embryo of cucurbit seeds frequent false positives by ELISA. Similar than the viruses transmitted through soybean declines during maturation of seed have also or pea seed. But more individuals were rated been reported, such as SMV in soybean cv. positive by ELISA than by the grow-out Merit (Bowers and Goodman 1979). test; even when derived from half embryos (c) Determination of Percent Seed Transmission with high positive values, the symptomless by Group Analysis:Wherethereislow seedlings were virus-free when cucurbit seed percentage of seed transmission, the number 126 6 Detection of Plant Viruses in Seeds

of embryos to be tested to have a good high vector intensities would experience probability of detecting virus in seed would major yield losses if more than one seed not be economically practical. A method of in 10,000 is infected. In case of LMV in determining transmission rate consists of di- lettuce, a tolerance limit of one infected viding a representative sample of the lot (are seed out of 1,000 has restricted the disease analysed) into ‘N’ groups of n seeds each. satisfactorily in France although it was not So, ‘N’ tests are then done instead of N x n determined to such a refinement (Marrou in single-embryo tests. Some mathematical et al. 1967). In California, for the same virusÐ elements allow estimating the most probable host combination, the limit chosen was five percentage of transmission, with confidence times lower, a test giving zero-infected seed intervals, as a function of the number of in 30,000 Ð for a seed lot a 99.9% probability ELISA-negative groups (Y) that were given of having less than 0.022% infection. This by Maury et al. (1987b). It is the magnitude limit gave an excellent control of the of confidence intervals for a given level of disease (Grogan 1980). Thus, geographical probability which accounts for precision. The variability is noted in seed transmission vis- magnitude is determined by four parameters a-vis` the vector prevalence. N, n, Y and the level of probability; it (e) Detection of Latent Infection:Itisalso keeps small values for Y D N but can be possible to detect the infection in seeds considerable for Y D 0 (all groups positive in collected from latent infected plants by using ELISA) (Maury and Khetarpal 1989). the technique. For example, in France some (d) Determining the Tolerance Limits:After of the pea plants infected with PSbMV under determining the per cent transmission of glasshouse conditions remained symptomless virus in mature seeds of harvested crops, and rarely exhibited a very faint leaf rolling, one requires some information on the and this weak and subminimal form of subsequent disease spread and degree of infection could be detected by ELISA five yield loss in a given agricultural context. The weeks after plant growth (Khetarpal and epidemiological study will help in getting the Maury 1990). information by taking into account the source The ELISA techniques will save time of virus, levels of resistance of the cultivars, and space. However, virus detection by this the date and intensity of transmission by method is very strain specific and probably vectors in relation to climatic conditions and not applicable unless an antiserum with the resulting percentage of infected plants in correct antibody is available. The techniques the field as well as yield reduction. In case are of little use in situations where numerous of legumes, the per cent seed coats infected serological variants of a virus occur. in a harvested seed lot gives an estimate of In developing countries, the availability the per cent infected plants. This observation of specific antisera ”-globulin, enzyme- can considerably simplify the studies on the conjugated IgG substrate and ELISA reader relationship between the seed-transmitted are limiting factors for large-scale application virus and resulting disease spread. However, of this technique for which regional and assessing the per cent infected plants in the international cooperation is very essential. field just before flowering is important since early season spread has greater influence on yields, the number of secondary sources and 6.4.4 Dot-Immunobinding Assay also the virus transmission through seed. (DIBA or DIA) The tolerance limit could reach 1% for seed sown in regions that regularly have An enzyme immunoassay (EIA) that has very low vector intensities early in the been applied to plant viruses is enzyme- season. On the other hand, regions with assisted immunoelectroblotting (IEB) (Towbin 6.4 Serological Techniques 127 et al. 1979; Rybicki and Von Wechmar 1982; 6.4.4.1 Application of DIBA Von Wechmar et al. 1984a) and was simplified For the first time, this technique was used by as dot-immunobinding assay (DIBA) by Hawkes Von Wechmar et al. (1984a) for the detection et al. (1982). This technique has been variously of Brome mosaic virus in seeds and seedlings described as dot-blot immunobinding assay of wheat. Subsequently, it was also applied (DIBA) by (Berger et al. 1984), immunoblot for the detection of BCMV in bean (Wang assay (Powell 1984), enzyme-linked immunoblot et al. 1985; Lange and Heide 1986; Lange assay (EIBA) (Wang et al. 1985), NC-ELISA et al. 1989), BSMV in barley (Lange and (Bode et al. 1984) and NCM-ELISA (Smith Heide 1986; Lange et al. 1989), PSbMV in and Banttari 1984; Banttari and Goodwin 1985). peas (Lange et al. 1989; Ligat et al. 1991;Ali However, this technique is popularly referred as and Randles 1997), Cucumber green mottle dot-immunobinding assay (DIBA) and also as mosaic virus (CGMMV) in cucurbitaceous crops dot-ELISA (Hawkes et al. 1982; Gumpf et al. (Shang et al. 2011)andBroad bean true mosaic 1984; Hibi and Saito 1985; Parent et al. 1985). comovirus (BBTMV) in broad beans (Makkouk The principle of DIBA is almost the same as et al. 1987; Makkouk and Kumari 1996; that of ELISA, except that antigen or antibody Khatab Eman et al. 2012). is bound to nitrocellulose membrane instead of The main advantages of DIBA are (1) rapid, polystyrene plate and that the product of the en- simple and economical; (2) highly sensitive and zyme reaction is insoluble. This technique is use- detects as low as 50Ð100 picogram of antigen; (3) ful because the dotted membrane can be stored requires only a single crude-specific antiserum and processed at convenience or may be sent each test virus and also a single generally appli- to other laboratories for processing if required cable enzyme conjugate; (4) cost of nitrocellulose facilities are not available with the worker. is less than that of plastic supports; and (5) part of DIBA is of two types, direct DIBA and the seed can be used for testing and the remaining indirect DIBA. The procedure for both the for sowing. methods is same as that of direct and indirect A drawback of this technique is that a ELISA. The extract from infected seed is spotted large volume (50 ml) of relatively concentrated on nitrocellulose membrane (NCM). After (1 mg/ml) antiserum is required but can also be incubating and washing the antigen, a blocking reused over a period of 6 months to test samples agent is added to saturate any unoccupied binding (Abdullahi et al. 2001). sites on the NCM. Then IgG rabbit antibody specific to the antigen to be detected is incubated 6.4.4.2 Tissue Blot Immunobinding with the NCM and unbound antibody is removed Assay (TBIA) by washing. Horseradish peroxidase-conjugated Tissue blotting printing technique is similar to anti-rabbit second IgG is added, incubated and dot-blot immunoassay but involves tissue in washed to remove unbound antibody. Finally, the printing on nitrocellulose membrane (NCM). substrate is added, that is, hydrolysed to form This technique does not involve disruption of water-insoluble coloured spots or dots on the tissue or extraction of antigen from the seed membrane. Horseradish peroxidase, the most sample. The fresh material or imbibed seed frequently used enzyme, produces purple spots material can be used as a imprint tissue on NCM after addition of substrate. Alkaline phosphatase (Lin et al. 1990; Hsu and Lawson 1991). In reacts with naphthol AS-MX phosphate mixed Syria, Makkouk and Attar (2003)havetested with 5-chloro-2-toluidinediazonium chloride the lentil seeds received from ICARDA gene hemizene chloride (fast red TR salt) to produce bank through TBIA and found out that CMV red dots or with diazotised 4-benzolamino-2,5- infection level was 7.4Ð35.8% in 2000/2001 and dimethoxyaniline ZnCl2 (fast blue BBN salt) to 7.0Ð64.2% in 2001/2002. When germinating produce blue dots on the white background of embryo axes of seeds collected from CMV- the NCM. infected lentil mother plants were tested by 128 6 Detection of Plant Viruses in Seeds

TBIA, the CMV infection range was 0.9Ð9.5% 6.4.5 Disperse Dye Immunoassay in 2000Ð2001 and 0.1Ð1.17% in 2000Ð2002. (DIA) Another example of this technique application is with Apple scar skin viroid (ASSVd) which This diagnostic method developed by Gribnau is seed transmitted in apple and pear (Hadidi et al. (1982) is as sensitive as ELISA and provides et al. 1991; Hurtt and Podleckis 1995). They an alternative for ELISA for large-scale indexing have collected 6Ð50 ripe fruits from each of of seed material. 17 cultivars, and tested by chemiluminescent DIA is a solid-phase immunoassay like ELISA tissue blot hybridisation with a cRNA probe for wherein the enzyme conjugate is replaced by ASSVd, 93% of the fruits from infected trees immediate dissolving of dye molecules with an were positive. However, none of 100 seedlings organic solvent, dimethyl sulphoxide (DMSO). grown from the seeds of infected trees showed After the addition of DMSO, the plates are ASSVd infection in tissue blot hybridisation shaken for a minute and colour intensity is read tests nor was ASSVd detected in any of the at 540 nm; this test was used to detect LMV and seedlings where they were graft inoculated to PEBV in the seeds of lettuce and pea, respectively the bioamplification host Virginia crab (Malus (Van Vuurde and Maat 1985). hybrid), and this host was tested for ASSVd by The advantages of DIA are (a) preparation of tissue blot hybridisation assay. These data may conjugate is simple and cheaper, (b) eliminates suggest that ASSVd is not seed transmitted at a substrate incubation step and (c) possibility of high rate in Asian pear even though the pulp of simultaneous detection of two different types of most of the pears on infected trees harbour the antigens (Gribnau et al. 1983). The disadvantage viroid and the tissue blotted antigen samples were of the test in comparison with ELISA is that a processed as dot-blot immunoassay. To remove higher amount of IgG is necessary to prepare the interference of seed tissues, detergents like the dye solution conjugate. This technique is Triton-X-100 or sodium hypochlorite can be unsuitable for crude plant extracts. used. The antigen-blotted membrane can be stored for several weeks at 4ıC and can be used effectively for processing of the bulk samples 6.4.6 Rapid Immunofilter Paper and also at field level. Makkouk and Kumari Assay (RIPA) (1996) and Makkouk et al. (1997) have studied TBIA application in ten virusÐhost combinations. RIPA is another simple, rapid, sensitive and Khatab Eman et al. (2012)havetestedthis virus-specific detection technique which has been technique to detect BBTMV in faba beans. This adopted for detection of a virus in plant tissues. In test was sensitive enough to detect the virus in this test, at the bottom of the Whatman glass filter all parts of the plant and at all growth stages. It paper, latex beads coated with virus antibodies is suggested that the test is useful for detecting were immobilised as a solid in a line. The bottom seed-transmitted viruses after seed germination end of the paper strip was dipped for 3 min and is more practical than ELISA. This test was in a mixture of virus-infected leaf/seed extract completedinlessthan4hwithout sacrificing and dyed latex coated with the virus antibody. sensitivity and is cheap and does not require A coloured band appears on the line where the sophisticated facilities. This technique is easily white latex has been immobilised and can be applicable to field sampling as tissue printings detected with the naked eye and the filter paper can be made in the field without the need to strips could be measured by chromatoscanner. collect leaf samples for sap extraction in the The filter paper strips coated with antibody can laboratory. be stored at room temperature for more than 6.4 Serological Techniques 129

1 year in a desiccator, and the coated strips can The main advantage of ISEM is its sensitivity, be used as easily and simply as pH test papers. which equals that of ELISA and local lesion The sensitivity of RIPA was demonstrated by assays (Baier and Shepherd 1978; Hamilton and Tsuda et al. (1992) with CMV and TMV plant Nichols 1978) and can be used for quantitative extracts, and attempts should be made to use it assay in crude seed extracts. It is quick, consum- for the virus detection in seeds. ing little antiserum, and also enables the use of relatively low-titre antisera. The grids can also be coated with mixtures of antisera for different 6.4.7 Immunosorbent Electron viruses, which allows detection of several viruses Microscopy (ISEM) at a time (Derrick 1973; Thomas 1980). However, the disadvantage of this method is that serologi- The visualisation of immunological reactions on cally distantly related strains may not be detected electron microscope grids is one of the most (Nicolaieff and Van Regenmortel 1980). sensitive serological techniques. Two different The ISEM technique has been applied approaches can be distinguished, depending on successfully in the detection of numerous whether the viral antigen in suspension is visu- elongated and isometric plant viruses in seed alised in thin sections of the infected tissue. In (Table 6.4) as well as identifying the presence recent years, viruses in suspension are visualised of double-stranded RNA in extracts of tobacco directly on the electron microscopic grid. This infected with TMV (Derrick 1978). When technique is unique in that it combines direct mixtures of different ratios of infected to visualisation of virus particles with the specificity healthy seeds were tested, these methods were of a serological reaction. found sensitive to detect one seed infected with Roberts et al. (1982) suggested two general PSbMV or LMV in 100 seeds (Hamilton and terms for this technique, that is, immuno-electron Nichols 1978) and one infected with TRSV, microscopy (IEM) and electron microscope SMV or BSMV in 1,000 seeds (Brlansky and serology (EMS). However, Derrick (1973)intro- Derrick 1979). duced this technique for the first time and termed it serologically specific electron microscopy 6.4.7.1 Clumping of Virus Particles (SSEM) which has been widely used in the When a virus preparation is mixed with literature (Paliwal 1977; Beier and Shepherd a suitable dilution of specific antiserum, 1978; Hamilton and Nichols 1978;Brlansky the formation of virusÐantibody complexes and Derrick 1979; Milne and Lesemann 1984; is visualised in an electron microscope by Rao and Singh 2008). However, since the term the appearance of clumps of variable size SSEM would also encompass the technique using (Ball and Brakke 1968; Milne and Luisoni ferritin-labelled antibody, it has been argued 1975; Roberts 1976). This method is simple, as an inappropriate term (Roberts et al. 1982). faster and suitable for verifying the presence All forms of immuno-electron microscopy are of viruses in seed extracts. The clumping ‘serologically specific’ and prefer to use a special phenomenon is especially valuable when the term for the technique in which virus particles virus concentration is too low for the particles to are attached to grids previously coated with an- be seen directly. tiserum. Roberts and Harrison (1979), therefore, However, the disadvantage of this method is introduced the term immunosorbent electron mi- that components of plant sap/seed extract and croscopy (ISEM) which is now popular in plant antiserum can interfere with staining and affect virology. In immunosorbent electron microscopy the image quality. Also, the attachment of virusÐ (ISEM), there are three general methods, namely, antibody aggregates to the grid is variable and clumping, antibody coating or decoration and some aggregates may be washed off during the trapping which are used for virus detection. staining process. 130 6 Detection of Plant Viruses in Seeds

Table 6.4 Application of immunosorbent electron microscopy (ISEM) for a detection of certain viruses transmitted through seed Virus Crop Reference Alfalfa mosaic Lucerne Lange et al. (1983), Mukhayyish and Makkouk (1983) Barley stripe mosaic Barley Brlansky and Derrick (1979), Lister et al. (1981), Lundsgaard (1985), and Lange and Heide (1986) Bean common mosaic Beans Jafarpour et al. (1979), Russo and Vovlas (1981), Lundsgaard (1983), Hagita and Tamada (1984), Lange and Heide (1986), Raizada et al. (1990), and Nalini et al. (2004) Green gram/blackgram Dinesh chand et al. (2004) Mung bean Jeyanandarajah (1992) Black eye cowpea mosaic Cowpea Taiwo et al. (1982) and Jeyanandarajah (1992) Blackgram mottle Blackgram Krishnareddy (1989) Broad bean stain Faba bean Anon (1983) Broad bean true mosaic Faba bean Anon (1983) Brome mosaic Wheat Von Wechmar et al. (1984a) Cacao Swollen shoot Sagemann et al. (1985) Cowpea aphid-borne mosaic Cowpea Kositratana et al. (1986) and Raizada et al. (1991) Cucumber green mottle mosaic Cucumber Mukhayyish and Makkouk (1983) Fig latent virus 1 Fig Castellano et al. (2009) Indian peanut clump Peanut Reddy et al. (1998a, b) Lettuce mosaic Lettuce Brlansky and Derrick (1979), Van Vuurde and Maat (1983), and Falk and Purcifull (1983) Maize dwarf mosaic Sweet corn Mikel et al. (1984) Pea seed-borne mosaic Pea Hamilton and Nichols (1978) Soybean mosaic Soybean Brlansky and Derrick (1979), Maury et al. (1983) Squash mosaic Melon Lange et al. (1983) Tobacco ring spot Soybean Brlansky and Derrick (1979) Vicia cryptic Faba bean Anon (1984)

6.4.7.2 Decoration or Antibody relatively low magnification due to an apparent Coating increase in their diameter (Martin 1985). Decoration of virus particles in electron micro- The efficiency of virus trapping on grids scope grids is similar to the clumping method coated with specific antibody or decoration of except that, prior to adding the antibody, the virus virus particles is 40Ð50 times greater than on is immobilised on the grid by dipping in the grids coated with non-specific serum (Derrick antigen or adding the seed extract homogenate to 1973). These procedures have been combined for the grid. After the grid is washed, a small drop use as a diagnostic tool (Milne and Luisoni 1977; of serum is added to it. Then it is incubated for Kerlan et al. 1981). Besides this technique offers 15 min in a water-saturated atmosphere, washed additional advantage to detect contaminant and again, stained and examined. Individual virus non-specific particles in the preparation. particles are coated with halo of antibody, if the Pares and Whitecross (1982) described gold- reaction is positive (Milne and Luisoni 1977; labelled antibody decoration (GLAD) with pro- Roberts et al.1982). tein A gold complexes using different strains of The decoration can be amplified by using a TMV as an antigen. With this technique, distantly double decoration procedure (Kerlan et al. 1981). related viruses are distinguished by qualitative In this method, an antibody specific for deco- analysis of the adsorbed gold particles, but such rating antibody is added after initial decoration analyses failed to distinguish between closely procedure. Virus particles are then observed at a related viruses. 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 131

6.4.7.3 Trapping in Electron Microscopy 6.5 Biotechnology/Molecular Biology-Based Virus The trapping of plant viruses to electron micro- Diagnosis scope grids coated with specific antiserum was first described and designated by Derrick (1973) 6.5.1 Introduction as serologically specific electron microscopy (SSEM); later it was renamed as immunosorbent Recent advances in molecular biology and electron microscopy (Roberts and Harrison 1979; biotechnology are being applied to the devel- Roberts et al. 1982). opment of rapid, specific and sensitive tools The method consists of allowing freshly for the detection of plant virus and viroid coated microscope grids with a parlodin carbon infections in seeds. Immunoassays have reached film to float on 10Ð50-l drops of diluted the point of practical application in agriculture, antiserum for about 30 min. Excess protein is and the DNA probe technology is established in then removed by floating these grids for several research laboratories. (Martin et al. 2000;Naidu minutes in a buffer solution. The coated grids are and Hughes 2003) Immunoassays are being drained by a brief contact with filter paper and routinely used to detect virus in vegetatively placed for 1 or 2 h on drops of virus extract. Salts propagated material and seeds for the purpose and contaminants are removed by washing with of quarantine, certification and maintenance. distilled water, and the particles are visualised Use of immunoreagents such as monoclonal after negative staining (Van Regenmortel 1982). antibodies in ELISA systems has the potential Use of protein A in ISEM increases two- for enhanced precision and sophistication of to sixfold in trapping viruses (Lesemann and detection. Paul 1980) and is specially suitable for low-titre When a virus is prone to antigenic variation antiserum (Milne and Lesemann 1984). Gold- or occurs without coat protein (Harrison and labelled protein A has been used successfully to Robinson 1978) and in viroids, immunological label several strains of TMV (Pares and White detection systems are unsuitable. Indirect meth- cross 1982). Protein A binds to the Fc region ods for comparing viral nucleic acids are routine of most immunoglobulins (IgG). Protein A may and have been used to detect viroids (Owens also be used to coat grids to provide a more and Diener 1984) or tobravirus (Harrison et al. uniform coating of grids with antibody (Shukla 1983). The nucleic acid-based detection systems and Gough 1979, 1984). which make use of cloned DNA or DNA probes The advantages of ISEM is its sensitivity and in a dot-blot or related assays have the potential its lack of strain specificity (Van Regenmortel to detect even single-nucleotide differences. The et al. 1980). It is ideal for small samples if ability to make DNA copies (cDNA) of a part or an electron microscope and the specific antisera the whole plant viral RNA genome has revealed are available. Direct ISEM of seed extracts may many new possibilities. The nucleotide sequence yield results in less than 2 h. The disadvan- of the DNA copy can be determined; it is a tages of ISEM are that, though sensitive, it is time-taking process, but it can be considered as not amenable for large-scale testing programmes a diagnostic procedure in certain special cases. where hundreds and thousands of samples are to Fundamentally, four approaches can be used to be tested as in many certification scheme agen- detect and diagnose the viral nucleic acids: (a) cies and quarantines (Martin 1985) and lack of type and molecular size of the viral nucleic acids, availability of the electron microscope facility at (b) cleavage pattern of viral DNA or cDNA, many seed testing and quarantine stations due to (c) hybridisation between nucleic acids and (d) high cost. polymerase chain reaction. 132 6 Detection of Plant Viruses in Seeds

Morris and Dodds (1979) developed a method case of viroid and RNA viruses or 32P cDNAÐ for the isolation and detection of dsRNA from DNA hybrid in case of DNA viruses. The extent virus-infected plants. The properties of dsRNAs of hybrid formation is a measure of the concen- associated with RNA viral infections have tration of viral or viroid sequences in the plant been used for diagnosis (Dodds 1993). These extract (Symons 1984). dsRNAs, which are very resistant to enzymatic Two procedures are available for the molecu- degradation, are not normally present in healthy lar hybridisation analysis of viroid or virus infec- plants. Simplification, together with improved tion, similar in principle but different in the way equipment for nucleic acid analysis, has made the 32P cDNAÐRNA hybrids are detected. the technique more practical and attractive to plant virus diagnosis. 6.5.2.1 Liquid Hybridisation Assay Application of each of molecular methods as In this method, nucleic acid hybridisation takes well as dsRNA and monoclonal antibody tech- place in solution. Most of basic studies were nologies for the detection and diagnosis of seed- performed using both the target and probe DNAs transmitted viruses and viroids are as follows: in solution, so it is called as liquidÐliquid hy- bridisation; there are also some data available for RNAÐRNA and DNAÐDNA interaction in 6.5.2 Molecular Hybridisation solution. Presently most of plant pathogen di- agnosis was involved by mixed-phase hybridi- Nucleic acid hybridisation is a powerful sation with the target immobilised on a solid technique for the diagnosis of many plant viruses matrix; the theory developed for liquidÐliquid hy- which are not easily detected by serological tech- bridisation is very relevant (Hull 2002). LiquidÐ niques. It is particularly effective in the detection liquid hybridisation has been used to examine of viruses occurring in low concentration in plant the relationship between plant viruses. The main tissues and also against viruses that are poor principle of the technique is to hybridise the immunogens or viroids which lack coat protein. target nucleic acid with the probe that has been Molecular hybridisation analysis also referred radioactively labelled than to digest away and to as the ‘nucleic acid hybridisation’ or ‘spot unhybridised ss probe, precipitate the ds hybrid hybridisation’ or ‘dot-blot’ technique has been on a filter membrane and count the radioactivity. shown to be a highly sensitive and specific It has been extensively used for indexing Avocado procedure for identifying RNA or DNA viruses sunblotch viroid (ASBV) disease of avocados (Abu Samah and Randles 1983; Gould and (Palukaitis et al. 1981; Allen and Dale 1981; Symons 1983; Maule et al. 1983) and plant Allen et al. 1981)andCoconut cadang-cadang viroids (Palukaitis and Symons 1978; Owens and viroid (CCCVd) in extracts of coconut and other Diener 1981). Basis of molecular hybridisation of palms (Randles and Palukaitis 1979; Randles viral nucleic acids was discussed by Hull (1993). et al. 1980; Boccardo et al. 1981) and also been The principle of hybridisation analysis for in- used to a very limited extent for detection of dexing of viroids and viruses is relatively simple. Chrysanthemum stunt viroid (CSVd) in extracts The first requirement is to prepare highly radioac- of infected plants (Palukaitis and Symons 1979). tive complementary DNA (cDNA) to the viroid This method has been used to assess relationships or viral nucleic acid using 32Por3H isotope. The between tombusviruses, and a sensitive nonra- cDNA is prepared enzymatically on the viral or dioactive procedure has been developed for de- viroid nucleic acid so that its sequence is exactly tection of BaMV and its associated satellite RNA complementary. When this 32Por3HcDNAis in meristem-tip cultured plants (Hsu et al. 2000). incubated with a nucleic acid extract of a test plant under appropriate conditions, it hybridises 6.5.2.2 Dot-Blot Hybridisation Assay to any viral or viroid sequences present to pro- This method is common for indexing of viroids duce a double-strand 32P cDNAÐRNA hybrid in and viruses and a popular method on large-scale 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 133 testing. The basic procedures have been described 1987). Hsu et al. (2000) developed dot-blot by Thomas (1980) and Owens and Diener (1981) hybridisation by using nonradioactive method which are extensions and modifications of ear- to detect the BaMV and its sat RNA. lier techniques, which are developed for use in general recombinant DNA technology. The major difference between liquid hybridisation and dot- 6.5.3 Double-Stranded RNA blot assay is that in the latter test nucleic acid (dsRNA) Analysis is immobilised on a cellulose nitrate sheet for hybridisation rather than being in solution. Almost 80% of plant viruses have RNA genomes The preparation of samples in this procedure that can be single stranded. During replication is quite simple. The crude seed extract samples of ssRNA viruses in plant cells, high molec- are simply spotted onto nitrocellulose filters and ular weight double-stranded RNA (dsRNA) is dried. The filters are subsequently hybridised produced as an intermediate product, and this with radiolabelled DNA probes which are copies dsRNA is called the replication form (RF) and of the viral genome. After hybridisation and is consistently present when plant is virus in- washing to remove the non-hybridised probe, the fected. Most of the dsRNAs are associated with presence of virus is detected by autoradiography plant ssRNA viruses. Double-stranded form of (Baulcombe et al. 1984). A modification of the the genome RNA accumulates that is twice the dot-blot assay, squash blotting has been used to size of the genomic RNA. It is known as replica- detect Maize streak virus (MSV) (Boulton and tive form (RF) or replicative intermediate (RI). Markham 1986). cDNA probes require the use of dsRNAs associated with RNA viral infections radioactive label (32P) which are not amenable for have been used for diagnosis (Dodds 1993). dsR- common use, and very limited success has been NAs are associated with plant reoviruses; cryp- achieved in their application. Prehybridisation toviruses have genome consisting of dsRNA and and the hybridisation steps are carried out in a in tissue infected with ssRNA viruses. Some specialised manner (Dijkstra and de Jager 1998). viruses have dsRNA genomes during replication; Salazar et al. (1983) and Bernardy et al. others produce dsRNA. Detection of high molec- (1987) have used this technique for the detection ular weight dsRNA has been used to study plant of PSTVd in mixtures of potato seed extracts diseases of suspected viral aetiology for which no equivalent to one infected seed among healthy virus-like particles could be identified (Chu et al. ones. This technique is being used in routine 1983; Dodds and Bar-Joseph 1983; Jordan et al. testing of seeds at International Potato Centre, 1983; Morris and Dodds 1979). Peru. Bijaisoradat and Kuhn (1988)useditto Double-stranded RNA (dsRNA), an indicator detect the presence of PMV and PStV in peanut for the presence of viruses in plants, can be seeds. Both the viruses have been detected readily isolated rapidly by simple methods from small in one mg of infected seed tissue and when tissue weights of seedlings grown out of in- extracts from seeds have been diluted to 1/62,500 fected seed and using relatively simple laboratory with water. One part of the infected seed can be equipment. The number, size, intensity and com- reliably detected when mixed with 99 parts of plexity of dsRNA detected by gel electrophore- healthy seeds. This sensitivity will be 8Ð10 times sis are distinctive and consistent for different greater than that achieved by the use of ELISA. virus groups and also aid in the diagnosis of a Besides sensitivity, rapidity and simplicity, this virus to a group or even a strain. Detection of test is highly specific. For example, PMV cDNA dsRNA of appropriate size may provide insight does not hybridise with PStV from infected into the cause of diseases of unknown aetiology peanut seeds and vice versa, even though these (i.e. little cherry, lettuce big vein, black currant two viruses share 60% nucleotide sequence reversion) (Dodds et al. 1984). Additionally, it homology when the hybridisation is performed was expected that detection of dsRNA molecules under stringent conditions (Sukorndhaman would improve awareness of latent infections in 134 6 Detection of Plant Viruses in Seeds apparently healthy plants because any suspect The need for rapid and reliable methods of dsRNA species could be labelled directly or af- diagnosing diseases and identifying pathogens is ter cloning. DNA copies can be multiplied in increasing as new technologies become available bacteria, thereby facilitating their routine use in to researchers and diagnosticians. Despite its ELISA-based systems or in nucleic acid hybridi- limitations, the procedure described by Valverde sation assays (Dodds 1986). The method for ex- (2008) could be used in most disease clinics traction of dsRNA from plant tissues, purification as a primary screening technique and as a and analysis is described by Valverde (2008). complement to other techniques to diagnose Plant virus infections so far tested have been viruses (Morris et al. 1983; Dodds 1986;Valverde in tobamo, cucumo, potex, carla, poty and clos- et al. 1990). Antibodies reacting non-specifically tero virus groups which contain disease-specific against dsRNAs were found in antisera prepared dsRNA, molecules of the size expected for the against phytoreoviruses. Non-specific antisera molecular weight of the genomic ssRNA. Bar- can be prepared by using synthetic poly (I) poly Joseph et al. (1983)showedTMVandCMV clonal antigens, but this technique is not well could readily be diagnosed in singly or double received. mAbs produced against dsRNA have infected plants by dsRNA analysis. been used in immunoblots to detect this form of This technique has several advantages over RNA in extracts from plants infected with viruses other methods for virus diagnosis. The dsRNA (Lukacs 1994). Polymorphisms in heteroduplex technique overcomes these problems: (1) The RNAs by adapting this method heteroduplexes technique is simple and relatively inexpensive. of RNA transcripts polymorphism were detected (2) dsRNA can be obtained regardless of the host between strains of PNRSV (Rosner et al. 1999). or the RNA virus. (3) Results are obtained in a relatively short time (8Ð12 h). (4) Interfering host components and instability of the virus or viral 6.5.4 Gel Electrophoresis RNA are two main problems encountered by plant virologists while using traditional methods. Electrophoresis is the migration of charged par- (5) The technique detects mixed infection, which ticles like proteins, nucleic acids or polysac- often go undetected with other methods and charides to either cathode or anode under the result in inadequate diagnoses. (6) Unlike most influence of an electric field. It is usually carried other diagnostic techniques, dsRNA analysis out in gels formed in tubes, slabs or on a flat is non-specific. It can be used to distinguish bed. In electrophoretic units, the gel is mounted not only different viruses but also strains of between two buffer chambers containing separate the same virus and satellite RNAs (Valverde electrodes so that the only electrical connection and Dodds 1986;Valverdeetal.1986). (7) The between the two chambers is through the gel. The purified dsRNA could then be used as a reagent sample may be run in agarose or polyacrylamide for inoculation, probe preparation or molecular or agaroseÐacrylamide composite gel. At the end cloning (Dodds 1986; Jordan and Dodds 1983; of run, the gels are stained and used for scanning Rosner et al. 1983). or visual recording of results. The dsRNA analysis has some limitations as The major useful systems for viruses and vi- only RNA viruses can be detected. The knowl- roids are (1) agarose gel electrophoresis and (2) edge about the number and size of viral RNAs polyacrylamide gel electrophoresis (PAGE). of different viral groups is required. Some plants contain cryptic viruses and/or cellular dsRNAs 6.5.4.1 Agarose Gel Electrophoresis that yield dsRNAs similar in size to those asso- The standard method used to separate, identify ciated with ssRNA viruses. Certain viral groups, and purify nucleic acid (RNA or DNA) frag- such as the luteoviruses and most potyviruses, ments is electrophoresis through agarose gels. yield very low quantities of dsRNA, making the This technique is simple and rapid to perform method impractical for their routine diagnosis and capable of resolving mixtures of nucleic acid (Valverde et al. 1990). fragments that cannot be separated adequately 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 135 by other methods, such as density gradient cen- RNAs and termed return PAGE (R-PAGE) has trifugation. Furthermore, the location of nucleic further facilitated viroid detection in various acids within the gel can be determined directly ways (Schumacher et al. 1986; Singh et al. 1988, by staining with low concentration of fluorescent 1992, 1993; Singh and Boucher 1987). or ethidium bromide dye and subsequent exami- In R-PAGE assay, nucleic acids are subjected nation of the gel in ultraviolet light. to two electrophoretic runs, one under non- The electrophoretic migration rate of nucleic denaturing and the other under denaturing acids through agarose gels is dependent on (a) conditions. Because of the denaturation, circular molecular size of the nucleic acid, (b) agarose RNAs lose their double-stranded configuration, concentration and (c) the applied current. become single-stranded covalently closed Several different designs of apparatus have circular forms, migrate much more slowly been used. Currently agarose gel electrophoresis in the second electrophoresis and thus are is being carried out with horizontal slab gels. well separated from non-circular molecules. Several different electrophoresis buffers are avail- The circular RNAs from the lowest bands able containing tris-acetate, tris-borate or tris- on the electropherograms render them easily phosphate, and among them tris-acetate is the distinguishable from noninfected plant extracts. most commonly used buffer. The procedure takes on a day and requires simple laboratory equipment and nonradioactive 6.5.4.2 Polyacrylamide Gel chemicals. Viroid concentrations as low as 15Ð Electrophoresis 20 pg can be detected reliably by R-PAGE (Singh Polyacrylamide gels are used to analyse and pre- 1989). For example, it detects viroids in one pare fragments of DNA/RNA and proteins. They infected potato leaf disc mixed with 500 healthy may be casted in a variety of polyacrylamide con- leaf discs and standard nucleic acid extracts centrations, ranging from 3.5 to 20%, depending diluted to 1:1,024 times. on the size of the molecules of interest. R-PAGE has been successfully utilised to de- Polyacrylamide gels are poured between two tect viroid from dormant potato tubers or from glass plates that are held apart by spacers. In this infected true potato seeds (TPS) singly or mixed arrangement, most of the acrylamide solution is with 100 healthy TPS (Singh et al. 1988). shielded from exposure to the air so that inhibi- It has been used to identify several viroids tion of polymerisation by oxygen is confined to a from various crop plants in developing countries. narrow layer at the top of the gel. Polyacrylamide A unique feature of the R-PAGE has been the gels can range in length from 10 to 100 cm, separation of viroid strains on the basis of their depending on the separation required, and are mobility on the gel, which has greatly aided invariably run in the vertical position (Sambrook the studies on cross-protection (Singh 1989). and Russell 2002). The other major usefulness of R-PAGE can be used to assess the PSTVd content PAGE system is determination of the molecular of various seed lots before planting either from weights of the polypeptides or nucleic acids and seeds or from in vitro seedlings, when germplasm detection of cryptic virus and dsRNAs in virus- is valuable and available in small quantities or viroid-infected seed material. (Singh et al. 1988, 1992).

6.5.4.3 Return-Polyacrylamide Gel Electrophoresis (R-PAGE) 6.5.5 Nucleic Acid-Specific Return-polyacrylamide gel electrophoresis (R- Hybridisation PAGE) has played a pivotal role in the diagnosis of diseases caused by viroids which have low Nucleic acid-based detection of the plant viruses molecular weight RNA (Singh et al. 1992). A is an important tool in molecular biology. Four modification of PAGE specially designed for important approaches are being used to detect the rapid and sensitive detection of circular and diagnose of the plant viruses: (a) type and 136 6 Detection of Plant Viruses in Seeds molecular size of the viral nucleic acid, (b) vi- in liquid but presently done by blotting the test ral nucleic acid cleavage pattern, (c) hybridisa- samples on membrane followed by hybridisation tion between nucleic acids and (d) amplification with a radiolabelled (32P) viral nucleic acid that studies of desired viral nucleic acid. Nucleic serves a probe. This technique was more sensitive acid-specific hybridisation techniques are being than ELISA for the detection of Potato virus used for testing plant viruses and it involves the X (PVX) and Potato leaf roll virus (PLRV) use of labelled complementary DNA (cDNA) infecting potato plants. The disadvantages or RNA (cRNA) prepared from purified viral of the radiolabelled probes are that they are nucleic acid as a probe or a recombinant clone of biohazardous. such viral nucleic acid as a probe. These labelled probes are used to detect the presence of plant 6.5.5.2 Non-radiolabelled Probes viral nucleic acid by forming hybrid with them. Due to biohazardous nature of radiolabelled Presently nucleic acid hybridisation includes the probes, the non-radiolabelled probes are formation of DNAÐDNA, DNAÐRNA and RNAÐ preferred to do the hybridisation studies RNA complexes (Singh and Dhar 1998). To de- for the viral nucleic acids. The most used tect the plant viruses and viroids, three types of nonradioactive-labelled compounds are biotin ‘molecular hybridisation’ tests are being used: (a) vitamin H and hapten digoxigenin (DIG). These solution hybridisation test, (b) filter hybridisation two compounds are detected by indirect way, test or dot-blot hybridisation or nucleic acid spot using labelled, specific-binding protein, either hybridisation (NASH) and c) in situ hybridisa- streptavidin (biotin) or anti-digoxigenin serum, tion test. and later visualised by a chemiluminescent or The NASH is one of the widely used tech- colorimetric reaction. The biotinÐstreptavidin niques to detect the plant viruses and viroids. In system is very sensitive and efficient, but we can this technique the target nucleic acid is immo- get positive or background occurrence. Genome bilised on a membrane (nitrocellulose or nylon) of the various viroids that are 246Ð375 bases long and then hybridised to a labelled specific nucleic is being used for the hybridisation studies. Non- acid probe. Crude leaf or seed extracts are applied radiolabelled hybridisation has been successfully to the membrane and labelled with specific probe used for detection of PSTVd in true potato seeds of DNA or RNA. Serological methods (ELISA) (Goldbach et al. 1992; Borkhardt et al. 1994). give little information on the virus detection, Advantages of non-radiolabelled probes are but nucleic acid hybridisation gives more genetic stability of probes, no radioactivity, inexpensive information on the viral or viroids nucleic acids. and rapid detection, and a disadvantage is Due to lack of coat protein in the viroids, the possible stearic hindrance during hybridisation nucleic acid hybridisation techniques are the only because of hapten presence. detection methods to characterise the genome. The identifying probes were made into two types: 6.5.5.3 cDNA Probes (a) radiolabelled probes and (b) non-radio la- cDNA technology utilises the specific recogni- belled probes. tion between the viral RNA and its comple- mentary DNA (cDNA) (Watson et al. 1983). As 6.5.5.1 Radiolabelled Probes majority of the plant viruses have single-stranded Several improvements have been made to RNA (ssRNA) as the genetic material, by using the application of complementary DNA or the reverse transcriptase, ssRNA can be copied RNA probes to detect viral pathogens. Mostly into cDNA which then by a recombinant DNA 32P labelled nucleotides were used to label technology can be cloned in a suitable cloning these nucleic acids radioactively, with specific vehicle. In this way, cDNA, specific to each virus, activities up to 1010 cpm/g, which can can be mass produced. detect 1 pg of target sequences. Nucleic acid DNA or RNA viruses can be detected in seeds hybridisation between DNA and RNA was done by using cDNA probes which are labelled with 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 137 radioactive markers (32Por3H) or nonradioactive 6.5.6.2 Microarrays markers such as biotin. Sensitivity of detection Microarray is a technique used in array tech- may be related to the probe size and larger probes nology, where the amount of sample used is give more sensitive assays. It is possible to make much smaller than macroarrays and the droplet diagnostic cDNA probes to regions of the certain size is less than 200-m space. The invention viral genome where the extent of sequence sim- of microarrays was chiefly for the detection of ilarity has been shown to distinguish the viruses differential gene expression patterns in cells, and and strains (Watson et al. 1983). This technique this was applied in case of plant viruses by Boon- was also proved to be effective in identifying ham et al. (2007)andBystrickaetal.(2005). the seed-transmitted infection of certain viroid Importance of microarrays in detection of plant diseases. For example, scar skin and dapple apple viruses was emphasised by Hadidi et al. (2004), viroids (Hadidi et al. 1991). Bystricka et al. (2005), Tomlinson and Mumford (2007) and Barba and Hadidi (2008). Microarray or DNA chip technology is used 6.5.6 Array Technologies generally for identification of differential gene expression patterns of an organism or tissue. It is chiefly used in functional genomics for under- Array technology has revolutionised the world standing the different types of tissues, develop- of viral diagnosis because of its efficiency in mental stages and disease conditions. The princi- screening a large volume of field samples in a ple is mainly based on nucleic acid hybridisation single array plate. Basic principle of array tech- and fluorescent dyes for labelling the hybridis- nology combines the binding of DNA on to a ation probes and hence termed as a process of solid support such as membrane filter or array in situ hybridisation. A microarray is a glass plate and followed by hybridisation technology slide, to which single-stranded DNA molecules with a specific probe that will detect the target are attached at fixed locations. Generally this DNA. This technology was first invented and slide is either made of glass or nitrocellulose that applied for gene expression studies; later it has can bind biomolecules covalently. Each spot will been used in various pathogens diagnosis includ- require 0.25Ð1 nl solution and diameter on the ing plant viruses (Boonham et al. 2007). There glass will be 100Ð150 m separated by a space of are mainly two types of arrays: (1) macroarrays 200Ð250 m. The samples are spotted on to the and (2) microarrays based on the volume of slide or chip in microvolumes in the form of an the sample and the droplet size used for the array. So the technology is defined as microarray analysis. technology which is a highly suitable technology for detection of plant viruses on a large scale. 6.5.6.1 Macroarrays High density nucleic acid samples are iso- Macroarray is a technique used in array technol- lated and purified which can be used as sam- ogy, where the amount of sample used is higher ples or else sometimes cDNA derived from re- than microarrays and the droplet size is more than verse transcription of expressed mRNA or RNA 200-m space. Principle involves simple blotting itself and sometimes termed as RNA microar- of oligoprobes of virus-specific sequences either rays. In some cases, oligonucleotides are syn- by dot-blot or slot-blot method followed by nu- thesised directly on to the microarray plates or cleic acid hybridisation with specific sample in the glass slides (known as in silico synthesis). which the virus is to be detected. This technique To print these samples, the scientists use high- was first applied in plant virology by Agindotan throughput robotic systems. These nucleic acid and Perry (2007) for detection of various RNA samples are then immobilised on to the substrate. viruses and also for detection of 11 potato viruses The next step includes hybridisation of probes. and a potato-infecting Potato spindle tuber viroid Probes are prepared from many sources, some (Agindotan and Perry 2008). from cell extracted nucleic acids, some are syn- 138 6 Detection of Plant Viruses in Seeds thesised oligonucleotides, some are from PCR- then alternated between hot and cold to dena- amplified products, some include the different ture and reanneal the DNA, with the polymerase cloned inserts, etc. Generally in order to identify adding new complementary strands each time. the expression patterns in genes, mRNA or cDNA In addition to the basic use of PCR, specially derived from mRNA is used as probes. In case designed primers can be made to ligate two dif- of DNA microarrays, DNA samples or fragments ferent pieces of DNA together or add a restriction are used as probes. The probes are isolated and site, in addition to many other creative uses. purified nucleic acids and they are labelled by Clearly, PCR is a procedure that is an integral two types of fluorescent dyes Ð one is CY3 that addition to the molecular biologist’s toolbox, and is a green channel excitation fluorophore and the the method has been continually improved upon other is CY5 that is a red channel excitation fluo- over the years (Candresse et al. 1998; Dietzen rophore. The labelled probes are hybridised to the 2001; Albrechtsen 2006). microarray plate in a hybridiser. After hybridisa- Polymerase chain reaction is a molecular tech- tion, the unhybridised probe will be washed out nique for the in vitro amplification of DNA or in with appropriate buffers. This microarray plate general terms known as photocopying of DNA. In is ready for scanning. Many microarray scanners this technique, a specific target DNA is amplified are available nowadays which apply briefly two into multiple copies by a set of oligonucleotides methods: (1) sequential scanning and (2) simulta- specific to the target DNA. Before understand- neous scanning. The image obtained by scanning ing the exact mechanism of how PCR works the microarray plate in a scanner is subjected to in amplification of target DNA, let us get into analysis by using softwares like ScanAlyze for the kinetics of how a DNA molecule replicates the expression pattern. in in vivo. In the first step of replication, DNA Similar principles could be applied in case of double helix strand is unwound into two indi- plant viruses where the microarray slide could be vidual strands by the enzyme helicase at the printed with oligo that are synthesised specific to site of replication initiation. The initial primer the plant viruses. At a time, two samples labelled required for the DNA replication initiation is with two different fluorophores could be used for synthesised by the enzyme primase. Then the hybridisation, and the presence of specific virus DNA polymerase synthesises the DNA strands or its genotype could be identified easily. by utilising the dNTPs present in the cytoplasm, and the synthesis is carried out which leads to the formation of Okazaki fragments in a replication 6.5.7 Polymerase Chain Reaction fork and this synthesis is completed by filling the (PCR)-Based Detection fragments in the replication fork. The basis of invention of PCR is to overcome 6.5.7.1 Polymerase Chain Reaction difficulties in amplification of the desired copy Basics of DNA. Old procedures included cloning and The advent of PCR by Kary B. Mullis in the replication of clone for multiplying the copy mid-1980s has revolutionised molecular biology. of desired DNA. The supplements of the DNA PCR is a fairly standard procedure now, and its replication are provided artificially to replicate a use is extremely wide ranging. At its most basic desired copy of DNA in PCR reaction. The most application, PCR can amplify a small amount of primary problem faced by molecular biologists template DNA (or RNA) into large quantities in in in vitro amplification of DNA is the stability a few hours. This is performed by mixing the of the DNA polymerases. Most of the DNA DNA with primers on either side of the DNA polymerases are not stable at higher temperatures (forward and reverse), Taq polymerase (of the like 90Ð95ıC where two strands of DNA get species Thermus aquaticus, a thermophile whose separated by heat denaturation. Studies have polymerase is able to withstand extremely high shown that certain hot spring living bacteria like temperatures), free nucleotides (dNTPs for DNA, Thermus aquaticus have polymerases that are NTPs for RNA) and buffer. The temperature is stable at higher temperatures. They are used in 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 139

PCR as a replacement to conventional DNA poly- number of copies of the target obtained after the n merases. In the PCR reaction, other requirements cycles will be 2n. Generally, these repetitions are are supplemented artificially like Taq DNA buffer done from 25 to 40 cycles depending upon the (which provides buffered environment for nec- amount of DNA copies. essary action of Taq DNA polymerase), MgCl2 (essential for activity of Taq DNA polymerase 6.5.7.2 Variants of PCR and Their and also aids by binding of dNTPs (dATP, dGTP, Applications dCTP and dTTP) for polymerisation), oligos PCR is a strong tool in molecular biology; af- (short stretch of DNA sequences that are comple- ter its invention, it was variantly modified to mentary to the target DNA, popularly known as serve many purposes and these modified types primers that will decide the range of amplifica- are termed as variants of PCR. The PCR variants tion), template DNA from which the amplifica- are RT-PCR, IC-RT-PCR, nested PCR, multi- tion of the specific target DNA is achieved and plex PCR, LAMP, direct binding PCR, IC-PCR, deionised water (to make up the concentration CF-PCR, PCR-RFLP, PCR-ELISA and real-time of the components). The PCR reaction is set in PCR. Application of these techniques in identi- polycarbonate tubes or strips or plates designed fying different seed-transmitted plant virus and for the purpose depending on the requirement viroids are presented in (Table 6.5). The advan- process. The kinetics of PCR are chiefly depen- tages of different PCR variants are discussed here dent on the concentrations of the components under. like MgCl2 concentration, dNTPs concentration, amount of enzyme used, concentration of primers 6.5.7.3 RT-PCR: (Reverse and amount of the DNA templates added into Transcription-PCR) the reaction mixture. All the components of the During the last two decades PCR and RT-PCR reaction mixture are mixed in a PCR tube; the are superb techniques for the isolation of the further reactions are carried out on thermal cycler target DNA or cDNA in a relatively short time, machines specially designed for the PCR. avoiding many of the time-consuming aspects of PCR reaction involves three basic steps that traditional gene cloning procedures. This tech- include denaturation, annealing and extension. nique will help in detecting the virus in picogram Denaturation step is carried out to separate dou- levels in infected seed or any other plant tissues or ble helix strands, generally done at approximately for confirmation of DNA insertion in transgenic 90Ð95ıC followed by annealing where the tem- plants. The high specificity makes PCR a prime perature of the reaction is lowered to ensure candidate for development of tests that allows the binding of oligos (primers) to the target. not only the detection but also the identification Generally these temperatures are lowered such of specific genetic sequences of the pathogens. that the annealing temperature is below the Tm Sambade et al. (2000) for the first time have of the primers (temperature of dissociation of developed one-step RT-PCR amplification pro- primer duplexes). The final step is known as cedure providing highly specific complementary extension, where the temperature of the reaction DNA from the plant virus RNA and being used by is adjusted to approximately 72ıC where most of a number of researchers. Till recently, more than the Taq DNA polymerases have their maximum 200 viruses and viroids are detected using PCR. activity extending the primer by polymerisation Using RT-PCR, the ability of specific primer of dNTPs towards the 30OH end thus forming the pairs to amplify from individual seed samples new copy of the target DNA completely from the were evaluated for Bean common mosaic virus template DNA. Additionally, initial denaturation (BCMV), Bean common mosaic necrosis virus is done for longer time to ensure proper and clear in bean, Soybean mosaic virus in soybean denaturation of template DNA molecules. Also and Pea seed-borne mosaic virus (PSbMV) final extension is done to fill the protruded ends in pea. Potyvirus group Ð specific degenerate of the incomplete fragments. During each cycle, primers which have potential in identifying new one copy of template DNA is doubled so the total or unknown potyviruses were used for their 140 6 Detection of Plant Viruses in Seeds

Table 6.5 Application of PCR-based techniques in the diagnosis of seedÐtransmitted plant viruses and viroids Virus Crop Reference Bean common mosaic virus Cowpea Hao et al. (2003) and Udayashankar et al. (2010) French bean Saiz et al. (1994) Bean common mosaic virus (PSt strain) Mung bean Choi et al. (2006) Chrysanthemum stunt viroid Chrysanthemum Chung and Pak (2008) Cocoa swollen shoot virus Cocoa Quainoo et al. (2008) Cowpea aphid-borne mosaic virus Cowpea El-Kewey et al. (2007), Udayashankar et al. (2009), and Salem et al. (2010) Peanut Gillaspie et al. (2001)and Salem et al. (2010) Cowpea mottle virus Cowpea Gillaspie et al. (1999, 2000) Cucumber green mottle mosaic virus Cucumber Hongyun et al. (2008)andShangetal.(2011) Cucumber mosaic virus Cowpea Gillaspie et al. (1998a), Abdullahi et al. (2001), and Salem et al. (2010) Lupin Wylie et al. (1993) Peanut Dietzgen et al. (2001) Pepper Akhtar Ali and Kobayashi (2010) Pumpkin Tobias et al. (2008) Spinach Yang et al. (1997) Dahlia mosaic virus Dahlia Pahalawatta et al. (2007) Hosta virus X Hosta Ryu et al. (2006) Lettuce mosaic virus Lettuce Revers et al. (1999) and Peypelut et al. (2004) Pea seed-borne mosaic virus Pea Kohnen et al. (1992), Phan et al. (1997), and Klem and Lund (2006) Peanut clump virus Peanut Lee et al. (2004) Peanut mottle virus Peanut Gillaspie et al. (1994, 2001) and Dietzgen et al. (2001) Peanut stripe virus Peanut Gillaspie et al. (1994, 2001), and Dietzgen et al. (2001) Peanut stunt virus Peanut Dietzgen et al. (2001) Pepper chat fruit viroid Pepper Verhoeven et al. (2009) Piper yellow mottle virus Black pepper Hareesh and Bhat (2010) Plum pox virus Stone fruits Thomidis and Karajiannis (2003) Subterranean clover mottle virus Clover Njeru et al. (1997) Subterranean clover red leaf virus Clover Bariana et al. (1994) Sugarcane mosaic virus Maize Li et al. (2007) Wheat streak mosaic virus Wheat Jones et al. (2005) White clover mosaic virus Trifolium Lee et al. (2004) Zucchini yellow mosaic virus Pumpkin Tobias et al. (2008) and Simmons et al. (2011) detection in RT-PCR. Bariana et al. (1994)have by using suitable primer pairs, simultaneous developed RT-PCR assays for the detection of detection of different viruses in seed is possible Subterranean clover red leaf luteovirus and by multiplex RT-PCR (Chalam et al. 2005). From Subterranean clover stunt virus, and this system Australia, Bariana et al. (1994) have detected helps in detecting all well-characterised viruses CMV, CYVV, AMV, BYMV and SCMoV which known to infect subterranean clover. The RT- are seed transmitted in subterranean clover by PCR assay detected all isolates tested for each a cocktail assay which detected all five viruses virus and was up to five orders of magnitude both individually and collectively. more sensitive than ELISA. The real-time RT- RT-PCR is widely used for detection of RNA PCR method was also useful in detecting BCMV viruses in plants. In RT-PCR, PCR is done in and PSbMV in single seed. The use of molecular two steps: (1) reverse transcription and (2) PCR. technique has special significance in detecting Reverse transcription is a process where cDNA latent infections and the detection of viruses is synthesised to an RNA template by involving occurring in very low concentration. Also, an enzyme known as reverse transcriptase. This 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 141 process also requires an important component for the Qiagen method (Gillaspie et al. 2001; Pinnow cDNA synthesis, that is, a primer possibly an et al. 1990). Earlier this test has been employed OligodT for the viruses with poly-A tail or a ran- against Subterranean clover mottle virus in dom primer or a reverse primer of the PCR itself. clover seeds for the routine testing of bulked The most commonly used and available reverse seed samples (Njeru et al. 1997). Even in fruit transcriptases are from viruses like, Moloney crops like plum, the seed transmission of Plum murine leukaemia virus (M-MuLV) and AMV. pox virus was confirmed by PCR test (Pasquini In this mechanism, the RNA is initially dena- and Barba 2006). RT-PCR is applied to detect tured, along with the primer, and snap chilled the viruses in the infected seed samples of the to unwind the super secondary structures present following virusÐhost combination by different in RNA and also to aid the primer annealing. researchers: Arabis mosaic virus, Bean yellow Then the other components like dNTPs, RT buffer mosaic virus, Cucumber mosaic virus, Clover and RNAse inhibitors are added and the reverse yellow vein virus, Subterranean clover mottle transcription is carried out at a specific temper- virus (Bariana et al. 1994) and in certain legume ature, generally 42ıC for most of the RTs, and seeds (Bariana et al. 1994; Njeru et al. 1997), a final step of termination is held by heating the Pea seed-borne mosaic virus in pea (Kohnen reaction mix to higher temperatures to inactivate et al. 1991, 1995), Wheat streak mosaic virus RT. The cDNA can be used as a template in in wheat (Jones et al. 2005), Blackeye cowpea the next step PCR whose details are discussed mosaic viruses in cowpea (Udayashankar et al. earlier. In case of viruses where a poly-A tail 2009), Dahlia mosaic virus in Dahlia pinnata is not present, either a reverse primer of PCR (Pahalawatta et al. 2007)andPeanut stripe and or a random primer (probably a hexamer) is Peanut mottle viruses (Gillaspie et al. (1999). used for cDNA synthesis. Reports of usage of As the Cucumber mosaic virus in spinach is terminal dinucleotidyl transferases to generate carried by the pollen and embryo infection takes ‘poly’-A tails for cDNA synthesis is also there in place during pollination, for virus detection in case of certain types of ilar- and bromoviruses. pollen grains of CMV-infected spinach, RT-PCR This technique was used in detection of Cow- is successfully used (Yang et al. 1997). RT-PCR pea aphid-borne mosaic virus by Gillaspie et al. has been used to detect Potato spindle tuber (2001), specific detection of Lettuce mosaic virus viroid in true seeds of potato (Shamloul et al. isolates by Peypelut et al. (2004), differentiation 1997)andColeus viroid in Coleus scutellarioides of peanut seed-transmitted potyviruses and cucu- (Singh et al. 1991a). During Boben et al. (2007) moviruses by Dietzgen et al. (2001), detection of have applied the RT-PCR for the detection Broad bean stain comovirus (BBSV) and Cow- and quantification of Tomato mosaic virus in pea aphid-borne mosaic potyvirus (CABMV) in irrigation water. faba bean and cowpea plants by EL-Kewey et al. Application of PCR in detection of various (2007) and detection of ZYMV and CMV in plant virus diseases, both seed-transmitted and Cucurbita pepo by Tobias et al. (2008). non-seed-transmitted is well known after its in- Gillaspie et al. (2001) processed Cowpea vention in the mid-1980s. PCR is used for de- aphid-borne mosaic virus-infected peanut seeds tecting plant DNA viruses like caulimoviruses, by taking 2- to 4-mm seed slices, which included geminiviruses, badnaviruses and nanoviruses. the seed coat as well as cotyledons, and produced more reproducible results by RT-PCR, and this 6.5.7.4 Duplex RT-PCR test has also been tested with peanut against Dietzgen et al. (2001) have developed duplex RT- Peanut stripe virus in peanut seeds (Pinnow et al. PCR for the detection of two potyviruses, PStV 1990). Individual peanut seeds were subsampled and PeMoV, and the two cucumoviruses, CMV by a modified non-destructive technique in which and PSV, that are seed transmitted in peanut. a slice was removed from each seed distal to Inclusion of an immunocapture step in the duplex the radicle with razor blade and the slices from RT-PCR assays may overcome inhibitors of PCR several seeds were combined and extracted by present in peanut seed extracts. 142 6 Detection of Plant Viruses in Seeds

6.5.7.5 Nested PCR 6.5.7.8 LAMP (Loop-Mediated It is a variant of PCR designed to get ampli- Isothermal Amplification) fication of desired fragment specifically. In this It is a modified, efficient and specific method technique, the product of the first few cycles for the amplification of the DNA templates. The (using a first primer set) is used as a template for reaction is performed with two sets of primers the second set of nested primers that are designed containing two specific regions of the target se- to amplify the desired fragment. It is designed to quence for amplification (Notomi et al. 2000). increase the specificity of detection and also to The process starts with annealing of the first enhance the copies of viral nucleic acid in case of primer set for synthesis of the complementary low virus titres. This technique was successfully strand that forms a loop out structure for further used in Vitivirus and Foveavirus species detection amplification by second primer set. The ampli- in grape vines (Dovas and Katis 2003)andBeet fication is carried out at isothermal conditions necrotic yellow vein virus detection in sugar beet (65ıC for 1 h) with Bst DNA polymerase that (Morris et al. 2001). has strand displacement activity. Target amplifi- cation is detected by measuring the turbidity of 6.5.7.6 Multiplex PCR the reaction mixture. This was used efficiently This variant of PCR is targeted for detection for detection of TSWV from chrysanthemum of multiple viral pathogens in a single reaction. (Fukuta et al. 2004) by combining LAMP with Multiplex primers are designed specifically for IC-RT steps; LAMP was used in diagnosis of different viruses but with same annealing tem- plant viruses (Wang et al. 2008). peratures and different sized product amplicons. Care is also taken that no secondary structure 6.5.7.9 IC-PCR formations are there between these set of primers. Immunocapture PCR is another variant of PCR When a PCR reaction is carried out from the that combines the capture of the virus particles template having a mixture of viruses (mixed in- by antibodies coated in a PCR tube or micro fections), only the viruses that are present are tube plate. The bound antigen (virus particle) amplified. When these products are analysed, is then lysed in a buffer that will release the basing upon the sized fragment obtained, the total viral nucleic acid content from the virion viruses are identified. This is done in case of into the medium, and PCR is carried out by many viruses, for example, six citrus viruses (Roy addition of other components to the medium. et al. 2005). For the six citrus pathogens that This method is very sensitive and specific to include CMBV, CTV, CLRV, ICRSV, CVV and the virus and is free of contaminants like plant Citrus exocortis viroid, multiplex primers were polyphenolic enzymes that inhibit PCR (Varveri designed and used successfully. This technique 2000). Though, this technique is used for the was used in detection of five seed-transmitted viruses which had polyclonal or monoclonal legume viruses by Bariana et al. (1994). antiserum and has been worked out against detection of seed-transmitted viruses like Pea 6.5.7.7 CF-PCR seed-borne mosaic virus in pea seeds (Phan It is a modified method of PCR which could be et al. 1997) and also BCMV infection in cowpea used to differentiate between the different strains seeds (Udayashankar et al. 2010). Gillaspie of virus where 50 fluorescent dye-labelled oligos et al. (1999) used immunocapture reverse are used for amplification. Upon obtaining the transcription-polymerase chain reaction (IC- amplicon, the dye fluoresces only in a double- RT-PCR) for large-scale assay of Peanut mottle stranded hybrid. This technique is used chiefly virus (PeMV) and Peanut stripe virus (PStV) to differentiate the viruses with divergence in 30 and could be reproducibly detected by RT-PCR end nucleotide sequences. This was chiefly used from a single seed from 100 seed composites to differentiate the multiple strains of the potato using smaller-sized (approximately 1 mm) seed viruses (Walsh et al. 2001; Webster et al. 2004). material. 6.5 Biotechnology/Molecular Biology-Based Virus Diagnosis 143

6.5.7.10 Direct Binding PCR 6.5.8.1 TaqMan Assay It is a method similar to the IC-PCR, but it In the TaqMan assay system, the real-time PCR involves direct binding of the virus particles from is carried out with the help of a set of primers the crude plant sap or seed extract to the PCR specific for an amplicon and a TaqMan probe tube, washing of the unbound particle and debris, that is also complementary to the target ampli- lysis of viral particles in a medium and PCR con. TaqMan probes depend on the 50-nuclease detection of the target. Although this technique is activity of the DNA polymerase used for PCR to simple and affordable, rate of success and level of hydrolyze an oligonucleotide that is hybridised to detection is lower than that of IC-PCR for many the target amplicon (Rao and Singh 2008). of the virus hosts with heavy polyphenolics. The TaqMan probe consists of two types of fluorophores linked to the 50 and 30 ends of 6.5.7.11 Reverse the probe, which are the fluorescent parts of Transcription-PCR–DBH reporter proteins (green fluorescent protein, GFP During 2009, from Iran, Bagherian et al. have often-used fluorophore). While the probe is at- described a new method based on a combination tached or unattached to the template DNA, be- of RT-PCR and dot-blot hybridisation (RT-PCRÐ fore the polymerisation starts, the quencher (Q) DBH); in this method, instead of using nucleic fluorophore, usually a long-wavelength coloured acid extracted directly from the plants, RT-PCR dye, such as red, reduces the fluorescence from products are subjected to dot-blot hybridisation. the reporter (R) fluorophore (usually a short- By using this method, Hop stunt viroid (HsVd) wavelength coloured dye, such as green) by the and Citrus excortis viroid (CEVd) were detected use of mechanism known as fluorescence res- from Washington navel orange plant samples. onance energy transfer (FRET), which is the This RT-PCRÐDBH method is over 1,000-folds inhibition of one dye caused by another without more sensitive than Southern or Northern blot and emission of a proton. The reporter dye is found over 100-folds more sensitive than electrophore- on the 50 end of the probe and the quencher at sis of product. High seed transmission is noticed the 30 end. First the probe binds to amplicon in six viroid diseases of plants (Hadidi et al. by following Chargaff’s base pairing rule to the 2003). This method which provides a highly sen- template and the primer set as well. When the sitive and specific means of diagnostic detection amplification is initiated by the Taq DNA poly- of plant viroid diseases is cost-effective and has merase from the primer 30 ends, the TaqMan been tried (Weissensteiner et al. 2004). probe is degraded due to the 50-nuclease activity of the DNA polymerase thereby releasing the reporter dye and the quencher free. As FRET no 6.5.8 Real-Time PCR longer occurs, the fluorescence increases by each cycle, proportional to the amount of amplicon It is a modified method of PCR where quantifica- obtained/probe cleavage (Ha et al. 1996). tion can be done while the amplification is under process. This is called ‘real-time PCR’ because 6.5.8.2 Molecular Beacons it allows quantifying the increase in the amount Molecular beacons also use FRET to detect and of DNA as it is amplified. Several different types quantify the PCR amplicons via a fluor coupled of real-time PCR systems are available having to the 50 end and a quench attached to the 30 their own advantages and disadvantages. RT-PCR end of an oligonucleotide. They slightly differ is performed on a real-time machine that has a from TaqMan probes as they are designed to function of both thermal cycling and also quan- remain intact during the amplification reaction titative fluorimetry that will quantify the amount and must rebind to target in every cycle for signal of DNA as it is amplified during the process (Rao measurement. Molecular beacons form a stem- and Singh 2008). loop structure when free in solution. So the fluor 144 6 Detection of Plant Viruses in Seeds and quencher molecule come nearer that prevents results in a wrong quantification of the target the probe from fluorescing. When a molecular concentration. For single PCR product reactions beacon is denatured and hybridised to a target, with well-designed primers, SYBR green can the fluorescent dye and quencher are separated, work extremely well, with spurious non-specific FRET does not occur, and the fluorescent dye background only showing up in very late cycles emits light upon irradiation. Molecular beacons, (Ma et al. 2006; Rao and Singh 2008). like TaqMan probes, can be used for multiplex The same real-time PCR principle can be used assays by using spectrally separated fluor/quench to detect and quantify the presence and titre of moieties on each probe. As with TaqMan probes, viruses in plant samples as well as the seed molecular beacons can be expensive to synthe- samples in case of seed-transmitted plant viruses. sise, with a separate probe required for each Real-time immunocapture RT-PCR was used for target (Ma et al. 2006; Rao and Singh 2008). detection of Pepino mosaic virus on tomato seed by Ling (2007). Multiplex real-time fluorescent 6.5.8.3 Scorpion Probes reverse transcription-polymerase chain reaction With scorpion probes, sequence-specific prim- was developed for detection of Potato mop top ing and PCR product detection is achieved us- virus and Tobacco mottle virus (Mumford et al. ing a single oligonucleotide. The scorpion probe 2000a), Pepino mosaic virus (Ling 2007)and maintains a stem-loop configuration in the un- Cherry leaf roll virus (Jalkanen et al. 2007). hybridised state. The fluorophore is attached to the 50 end and is quenched by a moiety coupled to the 30 end. The 30 portion of the stem also 6.6 Molecular Markers contains sequence that is complementary to the extension product of the primer. This sequence is Molecular markers are any kind of molecule linked to the 50 end of a specific primer via a non- indicating the existence of a chemical or a amplifiable monomer. After extension of the scor- physical process. Molecular markers include pion primer, the specific probe sequence is able biochemical constituents (e.g. secondary to bind to its complement within the extended metabolites in plants) and macromolecules (e.g. amplicon thus opening up the hairpin loop. This proteins and deoxyribonucleic acid). Strauss et al. prevents the fluorescence from being quenched (1992) distinguished the molecular markers into and a signal is observed. The advantage of this two classes. Biochemical molecular markers configuration is that the unimolecular nature of derived from the chemical products of gene the primerÐprobe scorpion probe allows for faster expression, that is, protein-based markers and hybridisation kinetics. This may be useful for molecular genetic markers derived from direct high-volume screening assays (Ma et al. 2006; analysis of polymorphism in DNA sequences, Rao and Singh 2008). that is, DNA-based markers. DNA markers can be used to diagnose the presence of the gene 6.5.8.4 SYBR Green Dyes without having to wait for gene effect to be seen. SYBR green provides the simplest and most The marker-assisted selection (MAS) permits economical source for detecting and quantifying the breeder to make earlier decisions about the PCR products in real-time PCR reactions. SYBR further selections while examining the fewer green binds double-stranded DNA and upon exci- plants. An added advantage in breeding for tation emits fluorescence. Thus, as a PCR product disease resistance behaviour is that this could accumulates, fluorescence increases. The advan- be done in the absence of pathogen including tages of SYBR green are that it is inexpensive, viruses once marker information is available. easy to use and sensitive. The disadvantage is that Many plant viruses are recognised as SYBR green will bind to any double-stranded emerging or re-emerging or new-emerging DNA in the reaction, including primer-dimers viruses which have to be studied rapidly and and other non-specific reaction products, which thoroughly for their aetiology, ecology and References 145 epidemiology. Emergence of new strains or new seed-transmitted viruses is a major thrust area of 6.7 Conclusions research since Brown’s development of molec- Molecular diagnostic assays should be viewed ular marker to track the movement of viruses in as tools to manage plant virus diseases. They the plant system and attempts were made in this are not designed to be used in a vacuum, but regard to locate genes conferring resistance to certain seed-transmitted viruses. For example, rather in conjunction with a knowledge of symp- Timmerman et al. (1993) determined the location tomatology, varietal responses to viruses and en- of Sbm-1 on the Pisum sativum genetic map by vironmental effects on disease development. As such, they will play a key role in crop man- linkage analysis with eight synthetic molecular agement systems, permitting accurate and early markers. Analysis of the progeny of two crosses detection of virus and viroid diseases and help confirmed that Sbm-1 is on chromosome six. The inclusion of Fed-1 (encoding ferrodoxin- in more efficient implementation of effective con- 1) and Prx-3 (encoding peroxidase) among the trol measures. markers facilitated the comparison of this map with the classical genetic map of pea. The Sbm- References 1 gene is most closely linked to RFLP marker GS 185 as a marker for Sbm-1 in breeding Abdullahi I, Ikotin T, Winter S, Thottappilly G, Atiri GI (2001) Investigation on seed transmission of cucumber programmes. The GS 185 hybridisation pattern mosaic virus in cowpea. Afr Crop Sci J 9:677Ð684 and virus resistance phenotypes were compared Abu Samah N, Randles RW (1983) A comparison of in a collection of breeding lines and cultivars. Australian bean yellow mosaic virus isolates us- In recent years, Smykal et al. (2010)have ing molecular hybridization analysis. Ann Appl Biol 103:97Ð107 followed marker-assisted pea breeding against Adams DB, Kuhn CW (1977) Seed transmission of peanut PSbMV and reported that resistance to PSbMV mottle virus. Phytopathology 67:1126Ð1129 is conferred by a single recessive gene e1F4E Afanasiev MM (1956) Occurrence of barley stripe mosaic marker localised on LG IV (Sbm1 locus). Even in Montana. Plant Dis Rep 40:142 Agarwal VK, Nene YL, Beniwal SPS, Verma HS (1979) in the breeding programmes of soybean, where Transmission of bean common mosaic virus through the Soybean mosaic virus is one of the major urdbean (Phaseolus mungo L.) seeds. Seed Sci Tech- problems, the molecular markers of all the three nol 7:103Ð108 resistance genes have been developed based on Agindotan B, Perry KL (2008) Macroarray detection of eleven potato-infecting viruses and Potato spindle tu- fine mapping with several molecular techniques ber viroid. Plant Dis 92:730Ð740 such as restriction fragment length polymorphism Agindoton B, Perry KL (2007) Macroarray detection (RFLP), random amplified polymorphism of plant RNA viruses using randomly primed and DNA (RAPD), amplified fragment length amplified complementary DNAs from infected plants. Phytopathology 97:119Ð127 polymorphism (AFLP), simple sequence repeats Ahlawat YS (2010) Diagnosis of plant viruses and allied (SSR) and single-nucleotide polymorphisms pathogens. Studium Press (India) Pvt Ltd, New Delhi, (SNP) (Yu et al. 1994; Hayes et al. 2000;Shi p 224 et al. 2008; Jeong et al. 2002; Jeong and Saghai Akinjogunla OJ, Taiwo MA, Kareem KT (2008) Immuno- logical and molecular diagnostic methods for detection Maroof 2004; Hwang et al. 2006; Shi et al. of viruses infecting cowpea (Vigna unguiculata).AfrJ 2008). Molecular markers were also used in Biotechnol 7(13):2099Ð2103 identifying the resistance genes for virus diseases Albrechtsen SE (2006) Testing methods for seed transmit- in vegetatively propagated crops, as seen in ted viruses: principles and protocols. CABI Publish- ing, Wallingford, pp 1Ð268 tristeza virus resistance programme in Poncirus Alconero R, Hoch JG (1989) Incidence of pea seed borne trifoliata (Mestre et al. 2007). Quarantines must mosaic virus pathotypes in the US national Pisum be strengthened by molecular marker methods to germplasm collection. 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Abstract This chapter describes how knowledge of epidemiology helps in under- standing the seed-transmitted virus disease spread and in framing suitable management measures. Seed infection is epidemiologically important as this is the primary source of inoculum and forms the starting point for the initiation of the disease. The epidemics of the virus diseases in a particular region are the result of complex interactions between various physical, chemical and biological factors, and major epidemics occur when conditions influencing the virus, host and its vector synchronise. The virus disease epidemics depend on the interaction of four components, namely, the pathogen, the vector, the plant and the environment. Among the seed-transmitted viruses, some have limited host range and some others have wide host range. Either annual or perennial or both are affected with virus diseases. Under field conditions, the weed and wild hosts are important as they are the reservoirs for the virus, vector or both. Survival of the virus in the seed for a long period also plays an integral role in virus perpetuation in off seasons. Seed-transmitted viruses are also vector specific, and aphid or beetle or nematodes or fungi play major role in causing epidemics based on the time of emergence, light, humidity, temperature and wind velocity, and abundance of vector population is a significant factor that determines spread of virus diseases both in time and space. Vectors also may have limited host plants or may be polyphagous. The time and number of vectors visiting the crop varies considerably with species over years. Pollen and seed transmission are closely related factors in virus epidemiology. A number of virus diseases spread horizontally through pollen in a number of fruit and vegetable crops. The epidemiology of virus diseases also depends on different pathogen strains which vary in virulence, host range and transmissibility. If the forecasting system is developed against a particular virus disease, it will help to operate an early warning system or to select growing season or areas for crop growing. Examples of how epidemiological information can be used to develop effective integrated disease management strategies for diverse situations are described.

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 7, 165 © Springer India 2013 166 7 Ecology and Epidemiology of Seed-Transmitted Viruses

has no known vector but still present in most 7.1 Introduction barley-producing areas of the world. No weed or wild grass is a significant reservoir of this Epidemiology of seed-transmitted viruses pro- virus, but seed transmission in a single plant vides a scientific modern theory of epidemic species, Hordeum vulgare L., is the most impor- development. Vander Plank (1963, 1982) has pro- tant factor for survival from year to year (Timian vided a unifying concept for its quantitative study 1974). In the United Kingdom, for the two beetle- and the use of mathematical models to describe, transmitted viruses, Broad bean stain and Broad interpret and predict the progress of epidemics bean true mosaic, which are seed transmitted, insight for effective disease management based the infected seed is the main source of spread on certain principles like environmental factors, for these viruses in spring-sown field bean crop sources of infection, vectors and their popula- (Cockbain et al. 1975). Seed-transmitted viruses tion dynamics. The value of epidemiological ap- transmitted by aphids are also of great concern proach lies in its ability to stimulate questions as infected seed serves as the primary source on ‘How many of the viruses are seed trans- of inoculum. Some of the most economically mitted? How are they spreading? How much important plant virus diseases of this category infection starts from sources other than seeds?’ a r e BCM V, L M V, CM V, PM V, PSt V, SM V a n d Vander Plank (1963, 1982) suggested that the Black eye cowpea mosaic viruses which are ex- relationship between the amount of initial in- tensively studied at different virusÐhost combi- oculum (Xo), average infection rate (r) and the nations (Irwin et al. 2000; Jones 2000;Sharma time (t) over which infection occurs determines et al. 2004; Dinesh et al. 2005; Coutts et al. 2009; the amount of disease (x) that develops. The Udayashankar et al. 2010). equation X D Xo ert helps in determining the rate The perusal of the Table 1.2 indicates that a of disease at a given moment of time. Zadoks number of seed-transmitted virus diseases occur and Schein (1979), Leonard and Fry (1986)and in majority of crop plants cultivated under varied Khetarpal and Maury (1998) have considered dis- environmental conditions. The epidemics of these ease control from an epidemiological standpoint virus and viroid diseases in a particular region and a primer as well as a base in advanced studies. are the result of complex interactions between various physical, chemical and biological fac- 7.1.1 Primary Inoculum Source tors, and major epidemics occur when condi- tions influencing the virus, host and its vector Seed infection is epidemiologically important be- synchronise. Hence, it is imperative to collect cause this is the primary source of inoculum information on different aspects like the nature and forms the starting point for the initiation of the virus, the percent seed infection as the of the disease. It ensures virus association with primary inoculum and proximity of the host crop the planted crop. As the infected seeds are ran- to the vector, the number and timing of vector domly dispersed in the field, the infected dis- appearance, the diurnal temperature range, the persed seedlings serve as sources of inoculum for rains, the RH of the air, the dew, the weed hosts secondary spread. When the infected seedlings of the vector and virus and the duration of the are the only virtually source of inoculum, seed crop and the nature of the produce fruit, seed transmission plays a critical role in virus epi- and tuber. This will help to draw reasonable pre- demiology as the seed-transmitted viruses appear dictions about disease epidemics and formulate to be the sole and or the primary source of suitable management measures. In a nutshell, the inoculum. In considering the inoculum threshold epidemiology of seed-transmitted virus diseases of seed-transmitted viruses (i.e. the maximum like in any other pathogenic diseases depends amount of inoculum that can be tolerated), this on interaction of four components: the pathogen, aspect is by far the most significant. For example, the vector, the plant and the environment. Exten- BSMV being seed transmitted to a high degree sive and detailed epidemiological studies made 7.2 Host Plant 167 in many crops over long periods have shown highly susceptible to viruses if extensively culti- the highly complex pattern of interacting factors vated with high rate of seed infection, and vector in the spread of seed-transmitted viruses. More presence results in maximum disease incidence. information can be obtained from review articles Among the seed-transmitted viruses, some and textbook chapters (Thresh 1974; Jones 2004; have limited host range like SMV, and others Maramorosch et al. 2006). have a wide host range (Irwin and Goodman 1981). Similarly, BCMV- and LMV-infected seedlings raised from the seed are considered 7.2 Host Plant to be the primary source for initiating new infections at the beginning of each season. New crops have been introduced for the first time These viruses spread to other plants through in new regions for their high yields and crop vectors, and plants will subsequently act as uniformity. These introductions will flourish in secondary sources of infection depending on their the new environment, where they will be free for susceptibility. In contrast, some seed-transmitted some time from virus diseases that were prevalent viruses of cucumovirus, tobravirus and nepovirus in the country of origin. In other instances, catas- groups have a very large number of natural and trophic losses have occurred when exotic crops experimental hosts comprising of annuals and were soon attacked by indigenous pathogens not perennials. For example, CMV can infect over previously encountered in the country of origin. 470 species of at least 67 families (Kaper and For example, when ‘Fasolt’, a hybrid cultivar of Waterworth 1981). Similarly, Tobacco rattle Brussel bread from Holland was introduced into virus has more than 400 experimental hosts England, it was severely affected by Turnip mo- belonging to 50 mono- and dicotyledonous saic and Cauliflower mosaic viruses (Tomlinson families (Schmelzer 1957). About 237 members and Ward 1981). of Gramineae have been shown as experimental The host plants and the type of vector involved hosts of various strains of BSMV (Jackson and also influence the efficiency of virus transmission Lane 1981). Arabis mosaic virus infects 93 out of as in the case observed by Jansen and Staples 136 species in 28 families and Tomato black (1970) with beetle-transmitted Cowpea severe ring virus has a host range of 76 species in mosaic virus (CpSMV). Both cowpea and soy- 25 families (Schmelzer 1963). Wherever the bean are susceptible when mechanically inoc- virus has a wide host range, the source of ulated. Beetles, however, transmitted this virus virus inoculum was necessarily another crop from cowpea to cowpea at high levels of effi- plant. For example, in Southern USA, peanut ciency but at very low level to soybean. CpSMV infected with PMV not only serves as a source was observed causing a very severe disease in of infection but spreads to nearby soybean crop soybeans in the fields at Puerto Rico (Thomg- (Demski and Kuhn 1974) and infected lupins meearkom et al. 1978) and Brazil (Anjos and Lin after peanut crop (Demski et al. 1983). Even 1984). The disease, however, occurred only in in beet and potato crops, a number of seed- few scattered soybean plants immediately adja- transmitted viruses are recorded, and the tubers cent to cowpea planting which was severely in- that escape during harvest or grown in waste fected with CpSMV. Apparently, the inefficiency fields serve as a source for the virus, the vector or of transmission of the virus by beetles from both (Wallis 1967). cowpea to soybean or soybean to soybean had Under field conditions, the weed and wild restricted the disease spread in soybean. hosts are important as they are the reservoirs for The growth habits of the crop plants vary in the virus, vector or both (Duffus 1971;Murant terms of height and lifetime, that is, annual or 1981a, b;Sastry1984a;Bos1981;Thresh1981); perennial. The transmission of seed-transmitted aid in virus perpetuation during the main or viruses has been recorded both in annual and off season; serve as inoculum source; and exert perennial crops (Table 1.2). Annual crops are great infection pressure. The survival strategies of 168 7 Ecology and Epidemiology of Seed-Transmitted Viruses viruses are just as diverse as their shape, size and nous herb Tephrosia villosa is a common host physicochemical characteristics. Harrison (1981) for CMMV in coastal Kenya (Bock et al. 1981). distinguished between ‘WILPAD’ viruses from Bean common mosaic was detected in legume tobravirus and nepovirus groups that seem par- weed Rhynchosia minima (Meiners et al. 1978). ticularly well adapted for survival in wild plants At Washington, PSbMV overwintered in hairy and ‘CULPAD’ viruses including members of vetch (Vicia villosa) and volunteer pea plants the tobamo and potex groups which seem to under field conditions (Stevenson and Hagedorn thrive best in cultivated plants. A majority of 1973). In South Carolina, pigweed (Amaranthus the seed-transmitted viruses infecting economic hybridus) formed the source of inoculum for crops have weed hosts widely which are dis- TRSV in squash crop (Sammons and Barnett tributed along irrigation channels, ditches, road 1987). In Hungary, the role of weed host on the sides, orchards, by the side of railway tracks, ecology of CMV in beans (P. vulgaris) has been in fallow lands and in neglected crops. Infected well studied by Horvath (1983). perennial weeds are more dangerous than annuals Hani (1971) demonstrated potentiality of the since they live longer. The chances of outbreak inoculum due to host and crops. It was noticed of disease epidemics will be more in areas where that the rates of infection of Stellaria media by infected weeds are abundant and support the mul- CMV were higher in certain areas of Switzerland tiplication of a particular vector. The importance with continuous cultivation of tobacco than in of source of virus near or especially within a regions with regular crop rotation. But the infec- crop has been demonstrated for many virusÐcrop tion of tobacco in each year mainly originated combinations (Heathcote 1970;Duffus1971;Bos from S. media, wherein the virus was also seed 1981;Sastry1984a, b; Jones 2004; Maramorosch transmitted. In fields where tobacco was grown et al. 2006). for the first time, the degree of S. media infection Within plantations, the infected weeds are par- rapidly increased during the season. Even for ticularly important since they exert the greatest Tomato ring spot virus which is seed transmitted infection pressure. Biennial or perennial weeds in peach and apple, the weed hosts like Tarax- play a significant role in facilitating the survival acum officinale, Rumex acetosella and Stellaria of viruses that attack annual plants in areas with sp. proved to be important sources of infections in growing seasons restricted by prolonged drought Indiana, New York and Pennsylvania states in the or cold. Examples of weed hosts as reservoirs USA, respectively (Mountain et al. 1983;Powell of seed-transmitted viruses are many. Tomlin- et al. 1984). son et al. (1970) reported that in Britain, CMV In certain cases, the weed hosts not only overwinters in chickweed (Stellaria media), a served as sources of virus infection during the source of infection for the following season’s let- crop season but also for the virus to overwinter tuce crop. CMV also overwintered on weeds like and aid in their dispersal to long distances Echinocystis sp. (9Ð55%) (Doolittle and Gilbert through man, wind and water. A large number 1919; Doolittle and Walker 1925), Stellaria sp. of viruses are thus transmitted through the seed (21Ð40%), Spergula sp. (2%) and Cerastium sp. of weed hosts which act as a major factor in the (2%) (Tomlinson and Carter 1970a, b). In Aus- ecology of seed-transmitted virus diseases. This tralia, lupin and clover infected by CMV strain factor enhances the chances of virus survival persist between growing seasons in eight alter- throughout the year and helps in virus spread native host species (McKirdy and Jones 1994). from crop to crop through the vectors. As early In Europe, overwintering groundsel (Senecio vul- as 1919, Doolittle and Gilbert emphasised the garis) was implicated as the main source of LMV role of wild cucumber (Echinocystis lobata) in lettuce, and the infected plants survived in the as a source for the spread of CMV which is winter (Kemper 1962). In USA, the leguminous also seed transmitted in this host. To cite a few weed Desmodium canum is the alternate host of more examples, TRSV was carried in the seed PMV (Demski et al. 1981). The woody legumi- of dandelion weed T. officinale to an extent 7.2 Host Plant 169 of 9Ð36% and caused severe mosaic diseases ring virus in 11 symptomless weed species, and of soybean and other crops (Tuite 1960), and many of these hosts also contained Raspberry the same virus in South Carolina was carried ring spot virus. Tuite (1960) recorded TRSV in the seed of pigweed, Amaranthus hybridus, in two symptomless weed hosts. AMV and to an extent of 21% (Sammons and Barnett Strawberry latent ring spot virus were also 1987). Murant and Lister (1967) listed several detected in a number of symptomlessly infected nepoviruses and the hosts in which they were weed species (Taylor and Thomas 1968). A seed transmitted. For example, four viruses were similar situation was recorded for CMV and carried by seed of infected Stellaria media plants LMV in a number of weed hosts (Ullrich 1954; (Arabis mosaic, Raspberry ring spot, Strawberry Kemper 1962; Tomlinson et al. 1970). ring spot and Tomato black ring viruses), two Even in wild germplasm, the virus diseases each by the seed of Capsella bursa-pastoris are carried through the off season, and their and of Chenopodium album (Arabis mosaic and spread through the exchange and transport from Tomato black ring viruses). It was also proved each country has been proven beyond doubt. For that even tobra group of viruses were carried example, true seed of Solanum spp. is infected through the seed of S. media and Myosotis with Andean potato latent virus (Jones and Fri- arvensis, but the extent of seed transmission bourg 1977), Potato virus T (Salazar and Harri- was very low (Murant 1970). son 1978)andPotato spindle tuber viroid (Diener As the virus is retained in the seed of certain and Raymer 1971). The reader can refer to the weeds over long periods, the chances of virus exhaustive compilation made in ‘Plant health and survival are more in the off season and also when quarantine in international transfer of genetic the crop is grown intermittently. In UK, the weed, resources’ (Hewitt and Chiarappa 1977). The risk Stellaria media, retained CMV for 21 months in of spreading viruses even between continents buried seed (Tomlinson and Walker 1973). Hani through the exchange of wild plants by botan- (1971) reported that the virus was infectious in ical gardens, germplasm collectors and through the same virusÐhost combination in the seed for breeding nurseries is very high due to latent 18 months. Even Tomato black ring and Rasp- infection and also lack of knowledge among the berry ring spot viruses were retained in seeds of in-charge personnel of botanical collections. weeds Capsella bursa-pastoris and S. media for Besides acting as a source of virus infection, over 6 years (Lister and Murant 1967). However, weeds and wild plants also act as breeding foci in the case of nematode-transmitted viruses, they for the vectors. In the Pacific Northwest, USA, were retained in the vector Longidorus spp. for overwinters on peach trees in only 8Ð9 weeks, and the presence of infected the egg stage and on weeds in drainage ditches weed seeds was essential for longer survival of as viviparous forms (Wallis 1967). Some of the some of the nepoviruses. However, viruses trans- nematode vectors like Longidorus elongatus mul- mitted by Xiphinema spp. were retained in the tiply in large numbers on some of the weed hosts vector for many months, and the role of weed like S. media, Poa annua and Mentha arvensis hosts seemed to be less important as the infected (Taylor 1967; Thomas 1969). In India, some of woody perennial hosts served as source of inocu- the weed hosts like B. arvensis harbour four to lum (Murant 1970). five aphid species like M. persicae, A. gossypii, Detection and management of seed-transmitted Brevicoryne brassicae and Rhopalosiphum pseu- viruses are more difficult when carried in dobrassicae. These are major vectors of mosaic the crop and weed plants without exhibiting diseases and affect economic crops in which any symptoms of the disease. For example, the virus is seed transmitted (Sastry 1984a, b). viruses like BSMV in barley and wheat and Similarly, Sorghum arundinaceum was a natural Avocado pear sunblotch in avocado were host for both Peanut clump virus and its fungal often symptomless (Neergaard 1977). Similarly vector Polymyxa graminis (Thouvenel and Fau- Murant and Lister (1967) detected Tobacco black quet 1981a, b). 170 7 Ecology and Epidemiology of Seed-Transmitted Viruses

7.3.1 Factors Influencing Vector 7.3 Vectors Movement Vector biology and behaviour are of paramount importance, since they have a profound influence Temperature, humidity, wind velocity and sea- on the temporal and spatial spread of virus dis- son are some of the factors which influence the eases. The extent of virus spread mostly depends vector movement. Generally, increase in relative on the density and movements of the vector popu- humidity to a higher level (above 80%) and high ı lation. These movements may be of a local nature temperatures (90 F) slowed down the flight ac- like between plants in a field or of dispersive type tivity of the aphids, while low temperature also from breeding areas/overwintering sites to crops. discourages aphid flights. Light intensity between Factors responsible for these movements involve 100 and 1,000 f.c. made little differences in flight, biotic factors like life history of the insect, its but below 100 f.c., flight activity declined rapidly. range and host preference. Long-distance dispersal of the viruliferous vec- Aphids are the vectors for a sizeable number tors takes place through wind currents. The flights of seed-transmitted viruses. Their host range, of the winged aphids would be discouraged when the time of emergence and abundance of wind speed is above 1 m/s (Carter 1962;Eastop winged forms among the population are the 1977). The importance of low level of jet winds in significant factors that determine spread of transporting aphids over a long distance has been aphid-transmitted virus diseases both in time related to disease outbreaks. Johnson (1967) sug- and space. Aphid species are mostly specific to gested that certain nonpersistent seed-transmitted one or several closely related host plants, while viruses can also be transported over relatively a few are polyphagous and infest hundreds of long distances of 60 km and that they might be plant species. Spread of nonpersistent viruses transported three times more distance on non- is greatly affected by the flight behaviour of stop flights. Planting over areas of upward wind aphid vectors. The take off of aphid vectors takes rather than downward wind helps in reducing the place either from the surface of the leaf or by virus spread. the insect dropping and kicking itself into flight. The total vector population and per cent in- Take off is influenced by light and temperature fective individuals in the population also play and does not occur at wind speed above 3 miles an important role in the spread of disease. For per hour (Kennedy and Booth 1963; Kennedy example, in Eastern Scotland, Apion vorax,anef- et al. 1962). ficient weevil vector of Broad bean stain (BBSV) The presence of an efficient beetle vector can and Echtes ackerbohnen mosaik viruses (EAMV be sufficient in spread of the virus resulting in se- syn. Broad bean true mosaic), is absent, and rious crop losses, even though seed transmission hence, no detectable spread of BBSV and EAMV levels are very low. In Eastern Scotland, seed- was observed. Therefore, there is a possibility of transmitted infection of Broad bean stain and producing Vicia faba seed free from these two Broad bean true mosaic viruses in commercial viruses with little effort in this area (Jones 1980). broad bean fields showed low levels of diseased However, the cumulative effect of a large popu- plants and no detectable further virus spread lation and high percentage of viruliferous vectors to healthy beans because of low population of causes catastrophic results. In California, where weevil vector, A. vorax (Jones 1978). In Southern aphids were numerous, lettuce seed with as low England, however, where A. vorax is present, the as an infection rate of 0.1% and below resulted in secondary spread of virus resulted in up to 90% high LMV infection (Zink et al. 1956). Similarly infection of these two viruses (Cockbain et al. in India, about 15% seed transmission of CpBMV 1975). resulted in 100% incidence in kharif and 27% in 7.3 Vectors 171 summer, supporting the role of aphid vectors in vectors P. graminis. Hence, the solarisation two different seasons with the same amount of effectively reduces clump disease. Even well- initial inoculum (Sharma and Varma 1983). cultivated soils profusely irrigated and covered In contrast, soil-borne virus diseases tend to with two layers of transparent polyethylene spread slowly after their initial appearance due sheets for at least 70 days during summer months to limited mobility of their vectors, namely, the reduce PCV incidence (Reddy et al. 1988). Dorylaimaida species of nematode Xiphinema, The timing and number of vectors visiting Trichodorus, Paratrichodorus and Longidorus; the crop vary considerably with species over the vectors of tobra and nepo group of viruses were years. For instance, Irwin and Goodman (1981) unable to carry the viruses to longer distances. and Irwin et al. (2000) while working with SMV Their migration is about 30 cm per year, and in soybean observed higher landing frequency of hence, the virus spread takes place either through Rhopalosiphum padi in spring 1976, 1977 and the infected vegetative planting material or 1978, but still, landing frequency was higher in through seed and pollen. Even the soil type the fall. Even landing rates of also can help in predicting the occurrence of were high in the spring of 1976 but low through- nematode-transmitted viruses like Tobacco rattle out the 1977 and 1978 seasons. In contrast, land- virus (TRV) with reasonable accuracy. In soil ing rates of Rhopalosiphum maidis were always surveys conducted in Britain, nematode vector, higher in the summer and fall and that of M. Trichodorus premitivus, occurs predominantly persicae and M. euphorbia were relatively low in sandy loams, whereas Paratrichodorus and concentrated in the middle of the season. It pachydermus was most prevalent in loamy was also observed that the SMV spread from the sand soils (Alphey and Boag 1976). In arable source outward was greater in downwind than soils of Scotland, about 80% of the trichodorid upwind. Since different species of vectors that population was found to carry TRV (Cooper transmit the seed-transmitted viruses alight with 1971). Similarly, Longidorus macrosoma occurs different frequencies at different times during the in clay with flint and alluvial soils in Southern growing season, a timing factor must be included England, and Longidorus attenuatus is found in to estimate economic losses. sandy soils mainly in East Anglia (Taylor and In virus epidemiology, accurate measurement Brown 1976). Even the soil temperature and of vector numbers and species alighting on plant soil moisture have an impact on movement and foliage is most essential. At present, different feeding of nematode vector. At low soil moisture, types of techniques such as use of yellow pan the nematodes are immobilised, and in dry soils, trap, vertical nets, cylindrical sticky thread they are killed due to desiccation, which is more trap and the JohnsonÐTaylor suction trap are likely to occur in the surface layers of soil than employed to monitor the vectors, depending on at considerable depths where a large proportion the crop growth and vector behaviour. The traps of the nematode population is found (Harrison should be mounted in such a way that they can 1977, 1981). be maintained at canopy level throughout the Even against fungal vectors like Polymyxa crop-growing season. graminis, the vector of Peanut clump virus In epidemiological studies, ELISA application (PCV), temperature plays important role in vector is essential for detecting the virus in the vec- transmission; when temperatures are less than tors. For example, Gera et al. (1978, 1979)re- 25ıC, only negligible PCV incidence is recorded. ported detection of CMV in single apterous Aphis The high temperatures that prevail in India and gossypii by ELISA. Aphids probing on plants West Africa during the main peanut-growing infected with a non-transmissible strain of CMV season (monsoon) are conducive to natural virus fail to acquire CMV,and short feedings of 1.5 min transmission. High temperatures during summer on healthy plants render viruliferous aphids non- in India followed by monsoon rains appear to reactive in ELISA tests. These results suggest the break the dormancy of resting sporangia of fungal use of ELISA for CMV epidemiological studies 172 7 Ecology and Epidemiology of Seed-Transmitted Viruses

(Gera et al. 1978). However, ELISA application size, morphology, viability and pollen tube size to winged aphids which acquire and carry the have been recorded (Yang and Hamilton 1974). virus under natural conditions has been demon- High level of pollen sterility resulting in poor strated in certain virus-host combinations. fertilisation has been observed in certain virusÐ host combinations (Ryder 1964). Pollen-carrying insects primarily honey bees 7.4 Pollen Transmission play a major role in transfer of virus-infected pollen to the stigma of flowers. Mink (1983) Infected pollen plays an integral role in the spread indicated the possible role of honey bees for long- of viruses of some woody plants. This topic distance spread of PNRSV from California to has been reviewed by the number of workers, sweet cherry orchards in Washington. namely, Shepherd (1972), Hardtl (1978), Phatak Generally, a greater percentage of seed- (1980), Mandahar (1981, 1983, 1985, 1986) transmitted virus transmission occurs when the andMink(1993). Transmission of plant viruses mother plant is infected than when pollen is the through pollen takes place both vertically and sole source of infection. Walter et al. (1992) horizontally. In case of vertical transmission of reported high percentage of seed transmission of viruses, the pollination and fertilisation of healthy Tobacco streak virus in bean plant when anthers ovules by infected pollen result in the formation from infected plants were used to pollinate of infected seeds which on germination produce healthy plants. infected seedlings. For effective horizontal spread In Europe,Rubus spp. particularly R. through infected pollen, the viruses invade and occidentalis and R. idaeus are infected with systematically infect the ovule-bearing mother Black raspberry latent ilarvirus Syn. Tobacco plant, and large quantities of infected pollen streak ilarvirus and are pollen and seed are released to infect the contemporary healthy transmitted. Virus transmission by pollen from plants. The virus transmission through pollen imported Rubus to local plants or propagation of is economically important in cross-pollinated imported Rubus would be the practical means of woody perennial plants than with annual crops establishment in Rubus in the EPPO region and wherein both vertical and horizontal transmission is considered as quarantine pest (OEPP/EPPO take place. 1994). In India, Tobacco streak virus (TSV) Transmission of certain seed-transmitted plant occurs in high incidence in sunflower, groundnut, viruses through pollen is prevalent in bromovirus, mung bean, soybean and some other crop and alfamovirus, cucumovirus and ilarvirus groups. weed hosts. The intensive seed transmission Virus particles are externally or internally pollen studies carried out by Prasada Rao et al. (2009) borne and have been observed in electron mi- have indicated that seed transmission was not crographs of infected pollen or pollen extracts noticed in any of the hosts tested. However, TSV as with BSMV (Gold et al. 1954; Carroll 1974), is transmitted in India through pollen assisted by TRSV (Yang and Hamilton 1974), TMV (Hamil- thrips. To quote few more examples of pollen ton et al. 1977) and PNRSV (Kelley and Cameron transmission, Vertesey (1976) demonstrated that 1986). The entry of the virus from the pollen in Montmorency sour cherry, seed transmission into the ovules takes place along with the male of PNRSV was 28% with pollen, 53% with gametes which move through the pollen tube that mother plant infection and 88% with pollen grows into the embryo sac. Of the two male and mother plant-combined infection. Similarly, gametes infected, one unites with the egg during Timian (1967) observed a high percentage (58%) fertilisation and the other unites with polar nuclei of seed transmission when both male and female giving rise to endosperm. Even Potato spindle barley parents were infected with BSMV, moder- tuber viroid (PSTVd) is pollen transmitted in ate (46%) with female parent and low (17%) with potato, tomato and Scopolia sinensis (Fernow male parent. This may become more significant et al. 1970; Kryczynski et al. 1988). The adverse if commercial hybrids are developed from male effects of virus infection on pollen in terms of its sterile barleys. Similar results were recorded 7.4 Pollen Transmission 173 earlier by Medina and Grogan (1961) while et al. 1980). For the first time, Mandahar and working with BCMV infection in French beans. Gill (1984) have brought out a review projecting Pollen and seed transmission are closely re- the epidemiological role of pollen in the spread lated factors in virus epidemiology. Most studies of viruses. Based on the available literature on pollen transmission of viruses indicate that a on the role of pollen in the epidemiology of greater percentage of seed contains virus when seed-transmitted viruses, the virus groups were the mother plant was infected than when pollen identified and presented in Table 7.1,andthe is the sole source of infection (Bennett 1969). details are discussed. In Montmorency sour cherry, PNRSV was seed transmitted to the tune of 28 and 53% when 7.4.1.1 Category A only the pollen and the mother plant were in- In this category, all externally pollen-borne fected individually and 88% with both pollen viruses and viruses causing pollen sterility are and the mother plants infected (Vertesy 1976). included (Table 7.1). No pollen transmission Similarly, in India, Sharma and Varma (1986), of these viruses was recorded since they while working with CpBMV in cowpea cv. Pusa are externally pollen borne and are of no Dophasli, recorded seed transmission of 22%, epidemiological importance. In case of viruses when both parents were diseased, and 19 and causing pollen sterility, the seeds are not 16%, respectively, when the female plant and produced, and the pollen infection becomes a the pollen-bearing plants were infected. Similar liability to the host plants. findings were also reported for Raspberry ring Some information is available on the detection spot (Lister and Murant 1967), Prunus necrotic of virus particles in pollen homogenates, but ring spot (Amari et al. 2007, 2009), Apple latent the viruses could not be pollen transmitted to spherical virus (Nakamura et al. 2011), AMV seeds, nor any abnormality in morphology or (Frosheiser 1974), BCMV (Medina and Grogan development of anthers and pollen grains was 1961)andTobacco ring spot virus and Tomato recorded, namely, Echtes Ackerbohnen mosaik ring spot viruses (Scarborough and Smith 1975) virus in Vicia faba, Potato virus T in Datura where minimum transmission took place through stramonium and Nicandra physalodes and SqMV infected pollen followed by infected ovules and in squash (Vorra-uraiand Cockbain 1977; Salazar then infected pollen and ovules. and Harrison 1978; Alvarez and Campbell 1978). Transmission by pollen may be the principal It appears that these viruses must be externally and only method for natural spread of the disease pollen borne on exine and should be regarded as under field conditions for some of the viruses such; none of these viruses are pollen transmitted belonging to the ilarvirus group (Davidson 1976). in their respective hosts. Pollen transmission is probably of little or no ecological significance when the host plant is mainly self-pollinated, as in the case of barley. 7.4.1.2 Category B The viruses included in this category are de- tected in the pollen extracts from infected plants, 7.4.1 Epidemiological Role but no experiments have been conducted either of Pollen-Transmitted Viruses in the greenhouse or under field conditions to find out whether cross-pollination of emasculated Some researchers maintain that virus transmis- flowers on healthy plants leads to the formation sion through pollen is of little consequence in of infected seeds. Hence, these viruses should nature since the infected pollen cannot compete not be considered as vertically pollen transmitted with healthy pollen during fertilisation (Shepherd unless more definite evidence is present in these 1972; Yang and Hamilton 1974). Others contend category of viruses and are of no epidemiological that the spread of some viruses through pollen importance for disease spread in nature. The list is rapid and widespread (Cameron et al. 1973; of viruses in this category with host plants are Davidson 1976; Daubeny et al. 1978; Mircetich cited in Table 7.1. 174 7 Ecology and Epidemiology of Seed-Transmitted Viruses

Table 7.1 Grouping of pollen-transmitted viruses into categories based on the importance of pollen transmission of plant viruses in disease spread in nature/greenhouse Virus Host Reference Category A (a) Externally pollen-borne viruses Apple chlorotic leaf spot Chenopodium quinoa; C. amaranticolor Cadman (1965) Potato virus X Petunia hybrida Neergaard (1977) Sowbane mosaic Atriplex coulteri Bennett and Costa (1961) Brome mosaic Phaseolus vulgaris Hamilton et al. (1977) Tobacco mosaic Vigna unguiculata Hamilton et al. (1977) Echtes Ackerbohnen mosaik Vicia faba Vorra-urai and Cockbain (1977) Potato virus T Datura stramonium; Nicandra Salazar and Harrison (1978) physalodes Squash mosaic Cucurbita pepo Alvarez and Campbell (1978)

(b) Viruses causing pollen sterility Beet yellows Beta vulgaris Larsen (1981) Datura quercina K. Datura stramonium Blakeslee (1921) Onion mosaic Allium cepa Cheremuskina (1977) Tobacco ring spot Glycine max Yang and Hamilton (1974) Tomato ring spot Pelargonium hortorum Murdock et al. (1976) Category B Bean southern mosaic P. vulgaris Crowley (1959) Bean yellow mosaic P. vulgaris Frandsen (1952) Cherry rasp leaf Prunus sp. Williams et al. (1963), Hansen et al. (1974) Cucumber mosaic Stellaria media Tomlinson and Carter (1970a, b) Onion yellow dwarf Allium cepa Louie and Lorbeer (1966) Pelargonium zonate spot Nicotiana glutinosa Gallitelli (1982) Category C Arracacha virus B Solanum tuberosum Jones (1982) Beet cryptic Beta vulgaris Kassanis et al. (1978) Broad bean stain Vicia faba Vorra-urai and Cockbain (1977) Cowpea aphid-borne mosaic V. sinensis Tsuchizaki et al. (1970) Cowpea banding mosaic V. sinensis Sharma and Varma (1984) Elm mosaic Sambucus racemosa; Ulmus americana Schmelzer (1966), Callahan (1957) Elm mottle Syringa vulgaris Schmelzer (1969) Lettuce mosaic Lactuca sativa Ryder (1964) Lychnis ring spot Lychnis divaricata; Silene noctiflora Bennett (1959) Potato virus T Solanum tuberosum Jones (1982) Raspberry ring spot Fragaria virginiana; Rubus sp. Lister and Murant (1967) Tomato black ring Rubus sp. Lister and Murant (1967) Tomato ring spot Pelargonium hortorum Scarborough and Smith (1975)

Category C1 Alfalfa mosaic Medicago sativa Frosheiser (1974) Barley stripe mosaic Hordeum vulgare Carroll (1974), Carroll and Mayhew (1976a), Hemmati and McLean (1977) Bean common mosaic Phaseolus vulgaris Medina and Grogan (1961)

Category D1 Cherry yellows Prunus cerasus Gilmer and Way (1960, 1963), George and Davidson (1963) Prune dwarf P. cerasus Gilmer and Way (1960), Ramaswamy and Posnette (1971) (continued) 7.4 Pollen Transmission 175

Table 7.1 (continued) Virus Host Reference Prune necrotic ring spot Cucurbita maxima Das and Milbrath (1961) Tobacco streak (Black raspberry latent strain) Rubus occidentalis Lister and Murant (1967)

Category D2 Cherry leaf roll Juglans regia Mircetich et al. (1980) Prunus necrotic ring spot Prunus cerasus Gilmer and Way (1960), George and Davidson (1963), Cameron et al. (1973), Davidson (1976) Raspberry bushy dwarf Rubus idaeus Cadman (1965). Barnett and Murant (1970), Murant et al. (1974), Daubeny et al. (1978), Jones and Wood (1979) R. occidentalis Murant (1976) R. ursinus var. Barnett and Murant (1970), Murant loganobaccus (1976) Source: Mandahar (1978)

7.4.1.3 Category C experiments that the viruses from this category The viruses included in this category are detected are transmitted through pollen and led to infected in pollen triturates and/or shown in glasshouse or seed production on emasculated healthy female field experiments on cross-pollination. They are plants. All these viruses are not only vertically vertically transmitted through pollen and led to transmitted but also horizontally transmitted the production of infected seed in emasculated through pollen to systemically back-infect the healthy female plants. However, their vertical pollinated (by hand or nature) mother plants. pollen transmission to produce infected seeds has Vertical pollen transmission of these viruses not been detected in nature, and the mother plant is also of little epidemiological significance does not get back-infected. In this category, there since their specific hosts are mostly perennial are 13 viruses which are pollen transmitted, and plants. However, based on horizontal pollen pollen transmissibility of these viruses may not transmission, these viruses are further partitioned pose any epidemiological threat (Table 7.1). into two subcategories.

7.4.1.4 Category C1 7.4.1.6 Category D1 In this category, the viruses are similar to those Horizontal pollen transmission of viruses like in ‘C’ except that their pollen transmissibility ap- Cherry yellows, Prune dwarf,PNRSVandTo- pears to be of some limited epidemiological value bacco streak was considered of limited epidemi- in disease spread of AMV, BSMV and BCMV; ological importance (Das and Milbrath 1961; however, these viruses cannot reinfect the female Gilmer and Way 1963; Lister and Murant 1967; parent. The limited epidemiological value of Converse and Lister 1969; Ramaswamy and Pos- vertical pollen transmission of these viruses is nette 1971). evidenced by the fact that, usually, pollen verti- Gilmer and Way (1963) reported that only two cally transmits the virus to more ovules. In other sweet cherry and three sour cherry mother plants words, it is somewhat less efficient than ovule were back-infected with Cherry yellows virus in transmission but still transmits virus to a con- the growing season after pollination. They con- siderable number of seeds (Medina and Grogan cluded that horizontal pollen transmission was 1961; Frosheiser 1974; Carroll 1974; Carroll and not a highly efficient means of disease spread. Mayhew 1976b; Hemmati and McLean 1977). Lister and Murant (1967) found that 5 out of the 12 hand-pollinated black raspberry plants got 7.4.1.5 Category D back-infected with Black raspberry latent virus, It was shown by embosoming and/or cross- astrainofTobacco streak virus, after 1 year pollination tests under greenhouse or field of pollination. Later, Converse and Lister (1969) 176 7 Ecology and Epidemiology of Seed-Transmitted Viruses concluded that pollen is the major source of Tomato ring spot viruses were established in three horizontal spread of this virus in Black raspberry randomised blocks of ten plants per block. RBDV (Rubus occidentalis)andBlackberry (R. ursinus). was first noticed in 1975 in 6 out of the 30 plants, Subsequent observations led to revise their earlier infected presumably from pollen of the nearby opinion and suggested caution for cataloguing infected cultivars, and increased to 11 and 14 in this virus among the known pollen-transmitted 1976 and 1977, respectively. Thus, about 50% of viruses. Costa and Neto (1976) reported thrips the plants became infected over a period of 2Ð (Frankliniella spp.) as a vector of this virus in 3 years, which is an alarming rate of horizontal Brazil. However, Converse’s own observation is spread. that this virus cannot back-infect the pollinated The disease always spreads to adjacent strawberry plants. More work is needed to resolve plants Ð a characteristic of pollen-transmitted this issue. It is therefore tentatively placed in D1 viruses. Murant et al. (1974) studied the spread category. of RBDV in raspberry (R. idaeus)cultivar‘Lloyd George’ in Scotland and found that after two 7.4.1.7 Category D2 flowering seasons, it infected 20 out of 24 test Horizontal pollen transmission of Cherry leaf plants in field plots containing infector plants roll,PNRSVandRaspberry bushy dwarf virus but none of the 24 test plants in plots without (RBDV) included in this category is of great infectors. However, in plots without infectors, the epidemiological importance in the spread of these disease appeared in 1 among 30 plants in 1971, diseases in nature. Cherry leaf roll virus caus- in 16 out of 75 plants in 1972 and increased to ing the blackline disease of English walnut in 28 in 1973. Thus, the disease spreads to most of the USA spreads horizontally by infected pollen the plants in the first two flowering seasons in and is a serious threat to the walnut industry in plots containing internal infection foci. In plots California (Mircetich et al. 1980). During 1975, without infector plants, it spreads very rapidly it was observed that among 361 walnut trees of even if a single plant gets infected from outside 10-year age propagated on Northern California source. Black root stock, only 58 trees showed disease; In Canada and USA, the infected pollen is the the number of diseased trees increased to 132 only means of natural spread of Prunus necrotic in 1979. This works out to be 5% increase per ring spot virus (PNRSV) in sour cherry orchards year. The second orchard, comprising of 1,044 which was always horizontal (Cameron et al. fifteen-year-old walnut trees propagated on para- 1973;Davidson1976). This was the first virus in dox root stocks, had 97 infected trees in 1977 but which pollen transmission was found to reinfect increased to 139 in 1979. This increase was 4% in some of the cross-pollinated mother plants. over a 2-year period. Thus, the disease spreads Way and Gilmer (1958) reported 5 out of 18 faster as more and more trees were infected as cherry seedlings (about 27% pollen transmission) pollen from diseased plants gain ascendancy over got infected from a cross between an infected the healthy pollen in the environment. male and a healthy female. George and Davidson Raspberry bushy dwarf virus (RBDV) hori- (1963) found 4 out of 14 emasculated mother zontally spreads naturally only through infected plants, fertilised by pollen from infected plants, pollen in Red raspberry (Rubus idaeus) orchards got systemically infected (about 28% pollen in Canada, England, New Zealand, Scotland and transmission). Subsequently, Cameron et al. USA (Cadman 1965;Murantetal.1974;Murant (1973) and Davidson (1976) conducted 7-year 1976; Daubeny et al. 1978; Jones and Woods test for epidemiological efficiency of horizontal 1979). In British Columbia (Canada), Daubeny pollen transmission of this virus. As many as et al. (1978) studied its spread in red raspberry 73.5 and 87.5% of the naturally flowering sour cv. BC 61-6-68 from 1973 to 1977. This selection cherry trees in orchards became infected with was propagated in 1973, and 30 healthy plants Prunus necrotic ring spot virus over the 7-year free of Raspberry bushy dwarf (RBDV) and period in Oregon state of USA and the spread 7.5 Viruses 177 of the same virus at Niagara Peninsula, Ontario, pollen so that their vertical transmission through Canada, respectively. The disease progress curve pollen plays a supplementary role in disease of this virus in sour cherry is a sigmoid curve spread by inducing greater production of infected (Davidson and George 1964), with exponential seeds. Thus, vertical virus transmission through increase during the 3rd to 9th year when all the pollen per se does not appear to play a major trees were infected by the virus. This happened role in disease spread under field conditions. due to infection of more and more plants by Although Cherry leaf roll virus is present in infected pollen possibly predominating in the walnut and gets transmitted vertically, it is the environs of an orchard. This is also the reason horizontal spread of the disease that threatens the why the timescale for the spread of horizontally walnut industry in California (Mircetich et al. pollen-transmitted viruses in plantation crops is 1980). always in terms of years. Viruses spread horizontally through pollen The virus particle of PNRSV occurs on the and invade and systemically reinfect the ovule- surface of the pollen of infected almond and bearing mother plants which, in turn, lead to cherry plants, and infectivity is retained for sev- the formation and release of larger quantities eral days. Cole et al. (1982) have speculated on of infected pollen. This cycle can be repeated this basis that foraging bees might mechanically during subsequent flowering seasons leading to spread and infect the flower parts with the virus rapid epidemiological build-up of infected pollen through abrasions caused by them. The virus and to disastrous consequences in a couple of then moves backwards and systemically reinfects years. This is the reason why only the horizontal the main plant through the plasmodesmata and transmission through pollen is epidemiologically phloem that connect the various flower parts important for viruses of categories D1 and D2 (except the male and female sporogenous cells) (Mandahar 1981, 1985; Mandahar and Gill with the sporophyte. 1984). In epidemiology of PNRSV-causing cherry rugose mosaic disease from some western states of USA, the mechanical transmission of the virus 7.5 Viruses by honey bees is important. About 50,000 bee- hives are shifted each year from Washington There are nearly 231 viruses which are to California for pollinating various stone fruit seed transmitted, and the frequency of seed orchards and then back to Washington for pol- transmission is more in potyvirus, nepovirus, linating sweet cherry (Mink 1983). Bees col- cryptovirus, ilarvirus, tobamovirus, potexvirus, lect and store huge quantities of PNRSV-infected comovirus, carlavirus, carmovirus, cucumovirus, pollen in their hives in California and continue sobemovirus, furovirus, bromovirus and ty- to store them on re-entry into Washington (Mink movirus groups (Table 2.1). The viruses are 1983). This factor is important epidemiologically spherical, bacilliform, rigid or flexuous rods in various ways: Even non-viable pollen can act (Table 2.2). Some of the viruses appear highly as a virus carrier and transmit virus to healthy specific affecting only one or two species or only plants through abrasion on flowers caused by species within one family like SMV, whereas bees, pollen from even non-compatible species others like TRSV or CMV may infect a large may be equally effective and the disease spreads number of species in many families, including rapidly over extensive areas. herbaceous and woody plants. Vertical pollen transmission does not play any The epidemiology of virus diseases also de- part in the spread of viruses which are grouped pends on different pathogen strains which vary in in A, B and C categories. In category C1, vertical virulence, host range and transmissibility. Viruses transmission through pollen seems to play some continue to change by mutation and selective epidemiological role in their spread. However, adaptation by passage through their hosts as in these viruses also reach the seed via infected case of CMV and BCMV strains. Variation may 178 7 Ecology and Epidemiology of Seed-Transmitted Viruses also result from pseudo-recombination as since, Another important aspect in epidemics is the some of them posses divided genomes or by het- introduction of resistant genotypes which led to erologous encapsidation. These phenomena are the emergence and spread of resistance-breaking both examples of direct interaction between virus strains. Experience with Sugarcane mosaic strains or viruses in mixed infections. in Louisiana and Soybean mosaic in Korea Variation in viruses is often apparent from illustrates the rapidity with which resistance- differences in such biological features such as breaking strains have become prevalent following symptom expression or host range and transmis- the introduction of resistant varieties (Pelham sion by vectors or through seed. The epidemi- et al. 1970) and also with the introduction ological importance of strain variation has been of TMV mild strain protection (Fletcher and recognised as in the studies on Sugarcane mosaic Butler 1975). These changes were monitored virus in western United States (Pelham et al. using isogenic tomato lines and suggested that 1970). Seasonal and other changes in prevalence the resistance-breaking strain did not compete of strains have also been recorded in studying successfully with the former one in susceptible the incidence of CMV in vegetable crops and varieties and declined rapidly when the resistant weeds in South-East France (Quiot et al. 1979). varieties were replaced. Similarly, limited The strains that predominated in spring-sown distribution of resistance-breaking strain of crops of tomato, celery and pepper were much Raspberry ring spot virus in Scotland may be more sensitive to heat than those isolated from due to its limited epidemiological competence, equivalent summer plantings (Thresh 1987). as it is less invasive and less readily seed Other complex interactions between the vir- transmitted than the common strain (Hanada ulence of strains and host response have been and Harrison 1977). observed with BSMV in barley. Sensitive barley varieties infected with virulent strains produce a large proportion of infected seed, although few 7.6 Conclusion seeds develop and tend to be small with poor viability. By contrast, tolerant varieties produce In a nutshell, the ecology and epidemiology of abundant viable seed, but few were infected. seed transmission are important because it helps Thus, the virus strains that persist readily for the virus to perpetuate during unfavourable envi- successive generations tend to be of intermediate ronmental conditions. It provides early and ran- virulence that infect substantial proportions of domised infection foci within a crop from which seed without drastic effects on fertility or on the virus spreads to other plants in a field either contact between plants (Timian 1974). mechanically through pollen or primarily through The above examples illustrate complex factors vectors depending on the virus. Because it is seed influencing the overall epidemiological compe- transmitted, the introduction and establishment of tence of viruses and their strains. However, much a virus into new and distant locations take place in of the evidence on variation has come from ‘taxo- nature. Generally, forecasting of seed-transmitted nomic’ or aetiological studies intended to provide diseases whose spread depends on interaction definitive descriptions of viruses and to deter- between virus, host and vector is difficult to frame mine their interrelationships. There have been as compared to other fungal or bacterial diseases. few comprehensive surveys of prevalence and Attempts at forecasting are justified because the distribution of various strains of even the most success indicates an understanding of the main important viruses. This is a serious limitation in factors influencing epidemiology. If the forecast- attempts to estimate crop loss or breed for disease ing system is developed against a particular virus resistance, as there can be greater differences disease, it will help to operate an early warning between strains in their ability to infect or in system or to select growing seasons or areas for their effects, with important interactions between special crops where infection is unlikely. The varieties and strains. quantitative epidemiological information has in a References 179 few cases allowed forecasting the development of Carroll TW, Mayhew DE (1976b) Occurrence of virions few seed-transmitted virus disease and helped in in developing ovules and embryo sacs of barley in preventing or reducing the crop losses. relation to the seed transmissibility of barley stripe mosaic virus. Can J Bothbox 54:2497Ð2512 Carter W (1962) Insects in relation to plant disease. 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Neergaard P (1977) Seed-borne viruses. Chapter 3. In: Schmelzer K (1957) Untersuchungen uber den Wirt- Seed pathology, vol I. Macmillan, London/Madras, spflanzenkeris des Tabakmauche-virus. Phytopathol Z 839 pp 30:281Ð314 OEPP/EPPO (1994) Certification schemes No. 10. Schmelzer K (1963) Untersuchungen an Viren der Zier- Pathogen tested material of Rubus. Bull OEPP/EPPO und Wildgeholze. 3. Mitt. Virosen an Robinia, Ptelea Bull 24:865Ð874 und anderen Gattungen. Phytopathol Z 46:235Ð268 Pelham J, Fletcher JT, Hawkins JH (1970) The establish- Schmelzer K (1966) Untersuchungen an Viren der Zier- ment of a new strain of tobacco mosaic virus resulting und Wildgeholze. 5. Mitteilung: virosen an Populus from the use of resistant varieties of tomato. Ann Appl and Sambucus. Phytopathol Z 55:317Ð351 Biol 65:293Ð297 Schmelzer K (1969) Das Ulmenscheckungs. Virus Phy- Phatak HC (1980) The role of seed and pollen in the topathol Z 64:39Ð67 spread of plant pathogens particularly viruses. J Trop Sharma SR, Varma A (1983) Effect of heat treatment Pest Manag 26:278Ð285 of infected seeds and granular application of insecti- Powell CA, Forer LB, Stouffer RF, Commins JN, cide on field spread of cowpea banding mosaic and Gousalves D, Rosenbergh DA, Hoffman J, Lister RM seed yield of cowpea. J Turkish Phytopathol 12(2Ð3): (1984) Orchard Weeds as hosts of tomato ring spot and 103Ð111 tobacco ring spot virus. Plant Dis 68:242Ð244 Sharma SR, Varma A (1984) Effect of cultural practices on Prasada Rao RDVJ, Jyothirmai Madhavi K, Reddy AS, virus infection in cowpea. Z fur Aker und Pflanzenbau Varaprasad KS, Nigam SN, Sharma KK, Lavakumar P, 153:21Ð31 Waliyar F (2009) Non transmission of Tobacco streak Sharma SR, Varma A (1986) Seed transmission of cowpea virus isolate occurring in India through seeds of some banding mosaic and cowpea chlorotic spot viruses crop and weed hosts. Indian J Plant Prot 37:92Ð96 through the seeds of cowpea. Seed Sci Technol Quiot JB, Devergne J-C, Cardin L, Verbrugghe M, Mar- 14:217Ð226 choux G, Labonne G (1979) Ecologie et epidemiologie Sharma OP, Sharma PK, Sharma PN (2004) Role of in- du Virus de la Mosaique du Concombre dans le Sud- fected and healthy seed in the epidemiology of French Est de la France. VII. Repartition de deux types de bean mosaic caused by bean common mosaic virus. populations virales dans les cultures sensibles. Ann Himachal J Agirc Res 30:78Ð84 Phytopathol 11:359Ð373 Shepherd RJ (1972) Transmission of viruses through seed Ramaswamy S, Posnette AF (1971) Properties of cherry and pollen. In: Kado CI, Agrawal HO (eds) Princi- ring mottle, a distinctive strain of prune dwarf virus. ples and techniques in plant virology. Van Nostrand- Ann Appl Biol 68:55Ð65 Reinhold Co., New York, pp 267Ð292, 688 p Reddy DVR, Nolt BL, Hobbs HA, Reddy AS, Ra- Stevenson WR, Hagedorn DJ (1973) Further studies on jeswari R, Rao AS, Reddy DDR, McDonald D seed transmission of pea seed-borne mosaic virus in (1988) Clump virus in India: Isolates host range, Pisum sativum. Plant Dis Rep 57:248Ð252 transmission and management. In: CooperJI, Asher Taylor CE (1967) The multiplication of Longidorus elon- MJC (eds) Developments in applied biology II. gatus (de Man) on different host plants with reference Viruses with fungal vectors, Wellesbourne, Warwick, to virus transmission. Ann Appl Biol 59:275Ð281 United Kingdom: Association of Applied Biologists, Taylor CE, Brown DJF (1976) The geographical distribu- pp 239Ð246 tion of Xiphinema and Longidorus nematodes in the Ryder EJ (1964) Transmission of common lettuce mosaic British Isles and Ireland. Ann Appl Biol 84:383Ð402 virus through the gametes of the lettuce plant. Plant Taylor CE, Thomas PR (1968) The association of Dis Rep 48:522Ð523 Xiphinema diversicaudatum (Micoletsky) with straw- Salazar LF, Harrison BD (1978) Host range, purification berry latent ringspot and arabis mosaic viruses in and properties of potato virus. T Ann Appl Biol raspberry plantation. Ann Appl Biol 62:147Ð157 89:223Ð235 Thomas PR (1969) Crop and weed plants compared as Sammons B, Barnett OW (1987) Tobacco ring spot virus host plants of viruliferous Longidorus elongatus.Plant from squash grown in South Carolina and transmission Pathol 18:23Ð28 of the virus through seed of smooth pig weed. Plant Thomgmeearkom P, Paschal EHH, Goodman RM (1978) Dis 71:530Ð532 Yield reduction in soybean infected with cowpea mo- Sastry KS (1984a) Role of weed hosts in the perpetuation saic virus. Phytopathology 68:1549Ð1551 of virus and virus-like diseases. In: Misra A, Polasa Thouvenel JC, Fauquet C (1981a) Further properties of H (eds) Virus ecology. South Asian Publishers, New Peanut clump virus and studies on its natural transmis- Delhi, pp 59Ð90 sion. Ann Appl Biol 97:99Ð107 Sastry KS (1984b) Management of plant virus diseases by Thouvenel JC, Fauquet C (1981) Peanut clump virus. No oil sprays. In: Misra A, Polasa H (eds) Virus ecology. 235 in Descriptions of plant viruses common Mycol. South Asian Publishers, New Delhi, pp 31Ð57 Lust. Assoc. Appl. Biol, Kew surry, England 4 pp Scarborough BA, Smith SH (1975) Seed transmission of Thresh JM (1974) Vector relationships and the develop- tobacco and tomato ring-spot viruses in Geraniums. ment of epidemics: the epidemiology of plant viruses. Phytopathology 65:835Ð836 Phytopathology 64:1050Ð1056 References 183

Thresh JM (1981) The role of weeds and wild plants in inoculum threshold levels for Bean common mosaic the epidemiology of plant virus disease. In: Thresh JM virus strain Black eye cowpea mosaic infection in (ed) Pests pathogens and vegetation. Pitman, London, cowpea seed. Afr J Biotechnol 9(53):8958Ð8969 pp 53Ð70 Ullrich J (1954) Untersuchungen uber Salat mosaik Nachr Thresh JM (1987) The population dynamics of plant virus Bl. dt. PflSchutsdienst 66:182Ð184 diseases. In: Wolfe MS, Caten CE (eds) Populations of Van der Plank JE (1963) Plant disease: epidemics and plant pathogens. Their dynamics and genetics. Black- control. Academic, New York/London, 349 p well, Oxford Van der Plank JE (1982) Host-pathogen interactions in Timian RG (1967) Barley stripe mosaic virus seed trans- plant disease. Academic, London/New York, p 207 mission and barley yield as influenced by time of Vertesy J (1976) Embryological studies of Ilar-virus in- infection. Phytopathology 57:1375Ð1377 fected cherry seeds. Acta Hort 67:245 Timian RG (1974) The range of symbiosis of barley and Vorra-urai S, Cockbain AJ (1977) Further studies on seed barley stripe mosaic virus. Plant Dis 64:342Ð345 transmission of broad bean stain virus and Echtes Tomlinson JA, Carter AL (1970a) Studies on the seed Ackerbohnen mosaik virus in field beans (Vicia faba). transmission of cucumber mosaic virus in chickweed Ann Appl Biol 87:365Ð374 (Stellaria media) in relation to the ecology of the virus. Wallis RL (1967) Some host plants of the green peach Ann Appl Biol 66:381Ð386 aphid and beet western yellows virus in pacific North- Tomlinson JA, Carter AL (1970b) Seed transmission of west. Ann Appl Biol 73:293Ð298 cucumber mosaic virus in chickweed. Plant Dis Rep Walter MH, Kaiser WJ, Klein RE, Wyatt SD (1992) Asso- 54:150Ð151 ciation between tobacco streak Ilarvirus seed transmis- Tomlinson JA, Walker VM (1973) Further studies on seed- sion and another tissue infection in bean. Phytopathol- transmission in the ecology of some aphid-transmitted ogy 82:412Ð415 virus. Ann Appl Biol 73:292Ð298 Way LD, Gilmer RM (1958) Pollen transmission of Tomlinson JA, Ward CM (1981) The reactions of some necrotic ringspot virus in cherry. Plant Dis Rep brussels sprout F1 hybrids and inbreds to cauliflower 42:1222Ð1224 mosaic and turnip mosaic virus. Ann Appl Biol Williams HE, Traylor JA, Wagnon HK (1963) The infec- 97:205Ð212 tions nature of pollen from certain virus infected stone Tomlinson JA, Carter AL, Dale WT, Simpson CJ (1970) fruit trees. Phytopathology 53:1144 (Abstract) Weed plants as sources of cucumber mosaic virus. Ann Yang AF, Hamilton RI (1974) The mechanism of seed Appl Biol 66:11Ð16 transmission of tobacco ringspot virus in soybean. Tsuchizaki T, Yora K, Asuyama H (1970) The viruses Virology 62:26Ð37 causing mosaic of cowpea and Azuki bean and their Zadoks JC, Schein RD (1979) Epidemiology and transmissibility through seeds. Ann Phytopathol Soc plant disease management. Oxford University Press, Jpn 36:112Ð120 Oxford Tuite J (1960) The natural occurrence of tobacco ringspot Zink FW, Grogan RG, Welch JE (1956) The effect virus. Phytopathology 50:296Ð298 of the percentage of seed transmission upon subse- Udayashankar AC, Nayaka CS, Kumar BH, Mortensen quent spread of lettuce mosaic virus. Phytopathology CN, Shetty HS, Prakash HS (2010) Establishing 46:662Ð664 Methods of Combating Seed-Transmitted Virus Diseases 8

Abstract The basic principles in the management of seed-transmitted viruses are generally more or less similar regardless of type of virus involved. There are five approaches, namely, (1) avoidance of the virus from the seeds, (2) prevention/minimising the rate of spread through vector manage- ment, (3) legislation, (4) production of healthy seeds through certifica- tion programmes and (5) modern diagnostic molecular and biochemical approaches. Avoidance of virus inoculum from infected seeds can be done by removal of infected seeds, chemical seed disinfection, seed disinfection by heat and by irradiation. Implementing the cultural practices like field sanitation, roguing, crop rotation, planting dates, barrier and cover cropping will also reduce the virus disease incidence. The management of virus spread under field conditions is also possible by the application of insecticides, pyrethroids and mineral oils, which reduces the vector population. The role of quarantines, ISTA, biosafety measures, resistant cultivars, healthy seed production and certification schemes for healthy seed production was discussed in detail.

and are very effectively transmitted through 8.1 Introduction vectors which are air or soilborne. Therefore, pre- venting their spread is a serious, complex prob- Seed-transmitted viruses cause enormous qualita- lem, and hence, epidemiology has a profound tive and quantitative yield losses, which are quite influence in combating virus diseases. However, evident from the examples given in Chap. 3.Be- detection and elimination of infected seeds is cause of the serious losses to agricultural crops, essential since it will eliminate the primary the virus diseases have acquired great importance source of infection and reduce the chances of es- in plant pathology and entrusted for effective tablishment of the virus in commercial plantings. control measures. They are not amenable to seed The basic principles in the management treatment like fungi and bacteria, and no com- of seed-transmitted viruses are generally mercial viricides have yet been developed. They more or less similar regardless of type of cause systemic infections after seed germination virus involved. There are five approaches:

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 8, 185 © Springer India 2013 186 8 Methods of Combating Seed-Transmitted Virus Diseases

(1) avoidance of the virus from the seeds, could be reduced by sieving out the smaller seeds (2) prevention/minimising the rate of spread (Inouye 1962). through vector management, (3) legislation, (4) Mechanical seed cleaning helps in bringing production of healthy seeds through certification down the initial virus inoculum under field con- programmes and (5) modern diagnostic molecu- ditions. In some instances, viruses do not ex- lar and biotechnological approaches. The control hibit any changes in the morphology of seed. measures that have been applied against different Even in certain virusÐhost combinations, the seed virusÐhost combinations are discussed in this coat mottling alone should not be considered chapter. as a reliable indication of seed-transmitted virus infection. The notable example is of soybean seed infected with Soybean mosaic virus (SMV), 8.2 Avoidance of Virus Inoculum wherein the seed coat mottling has been viewed from Infected Seeds as generally inconsistent and an unreliable indi- cator of the presence of virus (Hill et al. 1980; 8.2.1 Removal of Infected Seeds Bryant et al. 1982;Laetal.1983). In such cases, the seed should also be tested through A careful and close look at the seed in cer- serological, molecular and biological techniques. tain cases gives an indication of the presence of virus(es). Seed morphological abnormalities like shrunkenness, shrivelled seed coats, discoloura- 8.2.2 Chemical Seed Disinfection tions, reduced size and weight, cracking, necrotic spots or bands are sometimes associated with the Seed disinfection helps in removing external presence of virus in the seed (Table 6.1). For ex- infection in fruit pulp remnants as in TMV on ample, pea seeds infected by Pea early browning tomato, chilli and brinjal seeds and Cucumber virus (PEBV) are wrinkled with the seed coat green mottle mosaic virus (CGMMV) on exhibiting a greenish grey discolouration (Bos cucumber seeds. The seed cleaning is usually and Van der Want 1962). Small and shrivelled done by fermentation of the seed containing seeds of barley, wheat and cowpea indicate the fruit pulp with pectolytic enzymes, detergents, possible infection of BSMV, BMV and CPMV, certain chemicals or by mechanical means. TMV respectively (Phatak 1974; Von Wechmar et al. infection in tomato seeds was greatly reduced 1984). SMV seed infection in soybean can be by treatment of the pulp with one quarter of its detected by characteristic seed discolouration and volume of concentrated HCl for 30 min, followed black bands or zones radiating from the long axis by washing and drying of the seeds (Howles of the hilum (Phatak 1974). 1957;Crowley1958; Broadbent 1965;McGuire Even in other virusÐhost combinations like et al. 1979). Even soaking the extracted seed peanut with PMV (Paguio and Kuhn 1974)and in 10% solution of teepol for 2 h or soaking in peanut stunt virus (PSV) (Culp and Troutman a 10% solution of trisodium orthophosphate or 1968) and soybean with soybean stunt virus 10% sodium carbonate solution helps in freeing (Koshimizu and Iizuka 1963), seed infection is the seed from TMV contamination (Alexander generally associated with reduction in seed size 1960; Nitzany 1960; Broadbent 1965;McGuire and mottled seed coat. Routine seed cleaning et al. 1979). Cordoba-Selles et al. (2007)have techniques minimise the extent of infection eradicated the Pepino mosaic virus infection but can never completely eliminate the viruses. from tomato seeds by immersing the infected For example, Stevenson and Hagedorn (1970) seeds in 10% trisodium phosphate for 3 h which reduced the transmission of Pea seed-borne do not hinder the germination. AVRDC scientists mosaic virus in a given seed lot from 10 to (Berke et al. 2005) have also suggested soaking 4% by removal of infected seeds with growth 2 g of chilli pepper seeds in 10 ml of 10% (w/v) cracks. Even BSMV inoculum in barley seeds trisodium phosphate (TSP) (Na3PO412 H2O) 8.2 Avoidance of Virus Inoculum from Infected Seeds 187 for 30 min, transferring them to a fresh 10% pepper. Salicylic acid was proved to be an TSP solution for 2 h, then rinsing in running effective inducer. Several reports are available on water for 45 min, and this treatment can be induction of resistance against plant viruses by done on freshly harvested or dry seeds. It is using chemicals, and one among them is salicylic also suggested to soak seeds for 4Ð6 h in 5% acid which is used to control TMV (Murphy and (v/v) hydrochloric acid, then rinse in running Carr 2002; Singh et al. 2004). water for 1 h and dry them for storage, or Elimination of some seed-transmitted viruses sow immediately for minimising Tobamoviruses in certain crops was also achieved by soaking including Tobacco mosaic virus (TMV), Tomato them in chemical solutions for varying periods. mosaic virus (ToMV) and Pepper mild mottle In India, malic hydrazide, NAA, 2-thiouracil, 2- virus (PMMV) in chilli pepper. 4-D and teepol were tested and soaking in malic Seed cleaning with pectolytic enzyme (pecti- hydrazide at 40, 100 and 400 ppm for 90 min; nase) and dilute HCL and drying at 80ıCfor 2-thiouracil at 500 and 700 ppm for 60 min; 24 h is found effective in bringing down TMV in- NAA at 40 ppm for 4 h and teepol (5 and oculum to a negligible level (Laterrot and Pecaut 10%) for 4 h completely inactivated Cowpea 1965). Tomato mosaic virus contaminated tomato banding mosaic virus (CpBMV) without affect- seeds were treated with 1% HCL and obtained the ing seed viability (Sharma and Varma 1975a, b). healthy crop by eliminating the virus contamina- Cherry leaf roll virus was inactivated in infected tion (Pradhanang 2009). seeds by giving a pre-germination imbibitions Gooding (1975) successfully eliminated the in an osmotic solution of polyethylene glycol TMV by treatment of tomato seed with trisodium (PEG) followed by high-temperature treatment orthophosphate and sodium hypochlorite. The during the early stages of germination (Cooper treatment comprised of soaking infected tomato 1976). Subsequently, Walkey and Dance (1979) seed in 1% aqueous solution of trisodium or- reported inactivation of Lucerne mosaic virus thophosphate for 15 min and in 0.52% sodium by imbibing diseased seeds in PEG and incu- hypochlorite for 30 min. The treated seed did bating for 6Ð10 days at 40ıC. Longer periods not lose viability. Even treating the seed of the of treatments at 40ıC impaired seed viability, Capsicum spp. infected with TMV, with 9% cal- while seeds treated for 9 days at 22ıC remain cium chloride or 10% trisodium phosphate (Na3 infected. Even Spinach latent virus was eradi- PO4) gave good virus elimination (Betti et al. cated from Nicotiana tabacum cv. Xanthi seed 1983). In the Netherlands, it was observed that by imbibing in PEG solution at 40¼C for 20Ð treating Capsicum seeds infected with Capsicum 40 days (Walkey et al. 1983). Certain dimethyl mosaic virus with 10% trisodium orthophosphate sulfoxide solutions reduced the BSMV symp- (100 g/l) solution for 2 h followed by dry heat toms in barley when the infected seeds were at 70ıC for 7 days helps in eliminating the virus treated (Miller et al. 1986). Disinfection of me- from seeds without affecting germination (Rast chanically transmitted Potato spindle tuber vi- and Stijger 1987). In contrast, polishing dry cu- roid from knives and other equipments could cumber seeds and treating them with detergents be achieved by soaking in sodium hypochlo- or chemicals did not completely eradicate cucum- rite solution at 2Ð3% concentration (Singh et al. ber virus 2 (Van Dorst 1967). 1989). Studies of Salamon and Kaszta (2000)and Svoboda et al. (2006) have indicated that capsicum seeds can be disinfected from Pepper 8.2.3 Seed Disinfection by Heat mild mottle virus by using 2% NaOH. Even Madhusudhan et al. (2005) have proved salicylic Most attempts to eliminate virus from seed acid (0.05 M) and neem oil (5%) seed/seedling by heat treatment have been done with high treatment was found to be effective in reducing temperatures for relatively short periods or at Tobamovirus concentrations in tomato and bell low temperatures for longer periods by means 188 8 Methods of Combating Seed-Transmitted Virus Diseases of hot water or dry heat treatments. CPMV in (1969) found that treatment at 70ıCformore cowpea seeds is inactivated when freshly infected than 1 day was sufficient to eliminate the same seeds were exposed to 30ıC for 4 days or by hot virus (CGMMV) from infected cucumber seeds. air treatment at 55ıC for 15 min followed by These seeds could tolerate dry heat treatment keeping the seeds at 25ıCfor4days(Verma with little adverse effect, although at 80ıCgermi- 1971). Sharma and Varma (1983) studied the nation was delayed and the cotyledonary leaves effect of dry heat on cowpea seeds infected were distorted. In a commercial trial, over 45,000 with CpBMV for 15 min at 65ıC; this was cucumber plants free from symptoms of the virus followed by incubating the seeds at 30ıCfor were raised from infected seeds treated at 70ıC 4Ð8 days or by hot air treatment at 55ıCfor for 3 days. Sharma and Chohan (1971) reported 15 min followed by keeping the seeds at 25ıC inactivation of Cucumis virus-1 in infected pump- for 4 days, which reduced the seed transmission kin (Cucurbita pepo) seed by hot air treatment from 13.1Ð24.7% to 0.9Ð3.3%. Heat therapy at 70ıCfor2daysor40ıC for 4 weeks. Hot of seeds also reduced the field spread of this water treatment at 55ıC for 60 min was found to virus to 4.7Ð9.1% as compared to 23.7Ð29.7% be effective in eliminating infection without any in control. Seed yield from heat-treated seeds adverse effects on seed germination. Similarly, was 19.3Ð22.4% higher than untreated diseased Cherry necrotic ring spot and prune dwarf (PDV) seeds. viruses were eliminated from freshly extracted TMV from infected tomato was successfully sour cherry seeds by exposing the soaked seed eliminated by subjecting the dried infected seeds to 60ıC for 1 h or dry seed to 90ıCfor1h to temperatures of 70ıCfor3daysor80ıCfor without any adverse effects on seed viability. In 1 day. This is one of the commonly used meth- another experiment, Necrotic ring spot virus in ods to eliminate the virus from seeds infected sour cherry seeds was inactivated by exposing internally (Broadbent 1965; Rees 1970;Howles at 55ıC for 6 weeks without affecting seed via- 1978). In Taiwan, dry heat treatment at 78ıCfor bility (Megahed and Moore 1969). Even Sharma 2 days against TMV was recommended (Green and Varma (1975b) recorded complete reduction et al. 1987). Heat treatment delayed germination, in seed transmission of CpBMV in cowpea by but the seeds did not lose their viability when hot water treatment for 40 min at 45ıCand only dried seeds were used. 20 min at 50ıC. Hot air treatment for 50 min Vov k ( 1961) reported disinfection of TMV by at 50ıC, 30 min at 60ıC and 20 min at 65ıC exposing infected tomato seeds at 50Ð52ıCfor also reduced infection, but viability of the seeds 2 days followed by 1 day at 78Ð80ıC. However, was considerably reduced at these temperatures. Howles (1961) could not eliminate TMV com- However, dry hot air treatment for 15 min at 65ıC pletely when infected seeds were subjected to followed by 2, 4 and 8 days of incubation at 30ıC 72ıC for 22 days, but further work has shown proved comparatively better in eliminating seed that most of the seeds can be freed from TMV transmission, while seed viability was affected to during 1Ð3 days treatment at 70ıCandLMVin a lesser degree. Similarly, the seed-transmitted lettuce seed by treatment with hot air maintained infection of ULCV was completely eliminated at 80ıC for 3 days without loss of seed viability. by treating the urd bean seeds in water bath LMV was also inactivated by dry air treatment for 30 min at 55ıC without affecting the seed for 80Ð120 days at 55ıC (Kristensen 1970). Ear- germination (Beniwal et al. 1980). However, heat lier, Rohloff (1963) reported that treatment of treatment did not always cure diseased seeds. mosaic-infected lettuce seeds at more than 100ıC For example, BSMV was not inactivated even reduced the percentage of seedlings carrying the when the seeds were subjected to 130ıCfor virus to 0.2% but seed emergence was only 29%. 30 min (Timian 1965). Similarly, no inactiva- Van Dorst (1967) reported complete inactivation tion of TRSV in the seeds of soybean without of CGMMV in cucumber seeds by hot air treat- loss of viability was recorded (Owusu et al. ment at 76ıC for 3 days. Similarly, Fletcher et al. 1968). 8.3 Reducing the Rate of Virus Spread Through Vector Management 189

8.2.4 Storage Effect at room temperature (25ıC). After storage for 2 years, both virus and seed viability decreased Some viruses survive in seed only for a few (Sharma and Varma 1986). months, and others persist for many years (Table From a commercial point of view, the stored 5.1). Long-term storage of infected seed also seed should have maximum germination, and so makes the seed virus-free, and the viruses which the method of eliminating the virus by storage are externally seed transmitted are often inacti- will be effective only when the virus is active vated than embryo-borne viruses which usually in seed only for a few months. For the viruses persist for longer periods. For example, BCMV retained in the infected seed for longer periods, was infective even after storage for 6 years (Polak alternative methods like chemical and heat treat- and Chod 1967)andBean (Western) mosaic virus ment should be worked out. for 3 years (Scotland and Burke 1961). Similarly, Bennett (1969) did not notice any decrease in Lychnis ring spot virus transmission in the seed of 8.2.5 Irradiation Effect Lychnis divaricata even after 9 years of storage. In some virusÐhost combinations, where the Gamma irradiation has been tried for the in- virus was seed transmitted, the loss of seed- activation of seed-transmitted viruses. Megahed transmitted virus was achieved with seed stor- and Moore (1969) reported the inactivation of age, and some of the successful attempts are as Necrotic ring spot and Prune dwarf viruses in follows. Substantial reduction in Brinjal mosaic Prunus spp. seeds after irradiation. Sharma and virus in brinjal was noticed by storing seeds Varma (1975a) failed to obtain promising results at room temperature (Mayee and Khatri 1975). in eliminating CpBMV from infected cowpea They have also observed a 72.5% reduction in seeds by gamma ray treatment at 20 KR. There virus incidence content in the first 3 months after was very little reduction in seed transmission of storage in the var S-16, and after 7 months, no the virus at higher doses (30 and 40 KR), but viral incidence was reported without appreciable viability of seeds was severely affected. Although loss in viability. Rader et al. (1947) recorded germination after treatment was good, a majority a decrease in mosaic virus infection (3Ð5%) in of seedlings died early due to a lethal muta- freshly harvested seed of muskmelon. It was tion induced by gamma rays. Treating infected observed that CGMMV was minimised by stor- seed with effective chemical inhibitors like 2- ing the infected seed for 2Ð3 years before sow- thiouracil or heat, followed by gamma irradiation ing. In tests conducted during 1961, infection of may yield better results in curing infected seeds. cucumber plants grown from seed obtained in 1960 was 44.0% and only 9.5% from a 1958 seed lot (Yakovleva 1965). Similarly, in 7 can- taloupe seed lots with 11Ð31%, Squash mosaic 8.3 Reducing the Rate of Virus virus (SqMV) transmission in 1967 showed no Spread Through Vector transmission in 1969 (Powell and Schlezel 1970). Management VanKootandVanDorst(1959) recommended 1- year storage for freeing cucumber seeds from the 8.3.1 Avoiding of Continuous same virus. Similarly, Prunus necrotic ring spot Cropping virus (PNRSV) and Prune dwarf virus (PDV) in the seed of Prunus spp. were lost after a 5-year Infection sources, generally from the same crop storage period, but seed viability decreased at a or from other crops which are susceptible and slower rate than that of virus infectivity (Fulton grown nearby, will lead to severe virus problems. 1964; Megahed and Moore 1969). Retention of Some of the crops like tomato, cucurbit, peanut CpBMV in cowpea seeds of cv. Pusa Dophasli and other legumes are grown throughout the year has been noticed when stored for up to 2 years without any break, and inevitably new crops are 190 8 Methods of Combating Seed-Transmitted Virus Diseases grown near old ones. By breaking the disease in the soil for long periods. In certain cases, the cycle, it is possible to minimise the spread of presence of weeds in the field becomes more virus diseases which have limited host range. dangerous as they are symptomless virus carriers Effective virus control would be significant only and consequently are difficult to assess. Control- if a crop-free period is enforced or voluntarily ling the weeds either manually or by weedicide agreed upon at a regional level. For example, a application in and around the fields has given celery-free period has been enforced in California encouraging results in crops like cucurbits and (USA) over four counties to control Celery mo- celery against CMV. Volunteer infected peanut saic virus, and this was also voluntarily imposed plants proved to be the source of PMV inoculum by growers in Florida (Zitter 1977). Similarly, for soybean crop in Georgia (USA), and the dis- in Salinas Valley (California, USA), a lettuce- ease spread was reduced by destroying volunteer free period in December along with seed certi- plants (Demski 1975). In India, high incidence fication programme has reduced Lettuce mosaic of PBNV is noticed in South India where ever incidence (Zink et al. 1956; Wisler and Duffus Parthenium weed population occurs in high pro- 2000). portion (Reddy et al. 1983). Similar situation Many aphid species will be drastically reduced was also recorded in sunflower in relation to in hot dry climates when the daily maximum TSV incidence (Prasada Rao et al. 2003, 2009). temperature exceeds 35ıC (Maelzer 1981). For All growers in a region should cooperate in a this reason, lettuce seed was grown free from weed control programme involving removal of mosaic virus during summer in Australia (Stubbs the weeds manually or by large-scale weedicide and O’Loughlin 1962). In Southeast Asian application. Even wild plants act as direct source countries where peanut is extensively cultivated, of viruses and vectors. Their removal eliminates PMV and PStV which are seed transmitted are the sources of infection, reduces virus spread in gaining importance since their aphid vector seeds Ð if the virus is seed transmitted Ð and also Aphis craccivora occurs in high proportions prevents vectors from breeding on them. in almost all seasons on a number of legume crop and weed hosts. Selection of virus-free seed and skipping peanut in one season may help in 8.3.3 Roguing reducing the virus spread. Certain nematode and fungal-transmitted viruses could also be reduced Even by roguing, the infected plants when the by avoiding planting of the susceptible crops in virus incidence is very low, especially in small fields infected with soilborne vectors. plantings, help in minimising the spread of virus. Inouye (1962) reduced the incidence of BSMV in two varieties from 85 to 12% and 46% to nil 8.3.2 Elimination of Weed, through early roguing. Zink et al. (1957) found Volunteer and Wild Hosts that roguing LMV-infected plants once soon after thinning had no effect on disease incidence at har- Weed and volunteer plants being major vest (81.7%) presumably because much spread components of the agro-ecosystem not only had already occurred, but roguing twice or thrice compete with crop plants for water and nutrients reduced the incidence, that is, from 73 to 43 and but also serve as source of inoculum, since they 36% and 17 to 9 and 6%, respectively, in two field harbour virus diseases (Duffus 1971;Thresh trials. In most countries, the commercial growers 1981;Sastry1984). rogue out the infected plants in seedbeds of crops It is well understood that weed hosts act as like lettuce and brassicas, but it is difficult to sources of virus inoculum for both the crop and implement roguing practice over large areas and the vector. In some of the weed hosts, the virus is also against the seed-transmitted virus diseases seed transmitted, and the infected seeds survive that spread rapidly and far from small initial foci. 8.3 Reducing the Rate of Virus Spread Through Vector Management 191

8.3.4 Crop Rotation removal of virus reservoirs (Raccah et al. 1980). At Davis (USA), Slack et al. (1975) reported Crop rotation has historically been a major means reduced spread of BSMV in spring-seeded barley of disease control in production of annual and by producing seed sources in fall-seeded areas. biennial crops. Generally fallowing is recom- At Pullman (Washington state, USA) wherein mended against soilborne nematode diseases. A the USDA Phaseolus germplasm collections are short period of fallow may or may not decrease maintained, Klein et al. (1989) could successfully the nematode vector population since they live for reduced the BCMV incidence by over 66% in 26 long periods. Encouraging results are available Phaseolus vulgaris accessions by propagation wherein the soilborne diseases are minimised in the greenhouse during the winter (January by crop rotation with non-susceptible hosts of through June) because of the absence of aphid virus/vector. vectors. The decline in BCMV incidence was TMV remains infectious even after 2 years in sufficient in most accessions as BCMV could the old infected root debris, and crop rotation is be eliminated from the important germplasm one of the ways of freeing the soil from TMV collections by elimination of infected plants satisfactorily, as the root debris and viruses are without affecting genetic diversity. In Nepal, eventually destroyed by fungi and bacteria in PSbMV incidence was higher in late planted pea cultivated soil. Tomato mosaic virus on tomato crops (January) than those sown in NovemberÐ crop was successfully eliminated by composting December (Dahal and Albrechtsen 1996). In tomato residues, and it was due to biological India, even Puttaraju et al. (2002) have noticed elimination of the virus or due to heat inactivation higher incidence of BICMV in cowpea sown in (Avegelis and Manios 1989). This suggests the March to May than in September to November. use of tomato compost in plastic houses since the At Ranchi (India), Prasad et al. (2007) found virus is inactivated during composting. that BCMV incidence in French bean would be lowest (5.4 and 7.3%) when sown on 5th and 20th September. Maximum BCMV incidence 8.3.5 Planting Dates (25.4%) was recorded in late sown crop, that is, 19th November. It was because of aphid Adjusting seeding/planting and harvesting time vector population which was least in September of the crops based on vector migration is a sown crop and maximum aphid population was strategy of control for some seed-transmitted recorded in November sown crop and BCMV virus spread. From Germany, Kemper (1962) incidence is directly related to aphid vector reported low incidence of LMV in lettuce population. plantings made during March when compared with the crop grown during JuneÐAugust, when the aphid vector population reaches its maximum. In contrast, in Jordan, the incidence of LMV 8.3.6 Plant Density increased from mid-January to end of February (Al-musa and Mansour 1984). In Delhi (India), High density of plants generally reduces the num- cowpea crop grown during MarchÐMay had ber of infections per unit area, and the planting considerably less CpBMV infection than that rate should be such that it should cover the soil sown in the month of July (Sharma and Varma without reducing the yield due to competition. 1984). In the coastal areas of Israel, bell peppers Some of the outstanding examples wherein high are not planted before mid-April to avoid heavy plant density decreased the incidence of seed- infections of CMV. From the beginning of May, transmitted viruses are PMV in soybean (Demski infection rates decreased in the vector population and Kuhn 1975) and CpBMV in cowpea (Sharma decreased because of drying of weeds and and Varma 1984). 192 8 Methods of Combating Seed-Transmitted Virus Diseases

8.3.7 Barrier and Cover Crops sativa) significantly decreased the incidence of Watermelon mosaic virus (WMV) in watermelon In developed countries, the protective cropping (Chalfant et al. 1977). There is scope for reducing of some commercial crops like lettuce, tomato SMV incidence by having sunflower as a barrier and cucurbits has been done by cultivating them crop in soybean (Irwin and Goodman 1981). in screenhouses/glasshouses. The scientists of the Virus-free nurseries of brassica, chillies, brinjal, Asian Vegetable Research and Development Cen- etc., could be raised by growing barrier crops like tre, Shanhua (Taiwan), found the tunnels of blue barley, wheat or sorghum around the seedbeds. plastic screen of no. 200 mesh (13.5 lines/mm) Choice of the distance between the barriers for placed over rows of Chinese cabbage was found providing the maximum protection will depend to be effective in greatly reducing Turnip mosaic upon many factors such as vigour, thickness and virus symptoms and in increasing yields (Anony- height of barrier crop and the environmental mous 1977b). Because of the high cost involved factors like the direction of the wind and its in glasshouse or screenhouse construction using velocity. plastic sheet or nets, simple methods like barriers of either plants or muslin cloth screens were used in virus control. 8.4 Integrated Cultural Practices The barrier crops should be generally of fast- for Seed-Transmitted Virus growing taller species, which would be not sus- Disease Management ceptible hosts for virus and vector, and hence, monocot plants are planted as barriers for dicot Cultural practices individually were proved to crops and vice versa. The barrier crops are more be effective in reducing the seed-transmitted effective against stylet-borne viruses, since the virus incidence in different crops. However, some aphid vectors lose the virus while probing the experimental results are available where in more barrier crop even for a brief period. The same effective virus management was achieved when principle also applies to cover crops. Devaux integration of different cultural practices were (1977) and Deol and Rataul (1978) reduced CMV followed. This approach was quite effectively infection in cucumber and chillies, respectively, proved by the experiments conducted by Bwye by planting the crop behind barrier rows of corn, et al. (1999) in lupins against Cucumber mosaic sorghum and pearl millet. virus. Seven experiments were conducted in In India, about 50% reduction of CpBMV Australia with lupin (Lupinus angustifolius) incidence in cowpea was achieved by raising 2 to examine the effects of cultural practices on rows of pearl millet at a distance of 3.75 and incidence of Cucumber mosaic virus (CMV). 5.25 m, but complete protection was achieved The factors investigated were row spacing, when the barrier rows were provided at 1.5 and banding fertiliser below seed, straw ground 2.25 m apart; however, this adversely affected the cover and tillage. The seed sown carried 5Ð growth of cowpea. Mixed cropping of cowpea 15% CMV infection. Seed-infected plants were with maize reduced the virus infection to 3.0%, the primary source for subsequent virus spread as against 12.6% in pure stand of cowpea crop by aphids. Incidence of seed-infected plants (Sharma and Varma 1984). and the extent of virus spread were gauged The least incidence of mosaic diseases was by counting numbers of lupin plants showing recorded in muskmelon by growing wheat as typical seed-transmitted and current-season a barrier crop (Toba et al. 1977). The wheat CMV symptoms. Due to greater competition with plants apparently provide additional probing other plants within wide than narrow rows, wide sites, so that incoming viruliferous aphids row spacing diminished the survival of seed- lose their virus on the non-host plants before infected plants by 46%. Increased plant growth probing susceptible cantaloupes. Similarly, at from banding superphosphate below seed did not Georgia (USA), a wide barrier of oat (Avena significantly decrease numbers of seed-infected 8.5 Crops Hygiene 193 plants surviving. Straw spread on the soil surface present on the seed coat adhering to the seedlings suppressed final CMV incidence by 25Ð40% or adhering to the root surface; at this point, and, when applied at different rates, diminished the seedling is only contaminated but not in- recorded CMV incidence more at 4 than 2 t/ha fected. When the seedlings of tomato/pepper are and least at 1 t/ha. Where there was no straw, pulled out from the ground for transplanting, CMV incidence increased faster with narrow root hairs are broken, giving way for entry of spacing than wide spacing. Soil disturbance TMV particles, and thus, the actual infection of from sowing seed with double discs instead of seedling takes place only at the time of trans- types significantly increased incidences of both planting. Seedlings are not infected when left seed-transmitted and current-season infection undisturbed even though they are raised from and diminished grain yield. Neither straw nor diseased seed. At USSR, it was found in field row spacing treatments significantly affected experiments that TMV incidence in tomato crop grain yield, but the decrease in CMV spread due was 10Ð15 times more when the crop was trans- to straw ground cover significantly increased planted and that cultivation without transplanting individual seed weight and overall yields were increased yield by 19% and also improved fruit greater with straw. Myzus persicae was the main quality (Nikitina 1966). Taylor et al. (1961) rec- colonising aphid species, but Aphis craccivora ommended direct sowing instead of transplant- and Acyrthosiphon kondoi also colonised the ing of tomato seedlings. Direct sowing has been lupins. There were significantly fewer colonising adopted by some growers to eliminate prick- M. persicae in plots with 4 t/ha of straw than ing out effect. This method is easier now as it in those with none. This work suggests that is possible to obtain pelleted seed. Goldin and stubble retention, minimum tillage and wide row Yurchenko (1958) observed infection of only 5 spacing should be included as components of an plants in a field in which tomato seed was di- integrated disease management strategy for CMV rectly sown in comparison to 71 infected plants in L. angustifolius crops. Effective management when they were transplanted. However, raising of nonpersistent aphid-transmitted viruses in the nurseries and transplantation is the popular Lucerne and CMV in lupins was achieved way of raising crops. The risk of mechanical by developing integrated disease management contamination could be greatly reduced, however, strategies by Jones (2001, 2004b). Similar by spraying plant beds of tomato and pepper type of integrated approach with modifications with milk just before plants are pulled out for depending on the locality and environmental transplanting. Dipping and washing hands with conditions, trails should be tried with different milk before handling the nursery is quite useful seed-transmitted viruses in other crops. since TMV is rapidly inactivated by proteins present in milk (Crowley 1958; Hare and Lucas 1959). Besides sterilisation, the virus contamination 8.5 Crops Hygiene on the farm implements can be reduced by washing with 10% trisodium phosphate or Against highly stable viruses like TMV and Cap- 2% formaldehyde plus 2% sodium hydroxide sicum mosaic virus in tomato and capsicum crops (Broadbent 1963; Patezas et al. 1989). The and also against certain viroid diseases, hygiene workers should not keep tools in their pockets is particularly important. Some of the recom- which might contain tobacco debris. Even mended measures to combat the spread of these preferring non-smokers or non-snuffers for jobs diseases during the cultural operations are as fol- which involve much handling of plant seedbeds, lows: For example, in tomato and pepper, TMV planting, etc., would minimise virus spread. occurs as a contaminant on the seed coat, and If not, every effort must be made to prevent as the radicle (root) grows, it comes into con- smoking or taking of snuff while work is in tact with the surface of the seed. TMV particles progress. 194 8 Methods of Combating Seed-Transmitted Virus Diseases

It has been demonstrated that packing (Fig. 8.1) which is followed as mentioned here. containers, if contaminated, also act as a probable Germination varies depending on variety, seed source of infection. The spread of CGMMV was quality and soil mixture. For optimum germina- attributed to the use of contaminated packing tion, sow seeds in a well-drained, sterile soilless containers used for harvest of cucumber fruits mix at 25Ð28ıC, and water daily. Under these and also for the transport of young plants (Van conditions, seeds will germinate in about 8 days. Dorst 1988). During transport, the leaves might Seeds will germinate in 13 days at 20ıCand have touched the contaminated sides of the 25 days at 15ıC; they may not germinate at all containers, resulting in early infection. Use of if temperatures are below 15 or above 35ıC. For low-cost disposable packing containers will help example, 1 g of chilli pepper contains approxi- in minimising contamination. mately 220 seeds. Approximately 150 g may be It is also established that TMV spreads from needed to transplant 1 ha at a density of 30,000 one crop to the next on contaminated trellis, plants/ha, assuming 90% germination and 90% of seedbed cloth, frame boards and wires. Nitzany seedlings are of good quality. (1960) reported retention of TMV for one and Fill the seedling tray with sowing medium, half to four months on trellis wires removed from such as peat moss, commercial potting soil, or infected fields when stored in a shaded store. a potting mix prepared from soil, compost, rice He also reported virus inactivation by dipping hulls, vermiculite, peat moss and/or sand. The infected trellis wires in 1% formalin for 5 min potting mix should have good water-holding ca- or 5% TSOP for 10 min or 0.1% caustic soda pacity and good drainage and recommend a mix- solution. In potato, PVX and Potato spindle tuber ture of 67% peat moss and 33% coarse ver- viroid diseases which are seed-transmitted spread miculite. For use of non-sterile components, it in the field during tractor operations. Washing the is recommended to sterilise the potting mixture machinery thoroughly with any one of the earlier by autoclaving or baking at 150ıCfor2h.If mentioned virus-inactivating detergents before it seedlings are to be grown on a raised soil bed, the is taken into the next healthy crop helps in reduc- soil should be sanitised by burning a 5-cm-thick ing the spread of the disease. layer of rice straw or other dry organic matter on In the glass/mesh houses which are used for the bed. This also adds small amounts of P and K tomato cultivation in countries like the USA, to the soil for the seedlings. Hungary, the UK, etc., and also for conducting Sowing one seed per cell of potrays (or broad- research work under controlled conditions, con- cast the seeds lightly in a seedbed) and covering tamination with TMV is a general problem as or sow them inside a greenhouse or screenhouse. even the small fragments of the infected leaf This provides shade and protects seedlings from debris either on the benches or the floor also serve heavy rain and pests, such as aphids, which trans- as source of virus inoculum. Thorough washing mit viruses. Use a fine sprinkler. Irrigate with a of glass or wire mesh houses with a disinfectant 0.25% (w/v) solution of water-soluble or liquid when they are empty between crops will ensure fertiliser (10-10-10) when two true leaves ap- that they do not become contaminated. pear. If damping-off occurs, irrigate with a 0.25% (w/v) solution of Benlate or similar fungicide. If the seedlings have been grown in shade, harden 8.5.1 Raising Transplants them by gradually exposing them to direct sun- light over 4Ð5 days prior to transplanting. On the A number of MNC seed companies and research first day, expose them to 3Ð4 h of direct sunlight. organisations are rising the transplants of crops Increase the duration until they receive full sun like tomato, capsicum, cucurbits, rice and other on the 4th day. The procedure described herein crops under protected environment to have good has been mentioned in one of the popular articles growth and protection from pests and diseases from AVRDC, Taiwan (Berke et al. 2005). 8.6 Control of the Vectors 195

Fig. 8.1 Raising transplants in protected environment (Source: R.A. Naidu)

In India, the spread of seed-transmitted virus 8.6 Control of the Vectors disease of cowpea was reduced by soil drenching with granular insecticides like aldicarb, phor- 8.6.1 Insecticides ate and dimethoate (Sharma and Varma 1972). Similarly, in the Primork region of the former Chemical control of vectors to avoid or limit USSR, the sprays of synthetic pyrethroid, L- introduction of virus and subsequent spread has cyhalothrin, at 5.0 and 7.5 g/ha induced aphid been in practice for long time. It proved to be suc- vector mortality and reduced the incidence of cessful in certain situations because of the ease cowpea viruses (Atiri and Jimoh 1990). SMV of application, ready availability, low cost and was minimised by 12.3Ð24.3% by management general effectiveness against insects. More than of aphid vectors through the application of aphi- 50% of seed-transmitted viruses are aphid-borne don and disyston and increased yields by 5Ð12% and nonpersistent type, wherein a few seconds of (Smirnov Yu 1975). In general, insecticides do acquisition and inoculation periods are required. not seem to have any effect on vectors of non- Adequate control methods are lacking since the persistent viruses which infect the crop from insecticides applied do not inactivate or kill the sources outside and have long continuous flight vectors within a short period before acquisition periods. A more effective method is to use the and inoculation, and before they die, the vectors insecticide against the vector either on the virus infect the plants. In recent years, photostable source outside the crop or on trap crops. synthetic pyrethroids have been prepared, some However, controlling the soilborne vectors by being found to be fast-acting insecticides and application of chemicals to infested soil is dif- have low mammalian toxicity (Naumann 1990). ficult as some of the nematode vector species However, very limited information is available on occur at considerable depths in the soil for long the effect of insecticide application in reducing periods. Another factor is that the virus could be the spread of some seed-transmitted viruses. transmitted to a plant by a single nematode. The 196 8 Methods of Combating Seed-Transmitted Virus Diseases efficient management of nematode-transmitted Germany. Some of the seed-transmitted virus virus disease requires application of a fumigant diseases which are effectively controlled by oil before planting to decrease nematode numbers. spray are CMV in cucumber (Loebenstein et al. Application of granular systemic nematicide at 1964, 1966), Pea mosaic virus in broad bean the time of planting is necessary as this will (Zschiegner et al. 1971), etc. At Georgia (USA), decrease the efficiency of surviving nematodes PMV and CMV were found to cause enormous and facilitate the establishment of the crop. This yield losses in peanut and soybean, and encour- has to be followed by repeated application of a aging results were achieved by spraying 0.75% systemic foliar spray or a drench to maintain a oil at weekly intervals (Kuhn 1969). toxic environment around surviving nematodes The cost of protecting a seed crop with oil throughout the life of the crop. Such a control could be about $35 per acre for material. Kuhn programme would be expensive and would re- and Demski (1975) estimated that peanut losses quire careful epidemiological and environmental from PMV in Georgia amount to $11,000,000 an- evaluation. The future of systemic nematicides nually. The cost of oil to protect the 30,000 acres that control nematodes transmitting plant viruses of crop needed for seed production and to plant will depend on the development of materials with the 512,000 acres of commercial peanut would greater direct nematicidal toxicity and/or greater be about $1,000,000 annually which provides persistence. Greater persistence could introduce the Georgia peanut growers an 11:1 return on the problem of toxic residues in the crop, so serial investment in virus control. While working with applications of short persistence materials could CMV, Loebenstein et al. (1966) also observed an be preferable. increase of 50% in the yield of marketable cu- Attempts were also made to control fungal- cumbers with low-volume sprays of summer oil. transmitted viruses by using biocides. The The use of oil as sprays has several advan- incidence of clump disease transmitted by P. tages. It is low cost, has good spread ability, is graminis in peanut crop could be reduced by easy to mix and has low toxicity to man and applying dibromochloropropane and furadon animals. Another important consideration is the at nematicidal dosages. Nevertheless, their fact that insect vectors have not yet developed application on small forms is unlikely to be any resistance to them. The efficacy of oils could economical (Reddy et al. 1988). be enhanced by judicious application at suitable concentration with the appropriate spray nozzle for the proper stage of the crop. A great disadvan- 8.6.2 Mineral Oils tage, however, is that these oils have to be sprayed at frequent intervals for effective control of virus Bradley (1963) showed that mineral oil sprays spread. Limitations to the use of oil sprays are impeded virus infection. Control of a large plant toxicity, volatility or viscosity of the oils, number of stylet-borne seed-transmitted virus adequate coverage of the leaves and removal of diseases with oil spray has been tried by the oil cover by rain or irrigation water. researchers with varying success for the last 25 years (Peters1977; Vanderveken 1977; Zitter and Simons 1980; Simons and Zitter 1980; 8.6.3 Repelling Surfaces Simons 1981, 1982; Sharma and Varma 1982a; Sastry 1984). Some of the seed-transmitted plant virus diseases Of the different kinds of oils in use, namely, of legumes, cucurbits and certain solanaceous vegetable, mineral, synthetic and essential oils, crops have insect vectors like aphids, beetles, effective control of virus diseases was achieved thrips and others which are responsible for only with mineral oils in countries like the USA, virus spread. Like insecticide and mineral the Netherlands, Israel, the UK, India and West oil application for vector control, even some 8.7 Virus Avoidance 197 repelling and attracting surfaces of aluminium, plastic and straw mulches were used in certain 8.7 Virus Avoidance virusÐhost combinations. Aphids transmit a 8.7.1 Exclusion number of seed-transmitted viruses, and the aphids respond differently to various wavelengths of light; the use of attractive colours as traps In various countries and territories, legislation or repellents to avoid landing of the vector on currently in force to prevent introduction of susceptible crops is advantageous in minimising important seed-transmitted diseases and pests the spread of virus diseases. The visible infrared involves the following quarantine regulations: (1) and ultraviolet lights emitted from different embargo and import permit, (2) inspection (field surfaces are responsible for vector repellency. inspection and laboratory testing) in the exporting It was first demonstrated that white surfaces country before shipment of the consignment, (3) reflecting ultraviolet or short wavelength which seed treatment for diseases and pests that can was unattractive to alighting aphids were even be eliminated by disinfection or fumigation with avoided by them (Moericke 1954). In number reasonable certainty, (4) post-entry growth in- of countries, aluminium mulches were used spection by the importing country in closed quar- for controlling virus diseases in crops like bell antine and (5) certification. A detailed discussion pepper, tomato, cucumber, lettuce and in certain of general quarantine principles and philosophy, ornamental crops. These mulches are effective except some information on seed-transmitted at least against 12 species of aphids (Smith virus diseases, is beyond the scope of this book. et al. 1964). In French beans, aphid-transmitted More information on quarantine aspects can be BCMV is effectively reduced by using silver obtained from the review articles of Hewitt and polythene film. Experiments conducted in Japan Chiarappa (1977), Kahn (1977, 1989), Neergard with reflective mulches in pea crop were effective (1980), Lovisolo (1981), Chiarappa (1981), in reducing Pea seed-borne mosaic virus. Reddy (1982), Raychaudhuri and Khurana A number of attempts were also made to (1984) Plucknett et al. (1987), Jalil et al. (1989), reduce the virus incidence by minimising the Frison et al. (1990), Kahn and Mathur (1999), vector population by using plastic mulches. The Ebbels (2003), Khetarpal (2004), Khetarpal and plastic mulches are cheaper than the aluminium Gupta (2006) and Munkvold (2009). mulches, and partial success has been achieved in Migrations, military conquests, voyages of many countries by reducing the virus incidence discovery, religious expeditions and germplasm by using different coloured plastic mulches. exchange have in many ways contributed to the Jones and Chapman (1968) tested plastic sheets spread of seed material. In addition, ship loads in nine different colours in addition to aluminium of grain for consumption or large quantities foil to determine their attractiveness to aphids. of seeds for direct sowing are imported by Yellow colour was most attractive, followed in many countries. Most countries differentiate order by pink, green, red and black, whereas between the seed imported for scientific purposes white, orange, light blue and dark blue colours and those imported for sowing or commercial attracted the fewest aphids. In certain crops, purposes. Since more than 231 viruses are seed straw of cereal crops was used as mulch and transmitted, there is a risk of introducing the proved to be effective in reducing the insect virus diseases which are not known to occur vector population and virus incidence (Cradock in a country if proper testing is not carried et al. 2001; Summers et al. 2005). The straw out. Even minute quantities of soil and plant as mulch will be effective for a short period debris contaminating true seeds can introduce when compared to aluminium or plastic mulches; the virus/vector, or both. Now a days chances however, due to biodegradation, the straw will of virus introduction are greater with quick mix with soil and enriches the soil. and efficient air transport enabling exchange 198 8 Methods of Combating Seed-Transmitted Virus Diseases of large quantities of seed material between plant the occurrence of high seed transmission of breeders and crop scientists around the world. BCMV in bean germplasm and CpAMV in Vigna Thus, man is the direct or indirect cause of most species is noticed in almost all countries. Klein epidemic imbalances that we know about, and et al. (1988) reported approximately 60% of the high virulence and extreme susceptibility of the 207 French bean accessions contaminated an epidemic situation is an unnatural imbalance with BCMV. Peanut stripe virus (PStV) was usually brought about by human disturbance introduced into the USA in the 1980s through (Stace-Smith 1985; Jones 2000; Degirmenci and peanut seed in germplasm imported from China Acikgoz 2005). (Demskietal.1984b). Even in India, the same The basic objective of plant quarantine is to virus was detected in peanut germplasm lines in check the introduction and spread of potentially Andhra Pradesh and Gujarat states (India) which dangerous pests and diseases imported along with was introduced through germplasm exchange germplasm from region to region by official reg- (Prasada Rao et al. 1988, 2004). During 2001, ulation. In various countries and territories, legis- PStV was intercepted in Australia from imported lation currently is in force to prevent introduction peanut seeds (Persley et al. 2001). At NBPGR, of important seed-transmitted diseases and pests. New Delhi (India), Chalam et al. (2005a, b)and There is need for systematic effort to reach Khetarpal et al. (2001) have reported viruses the goal of virus-free germplasm collection and which were intercepted in germplasm of different maintenance. Often the virus affected germplasm crops like cowpea, mung bean and broad bean in collections are received at quarantine stations the quarantine section, and the list of viruses is as in cases like soybean with SMV (Goodman presented in Table 8.3. and Oard 1980) pea and lentil with PSbMV Chalam et al. (2007a) have screened the (Hampton 1982, 1983) and; Coutts et al. (2008), imported seed material by following the French beans with BCMV and CMV (Hampton advanced serological techniques and could find and Braverman 1979; Davis et al. 1981). In post- five exotic viruses which were not recorded in entry quarantine stations in peanut germplasm, India, namely, Barley stripe mosaic virus in PStV virus was intercepted (Demski et al. 1984b; barley and wheat; Broad bean stain virus in Persley et al. 2001). Urd bean with ULCV (Beni- faba beans; Cherry leaf roll virus in soybean and wal et al. 1980) and the viruses involved were French bean; Cowpea mottle virus in cowpea and commonly detected in routine seed evaluation Raspberry ring spot virus and Tomato ring spot tests. Hampton et al. (1982) listed some of virus in soybean. Hence, every care should be the viruses that could be expected to occur in taken to minimise the seed-transmitted incidence germplasm accessions of major crops in the USA. of viruses in accession seeds since it will be a Seed-transmitted infections by PSbMV in source of contamination of breeding stock as pea germplasm, sometimes reaches 90% or well as a cause of yield losses commercially. Its more, are the main sources of its distribution management in germplasm collection conditions over long distances (Hampton and Mink 1975). should be taken into account for the possible The germplasm collection such as the USDA loss of genetic diversity of some accessions pea collection at the Northeast Regional Plant (Alconero et al. 1985). Procedures used in the Introduction Station, Geneva, New York, is not elimination of the virus should be consistent only a source of valuable pea characteristics but with the need to maintain genetic characters of also a source of seed-transmitted pathogens such the accessions. Therefore, FAO/United Nations as PSbMV. It has been estimated that 23% of the Environment Programme/International Board of accessions in the collection were infected by the Plant Genetic Resources in a conference during virus (Hampton and Braverman 1979). Similarly, 1981 have recommended that all germplasm Alconero and Hoch (1989) have recorded 189 exchanges should take place through National isolates of PSbMV from seedlings of 435 pea Quarantine Services. They have suggested that germplasm introductions. Similar situation of national and regional laboratories be established 8.8 Resistance 199 to expedite the passage of germplasm through plant genes while lowering the chances of inad- National Quarantine Services. The seed lots vertently exchanging some of the serious seed- received at the quarantine stations should be transmitted viruses. initially evaluated by growing-on tests, indicator- Even though strict legislative measures are inoculation tests, serological tests, radiography, enforced, still a few new virus diseases are in- etc. In recent years, serological tests like ELISA, troduced into countries and are published as new RISA, SSEM, cDNA, PCR, RT-PCR, and records either to the host or virus. Some reasons western blot have been developed and are used attributed to this are as follows: The methods for quick and judicious detection of the seed- may not be sensitive enough to reveal latent transmitted infection to avoid the introduction of infection and the lack of adequately trained per- new viruses or severe strains of certain existing sonnel. Those responsible for quarantine mea- viruses. Hence, strict quarantine treatments are sures should be properly trained and experienced. applied in almost all countries to prevent the entry There is need for professional workers and the of new viruses into an area where it is not known public to cooperate on an international scale for to occur Ð or is of limited distribution. Quarantine effective operation of quarantine regulations. The treatments can be divided into three groups: (1) importance of quarantine has increased by many chemotherapy, (2) thermotherapy and (3) cultural folds in the WTO regime and by adopting not procedures. The impact of these treatments only the appropriate techniques but also right in different virusÐhost combinations has been strategies for virus detection would go a long way discussed earlier in this chapter. In almost all in ensuring virus-free seed trade and exchange of countries, quarantine stations are located near germplasm. airports or seaports, wherein vigorous testing of the seed lots is carried out despite the delay and inconvenience involved. 8.8 Resistance From the facts cited above, it is clear that in general the quarantine policy towards seed is not Plant resistance in crop plants having virus trans- adequate without sound seed health testing fa- mission through seed has the great advantage cilities for detection of seed-transmitted viruses. for a component that it usually enhances the There is need for international cooperation, par- effectiveness of other virus management mea- ticularly in the alignment of seed health testing sures. Plant resistance can reduce virus infection methods. Emphasis has to be laid on seed crop and disease development, and a large number of certification with a view to supply virus-free seed disease resistance genes were identified during material to growers of all countries. 1986Ð1996. Although the scientific and technical In order to lower or minimise the virus risk as- advances have been rapid, additional studies must sociated with the import of seed, safeguards like be carried out for the better understanding of the regulations themselves, phytosanitary certifi- the molecular basis of resistance of producing cates with or without added declarations, permits, transgenic plants. inspection upon arrival, treatment, quarantine, It is well documented that host plant and vec- etc., are to be undertaken. tor resistance are the most effective control mea- Embryo culture methodology is being used sures against certain seed-transmitted diseases. even though it has certain limitations as a safe- Usefulness and success of the resistance strategy guard in the international transfer of plant genetic depends on our knowledge of the mechanism(s) stocks. Kahn (1977, 1989) has developed this of resistance and its effects on the virusÐvectorÐ technique by using tissue culture methodology. host interactions. More complex and probably In this technique, instead of complete seed, more durable resistance is more difficult to es- only embryo axis is excised and used for cul- tablish and certainly more difficult to breed. This turing, and it can be mailed without any risk. does not preclude their existence or possible The objective is to facilitate the exchange of future utilisation. Paradoxically, application of 200 8 Methods of Combating Seed-Transmitted Virus Diseases knowledge of the genetics of major gene resis- disease resistance. Interestingly, it has been tance in breeding programmes may have mili- shown that variation induced by callus culture tated against breeding for polygenic resistance by may be under monogenic or polygenic control. more empirical approaches. Culture techniques may provide a convenient, It is clear that before horizontal or polygenic non-hybridisation method for selecting and resistances can be exploited in crop protection, handling polygenic resistance. there should be an attempt on cost–benefit as- The technique that leads to production of hap- sessment. The evidence for horizontal resistance loids, although spontaneous or chemically in- against plant viruses is still sparse. It may be more duced doubling of the chromosome number can effective to combine known major genes which give dihaploids. Production of a number of dihap- are genetically well understood and therefore can loids can expose variation present in the parent, be handled on a rational rather than empirical by eliminating dominance and complex hypo- basis, to construct robust oligogenic systems. static effects, and by offering a number of ‘cross In some of the crops where seed transmission sections’ of genetic variability which then can of virus is noticed, resistant/tolerant varieties be usefully combined in accelerated breeding were identified namely, soybean lines against programmes. The process of haploidisation also Soybean mosaic virus (Almeida 1995; Provvi- may be mutagenic and leads to the association denti 1977; Pedersen et al. 2007), muskmelon of virus resistance with other desirable charac- lines against Cucumber green mottle mosaic ters. virus (Rajamony et al. 1990), pea lines against The basic techniques for transferring plant Pea seed-borne mosaic virus (Khetarpal et al. genes between taxonomically unrelated species, 1990), cowpea against cowpea viruses (Sharma and for ensuring their expression in the recipient and Varma 1981) and soybean against Soybean species, are now established. Many problems mosaic virus (Cho and Goodman 1979;Lim remain to be solved, but these are perhaps matters 1985;Suteri1986; Arif and Hassan 2002). of degree rather than of fundamental principle. Novel methods for revealing or creating vari- For resistance genes, the major difficulty remains ation and for transferring it between genotypes in the isolation of useful genes which have been by nontraditional methods are already available successfully transferred and coded. Known major and are increasingly being applied to resistance proteins produced in large amounts, and these genes. factors have undoubtedly eased gene identifica- The inability of even quite closely related tion. Knowledge of gene products and biochem- species to hybridise creates a major barrier to istry of action is so scanty that isolation of re- transfer of potentially useful resistance genes. sistant genes is going to be a major difficulty. This may be especially relevant to wild relatives Unless suitable ‘shotgun’ approaches to selection of crop species. Somatic hybridisation by pro- of resistance genes from gene libraries can be toplast fusion has already shown its ability to developed, there will remain a need for more transfer virus resistance genes between species, fundamental research on mechanisms of gene where transfer by conventional breeding tech- activity. niques has failed because of sexual incompatibil- On the other hand, the demonstrated genetic ity. At present, transfer of genes between genera simplicity of most of the presently understood by somatic hybridisation is more difficult than mechanisms makes them primary targets for ge- between species in one genus. netic manipulation. Furthermore, many viruses Callus culture can induce mutation. Clones cause serious disease in several crop species, regenerated from callus cultures have shown so a resistance gene made accessible to genetic an encouragingly high level of variability manipulation might possibly be utilised several (somaclonal variation) in useful characteristics, times. Care should be required, however, to en- with many clones outperforming the parent lines. sure that this did not lead to placing the genetic Several of these instances involve improved control of virus diseases on too narrow base. 8.8 Resistance 201

Manipulation of resistance genes from several 8.8.1 Host Resistance sources against the same virus could be used to develop artificial polygenic systems. It seems un- Resistance to viruses manifests itself as absence likely that the current art of genetic manipulation of symptoms and/or restriction of virus mul- could deal easily with natural polygenic systems tiplication and spread within the plant and is or horizontal resistance, unless individual compo- an effective measure to control seed-transmitted nents could be recognised. virus diseases. It may not often be practicable It is established that many genetically under- to adopt cultural and chemical control measures stood resistance mechanisms involve gene dosage against seed-transmitted viruses in time because effects. It is possible that an increased effective- of cost and other considerations. However, use of ness of resistance within a cultivar could be ob- resistant/tolerant varieties has been found to be tained by artificial amplification of gene number. the most efficient and economical method, pro- Further reduction in virus multiplication might vided stable sources of resistance are obtained. act to limit the frequency of evolution of viru- A precondition for the development of virus- lence. resistant varieties is that the plant breeder should Some authors have suggested that ability to be aware of the viruses which he is confronting transfer genes between non-hybridisable species and also with its host plant. There are instances of might allow non-host immunity to be used for breaking down of resistance due to the evolution crop protection. This might be possible for non- of new virus strains, and hence, the plant breeder host immunity, although it would be difficult if a aims to introduce disease resistance into cultivars number of genes had to be transferred. Non-host that will provide useful disease control for a immunity based on the ‘negative’ model would period long enough to ensure that the commercial be impossible to transfer, being based on lack life of a cultivar is not curtailed. The major advan- of a susceptibility gene. It does raise the idea of tage of breeding for resistance to viruses is that conferring resistance by deleting or nullifying a once a resistant cultivar is developed, no specific susceptibility gene in the normal host. action is required by farmers to achieve control. In contrast to the complexities of the plant More information on virus disease resistance can genome, the genomes of viruses are very sim- be had from review articles of Fraser and Van ple to analyse and manipulate. The near future Loon (1986), Nene (1988), Boulcombe (1994), should see molecular explanations of the nature Khetarpal et al. (1998), Michael Deom (2004), of virulence, which may provide valuable clues to Kang et al. (2005)andGomezetal.(2009). mechanism of resistance gene function and hostÐ virus interactions. Isolation and characterisation of the genes to 8.8.2 Sources of Resistance be introduced are crucial steps in transforma- tion, and various methods are currently available The examples of durable resistance include both for the introduction of exotic genes into plant monogenic and polygenic resistance, whereas cells. Transformation using the Agrobacterium theoretically polygenic resistance should be more Ti-plasmid system is widely used directly in di- durable. Some of the resistant sources identified cotyledonous plants. The utilisation of virus coat against seed-transmitted diseases are discussed protein genes for the protection of virus infection below. is successful in crops like tomato, cucumber, capsicum, etc. The analysis of gene structures of 8.8.2.1 Resistance in Cultivated Species economically important seed-transmitted viruses Breeding for resistance to virus diseases which in commercial crops is to be carried out in order are seed transmitted has been successful in cer- to use the information on the nucleotide sequence tain cases and is presented below. of the viruses and their satellite components for TMV in tomato (Honma et al. 1968; Besedina the cross protection. 1985)andCapsicum species (Provvidenti 1977; 202 8 Methods of Combating Seed-Transmitted Virus Diseases

Rast 1982;Sowell1982; Patezas et al. 1989); 1975; Lecoq and Pitrat 1983;Yangetal.1986); Tobacco streak virus (Kalyani et al. 2005, mung bean (Sittiyos et al. 1979); and cowpea 2007); PMV in peanut (Kuhn et al. 1968)and (Mali et al. 1987); ULCV in urd bean (Indu soybean (Demski and Kuhn 1975); BCMV in Sharma and Dubey 1984;Bashiretal.2005; peas (Provvidenti 1991) and beans (Temple Ashfaq et al. 2007); Blackgram mottle virus in and Morales 1986; Allavena 1989; Gupta and urd bean (Krishnareddy 1989); LMV in lettuce Chowfla 1990; Ogliari and Castarro 1992; Kelly (Ryder 1970, 1976;Walkeyetal.1985;Pavan 1997; Jones and Cowling 1995). BYMV and et al. 2008); BYMV in yellow lupin (Pospieszyn pea virus II in French beans (Provvidenti and 1985). Alfalfa mosaic in Lupins (Jones et al. Schrolder 1973); Bean pod mottel virus in 2008) and CMV in lupins (Jones and Latham Soybean (Hill et al. 2007); Soybean mosaic 1996). virus in soybean (Lim 1985; Arif and Hassan 2002; Cho and Goodman 1982; Hill et al. 8.8.2.2 Resistance in Graminaceous 2007; Pedersen et al. 2007; Domier et al. 2007) Cereal Crops BYMV in soybean (Provvidenti 1975) and lupins Some of the examples for resistance for virus (Mckirdy and Jones 1995a); subterranean clover diseases of graminaceous crops are Sugarcane (Mckirdy and Jones 1995b) and peas (Musil mosaic virus in sorghum (Teakle and Pritchard and Jurik 1990); PSbMV in lentils (Haddad 1971;Henzelletal.1982), BSMV in barley et al. 1978; Latham and Jones 2001a; Anjum (Timian and Sisler 1955; Jackson and Lane 1981; et al. 2005); faba beans (Fagbola et al. 1996)and Catherall 1984; Timian and Franckowiak 1987; peas (Hampton 1980; Hampton and Braverman Edwards and Steffenson 1996) and MDMV in 1979; Muehlbaur 1983; Baggett and Kean 1988; maize (Miao-Hongqin et al. 1998). Provvidenti and Alconero 1988; Khetarpal et al. 1990; Maury et al. 1992; Thakur et al. 1995; 8.8.2.3 Resistance in Fruit Crops Dhillon et al. 1995; Lebeda et al. 1999; Kraft Raspberry bushy dwarf virus (RBDV) is seed and Coffman 2000; Latham and Jones 2001a, transmitted in Rubus idaeus to the extent of b); SMV in soybean (Koshimizu and Iizuka 22Ð60% (Cadman 1965;Converse1973) and can 1963;Ross1977;KwonandOh1980;Choand be controlled through the use of gene Bu that Goodman 1979, 1982; Bowers and Goodman confers resistance to D-type isolates of RBDV. 1982, 1991;Hashimoto1983; Buzzell and Tu In Rubus, new sources of virus resistance derived 1984;Suteri1986); Cowpea mottle virus in from pathogen themselves have been developed cowpea (Allen et al. 1982); CPMV in cowpea for RBDV in Scotland and North America. How- (Wells and Deba 1961; Robertson 1966; Beier ever, whether fruits produced from transgenic et al. 1977); TRSV and CPMV in cowpea (Mali plants will be a susceptible socially remains to be et al. 1987; Ponz et al. 1988); CpAMV in cowpea determined (Martin 2002). Resistance gene fac- (Ladipo and Allen 1979;Malietal.1987); Black tors for other virus diseases in certain important eye cowpea mosaic virus in cowpea (Ndiaye fruit crops are also well documented. et al. 1993;Bashiretal.2002; Lovely et al. 2006; Kamala et al. 2007); BCMV in French 8.8.2.4 Resistance in Wild Species beans (Zaumeyer and Meiners 1975; Provvidenti Certain wild species of crop plants have been 1977; Walkey and Innes 1979; Sastry et al. screened when the resistant source is not 1981; Naderpour et al. 2010); and Phasemy identified among the cultivars. For example, bean (Provvidenti and Braverman 1976); Melon Culver et al. (1987) identified resistant sources mosaic virus in cucumber (Cohen et al. 1971); to Peanut stripe virus in Arachis diogio (PI- CGMMV in muskmelon (Rajamony et al. 1990) 46814 and PI-468142), A. helodes (PI-468144), CMV in vegetable marrow, Cucurbita pepo (Pink Arachis sp. (PI-468345) and of the rhizomatosae and Walkey 1984; Walkey and Pink 1984); section (PI-468174, PI-468363 and PI-468366). melons (Webb and Bohn 1962; Karchi et al. In India, peanut accessions of the Arachis 8.8 Resistance 203 section A. cardenasii (PI-11558) were absolutely gene closely linked with resistance genes to other resistant to PStV even after sap, aphid and potyviruses BCMV (BICMV strain), CABMV, graft inoculation, while A. chacoense (PI- SMV and WMV-2 (Kyle and Provvidenti 4983), A. chiquitana (PI-11560), A. cardenasii 1987). (PI-11562 and PI-12168) and accessions of Resistance may also be conferred by recessive Erectoides section, A. stenophylla (PI-8215), bc genes (Drijfhout 1978). Presently, resistance and A. Paraguariensii (PI-8973) were resistant programmes have been achieved in developed after aphid and sap inoculation only (Prasada Rao countries giving satisfactory results. However, et al. 1989, 1991). Similarly, sources of resistance necrotic strains of BCMV induced severe epi- to Peanut mottle virus in eight peanut entries of demics in the USA (Provvidenti 1990). Further Rhizomatosae and Arachis sections were also studies also indicated that the virus inducing such identified (Melouk et al. 1984). Jones et al. necrosis was classified as a potyvirus different (2008) have reported that Lupinus angustifolius from BCMV and named Bean common mosaic exhibited milder symptoms on sap inoculation necrosis virus (BCMNV) (Vetten et al. 1992b). with Alfalfa mosaic virus and seed transmission BCMNV is rare in bean crops in Europe and was only 0.8% in this host. From Canada, Singh North America. The I gene-carrying cultivars re- (1985) reported clones 1,726 and 1,729 of PI- sistant to BCMV are susceptible to BCMNV, but 473340 of Solanum berthaultii to be resistant they would not transmit it through seed (Morales to PStVd, which has high seed transmission. and Castano 1987). However, a virulent strain K Provvidenti et al. (1978a) identified Cucurbita causing mosaic on these I gene-carrying cultivars ecuadorensis and C. foetidissima to be resistant to instead of necrosis has been identified (Buruchara CMV. Kalyani et al. (2007) identified 8 resistant and Gathuru 1979). Finally, I gene-carrying cul- accessions against Tobacco streak virus (TSV) in tivars are resistant to both BCMV and BCMNV wild Arachis, which are cross compatible with A. when they possess also the recessive bc resistance hypogaea for utilisation in breeding programme. genes bc3 or bc22 (Davis et al. 1987). Regarding TSV, both positive and negative seed It is also interesting to analyse the situation transmission reports are existing (Sharman et al. in Africa, the third largest producer of bean: 2009;PrasadaRaoetal.2009). Africa often grows beans as traditional mixtures of 10Ð20 cultivars selected for taste, seed colour, speed of cooking and global resistance to the 8.8.3 Conventional Breeding different pathogens. A survey in Rwanda indi- of Natural Resistance Genes cated that 96% of farmers preferred planting mixtures because they resulted in higher and 8.8.3.1 Bean/BCMV more predictable yields. In this country, as well Bean is originated in Latin America which is as in Burundi, bean provides 45% of the total the largest producer. According to FAO (1990), protein of human diet, which is the highest world the area of production in the world covers 24 percentage. Bean is also important in 22 other millions ha, North America being the second African countries. However, average bean pro- largest producer followed by Africa. BCMV was duction in Africa is 660 kg/ha and may be as low found throughout the world wherever beans are as 214 kg/ha, to be compared with 1,500 kg in grown. BCMV is also seed transmitted in other North America and 2,000Ð3,000 in some parts of important legume crops like cowpea (previously Europe (FAO 1990). BICMV Ð Blackeye cowpea mosaic virus)and BCMV/BCMNV is by far the most important peanut (previously PStV Ð Peanut stripe virus). virus disease of bean in Africa. Since the first bean cultivar resistant to BCMV BCMV was frequently isolated as seed trans- (Spragg and Down 1921) improved cultivars of mitted from local farmers’ landraces of bean and bean could be obtained by introduction of a was found prevalent in western Kenya. However, dominant I gene conferring hypersensitivity, a the widespread occurrence and predominance of 204 8 Methods of Combating Seed-Transmitted Virus Diseases

BCMNV was clearly demonstrated in central programmes of incorporation of natural resis- eastern and southern Africa not only on beans but tances have been achieved only in some coun- also, interestingly, on wild species of legumes of tries. Such type of resistance is not completely genera Cassia, Crotalaria, Macroptilium, Vigna satisfactory as the virus multiplies and virulence and Rhynchosia, which also transmitted this virus has been observed. Other approaches are neces- through seed (Spence and Walkey 1993). sary for controlling LMV. BCMNV is not thought to occur naturally in South America, the centre of origin of P.vulgaris. 8.8.3.3 Barley/BSMV When this virus has been recorded there or in Some sources of resistance to BSMV exist, and North America, its occurrence has been traced they are being mapped in relation to molecular to infected imported seed (Spence and Walkey markers, in view of being included in barley 1993). In contrast, the widespread occurrence and breeding programmes (Edwards and Steffenson predominance of BCMNV infecting both beans 1996). and wild species of legumes could qualify Africa as the geographical centre of origin of this virus. The observation that BCMNV is often latent on 8.8.3.4 Soybean/SMV wild species of legumes would suggest host adap- Soybean mosaic potyvirus is widespread in areas tation after long-term exposure and add support where this crop is grown. In the USA, six resistant to such a hypothesis. soybean cultivars were used as a reference for To conclude about this system, the sources of classifying SMV strains into seven pathotypes. resistance to BCMV/BCMNV are thus identified Several dominant resistance genes and also a re- and satisfactorily used in industrialised countries; cessive resistance gene have been identified. Par- in Africa, a problem of virus diversification and ticularly, a dominant gene Rsv2 conferred resis- a problem of ground to make up for controlling tance by hypersensitivity to all the seven patho- the same disease, the regional crop improvement types of SMV identified in the USA (Buzzell programmes for providing African farmers with and Tu 1984). However, in Japan, several strains agronomically adapted resistant cultivars are not of SMV had virulence properties different from achieved. This situation thus appears as most crit- strains identified in the USA. Another type of re- ical in countries which have a nutritional depen- sistance, preventing the long-distance movement dence on this crop: It calls for other approaches of SMV in the soybean plant and effective against for controlling this disease in the short term. all Japanese strains, has been detected in three cultivars (Lizuka and Yunoki 1983). 8.8.3.2 Lettuce/LMV Two recessive genes g and mo govern a type of 8.8.3.5 Tomato/TMV resistance named tolerance to LMV; that is, the TMV and ToMV cause significant losses, in nor- virus multiplies but does not induce symptoms; mal and soilless culture conditions because both seed transmission is also much reduced. The gene are highly infectious and stable and easily trans- g was largely introduced in European varieties; mitted by mechanical inoculation. Use of virus- in North America, a few varieties incorporate g resistant varieties has provided effective control or mo. These two genes, considered as identical, of these two tobamoviruses. would rather be closely linked genes, the gene mo Gene Tm-l from Lycopersicum hirsutum and conferring a resistance to a larger range of strains. gene Tm-2 or Tm22 from L. peruvianum are two Virulence of LMV with respect to both genes well-known sources of resistance. Particularly, has been observed, and seed transmission too. Tm-22 has proven very effective against TMV Other sources of resistance have been discovered and ToMV wherever used (Watterson 1993). in cv. Ithaca (single dominant gene) and in wild However, breeding for resistance has been Lactuca species that would help improving the laborious due to the tight linkage of Tm-22 with control (see Dinant and Lot 1992). For this crop, undesirable traits. 8.8 Resistance 205

8.8.3.6 Pepper/TMV, ToMV and PMMoV cases where resistance sources are not available TMV, ToMV and PMMoV cause significant or to pyramid natural and engineered resistance losses. Four different tobamoviral resistance in the search for durable resistance. genes are known in the Capsicum spp., namely, Ll from C. annuum, L2 from C. frutescens, L3 from C. chinensis and L4 from C. chacoense 8.8.4 Cultivars with Low Seed (Watterson 1993). These genes govern a Transmission hypersensitive response. Particularly, L3 confers a very effective resistance. Certain isolates of Attempts have also been made to locate cultivars PMMoV are the only tobamoviruses able to in which the virus transmission through seed overcome the L3 resistance. is either low or nil, since the reduction of seed transmission has a major impact on virus 8.8.3.7 Pea/PSbMV spread as they form the primary source of Three recessive genes sbml, sbm3 and sbm4 virus inoculum from plant to plant spread in confer immunity against a large range of strains the field. Soybean lines PI-86736, improved of Pea seed-borne mosaic virus (Provvidenti Pelican and UFV-1 were found to possess low and Alconero 1988). These three sbm genes rate of seed transmission of SMV with superior were grouped with resistance genes to other agronomical qualities (Goodman and Oard 1980; potyviruses of pea and conferred to these lines Irwin and Goodman 1981). Against the same an excellent level of resistance to experimental virus, Goodman and Nene (1976) also identified check. Therefore, this resistance is being 12 lines of soybean in which seed transmission introduced in improved cultivars. However, lines was not noticed. At Columbia (USA), the lowest having the 3 sbm genes could be experimentally incidence of BCMV (0Ð1%) was recorded in infected at low frequency in the glasshouse by French bean lines, namely, Pinto-114, Imuna and a strain of a virulent pathotype (Khetarpal et al. Great Northern 123 and 31, while in susceptible 1990, 1997). lines, the extent of seed transmission was up to 54.4% (Morales and Castano 1987). PMV was 8.8.3.8 Other Crops not seed transmitted in the peanut lines EC-76446 No resistance to PeMoV and PStV is known (292) and NCAC 17133 (RF), and these are in cultivated peanut. Besides, sources of used in resistant breeding programme (Bharathan resistance are available for a number of seed- et al. 1984). In Montana (USA), Mobet barley transmitted viruses of which important one are germplasm (PI-467884) was developed for its BCMV (BICMV)/cowpea, CABMV/cowpea, resistance to seed transmission of three isolates SBMV/French bean, SqMV/cucurbits, ULCV/ of BSMV (Carroll et al. 1983). In Poland, yellow mung bean and urd bean. lupin variety Topaz had the least tendency to To summarise, conventional breeding of nat- transmit BYMV through its seeds (Pospieszyn ural resistance genes is playing a significative 1985). Combined resistance to seed transmission role in the control of seed-transmitted viruses of four viruses, namely, BICMV, TMV, CpAMV but far from a generalised implementation, either and CMV, in Vigna unguiculata was identified because in some cases, no source of useful resis- by Mali et al. (1987) in genotypes like CoPusa-3, tance is available or because the natural available N-2-1 and V-16. genes of resistance have not been incorporated While screening the germplasm, locating a in cultivars growing in important areas, or be- resistant line against a number of virus strains cause virulent isolates overcome such natural re- with good agronomical characteristic is a major sistances. The biotechnological approach, known objective. Often the varieties found resistant to for maintaining the important genetic traits of a one virus strain turn out to be susceptible to cultivar to be transformed for resistance, is an another virulent virus. For example, several cul- exciting new field which has come to rescue the tivars of pea were reported to be resistant to an 206 8 Methods of Combating Seed-Transmitted Virus Diseases isolate of Pea early browning virus at one site in 201, 410 and 3273), although resistant to aphid Britain, whereas at another site, all of them were (A. craccivora), could not establish resistance susceptible (Harrison 1966). Plant breeders are for CpAMV (Atiri et al. 1984). In India, Mali therefore advised not to rely on results from only (1986) reported that cowpea genotype, P-1476, one site in making selections for resistance. was resistant to A. craccivora. As resistance to a vector may result in an increased level of virus spread, it is incumbent 8.8.5 Vector-Resistant Cultivars upon those breeding for resistance to consider the probable effect of vector resistance on virus Resistance to a virus vector is likely to exert spread. In addition, since resistance to one arthro- a complex influence on virus spread. Since pod species may be associated with altered levels non-preference, antibiosis and tolerance are of susceptibility to other species, the potential often combined into a single resistant cultivar, impact on virus spread of cross resistance to a their relative contribution to the resistance as vector species should not be ignored. Despite well as the overall magnitude of the resistance these dangers, the potential for controlling certain will influence the effect of resistance on virus types of virus diseases through vector resistance spread. In addition, observed virus spread is considerable. There exist a sufficient number is the result of both primary and secondary of examples wherein vector resistance has con- spread and the relative importance of these tributed to disease reduction to justify continued and the effect of the resistance on them is efforts in this area. Research in vector behaviour important. The complexities involved are such (e.g. alighting and probing) as it is influenced that without a thorough understanding of the by the host genotype may provide further insight ecology of the virus and vector and the biology into this aspect of resistance to vectors and its of vector resistance, it would be impossible to relationship to virus spread. predict a priori the effect of vector resistance In almost all the crops, resistance sources on virus spread. Each combination of virus, were identified and used in breeding programmes vector and host resistance must be considered against majority of the seed-transmitted virus and separately. vectors, and the approach is likely to be used in- The effectiveness of a vector-resistant cultivar creasingly in the future. However, resistance was for the spread of seed-transmitted plant viruses broken in certain instances due to introduction of will mostly depend on the type and effectiveness newer virus strains. Hence, resistance developed of resistance, its relative importance in primary to strains in one location cannot be expected (introduction of virus from outside the crop) and to operate against strains occurring elsewhere, secondary (spread of virus within the crop) virus and growing a cultivar in a new area may lead spread and virusÐvector relationship (Kennedy to serious epidemics. Furthermore, evidence is 1976). The cumulative interaction of these factors required on the durability of different types of may result in eventual differential effect on the resistance, on possible gene-for-gene relation- spread of seed-transmitted viruses. For example, ships, on the relative merits of major and minor Wilcoxson and Peterson (1960) found less inci- genes and on the possible advantages of using dence of Pea seed-borne mosaic virus in fields mixtures of different genotypes. At least, some of aphid-resistant red clover cv. Dollard than in of this information may soon be forthcoming adjacent fields of the relatively susceptible cv. because of the increased attention being given Wegner. Lecoq et al. (1979) identified Cucumis to breeding for some form of resistance against melo Songwhan Charmi (PI-161375) to be resis- seed-transmitted viruses at many establishments tant to CMV when tested with aphids (M. persi- and especially at the International Research In- cae and A. gossypii), and the mechanism was not stitutions. The inheritance pattern reported in dif- specific to any virus strain. Studies conducted in ferent crop species against major virus diseases is Nigeria indicated that cowpea lines (TVU 408Ð2; presented in Table 8.1. 8.9 Immunisation 207

Table 8.1 Seed-transmitted viruses: inheritance pattern in different crop species Type of Virus Crop resistance Reference Alfalfa mosaic Alfalfa Monogenic Fraser (1986) Barley stripe mosaic Barley Monogenic Carroll et al. (1979b) Bean yellow mosaic Cowpea Monogenic Reeder et al. (1972) Blackeye cowpea mosaic Soybean Monogenic Provvidenti et al. (1983) Blackeye cowpea mosaic Cowpea Monogenic Taiwo et al. (1981) Cowpea mosaic Cowpea Monogenic Raj and Patel (1978) Cowpea aphid-borne mosaic Soybean Monogenic Provvidenti et al. (1983) Cucumber mosaic Cowpea Monogenic Khalf-Allah et al. (1973) Cucumber mosaic Mung bean Monogenic Sittiyos et al. (1979) Pea seed-borne mosaic Pea Monogenic Hagedorn and Gritton (1973)and Provvidenti and Alconero (1988) Pea seed-borne mosaic Lentil Monogenic Haddad et al. (1978) Soybean mosaic Soybean Monogenic Provvidenti et al. (1982), Buzzell and Tu (1984), and Provvidenti and Hampton (1992) Tobacco ring spot Cowpea Monogenic Fraser (1986) Cowpea chlorotic mottle Soybean Monogenic Boerma et al. (1975) Peanut mottle Soybean Monogenic Boerma and Kuhn (1976)and Provvidenti and Hampton (1992) Bean southern mosaic Bean Monogenic Zaumeyer and Harter (1943) Bean common mosaic Bean Monogenic Provvidenti and Hampton (1992)

soybean seedlings grown from these seeds were 8.9 Immunisation widely transplanted in growers fields in 1991 (45 ha) and 1992 (50 ha) in Wachi, Kyoto (Japan). When field application is considered, the pro- The incidence of virulent strains of SMV in late tecting strain is generally an isolate which in- July was found to be 1.3% in 1991 and 0.8% in duces mild symptoms and does not affect the 1992, compared with the rates of >60% in 1989 marketable yield of the crops. However, when and 1990. The use of seeds from A1 15-M2- losses from the virus disease are great, some yield inoculated plants was also found to be effective reduction induced by the protecting strain might for the control of SMV in other areas during be economically tolerable. Successful application 1992. of immunisation/cross protection in some seed- In peanut, PStV is economically important, transmitted viruses in certain host plants is dis- and Wongkaew and Dollet (1990) categorised 24 cussed herein. isolates from eight different countries into eight In Japan, the effects of Soybean mosaic virus strains based on disease reactions on specific (SMV) were reduced by immunisation technique hosts and serology. Possibility exists for immu- under field conditions (Kosaka and Fukunishi nisation phenomenon at field level. Preliminary 1994). In 1990 and 1991, c.20,000 and 78,000 information is available on the use of this tech- cv. Shin seedlings, respectively, of black soybean ( nique against PStVd, which is seed transmitted in Tambaguro) were transplanted 1Ð3 days after true potato seed (Singh et al. 1990, 1993). Perring protective inoculation with an attenuated isolate et al. (1995)andWalkey(1992) demonstrated of SMV, Aa 15-M2. In the plants that received that in vegetable marrow Zucchini yellow mosaic protective inoculation, virulent strains of SMV virus, incidence could be reduced by mild strain were effectively suppressed. Seeds virtually free inoculation at seedling level prior to aphid trans- from SMV were produced in 1990 (971 kg/ha) mission of severe strain. and free from SMV in 1991 (4,740 kg/ha). Black 208 8 Methods of Combating Seed-Transmitted Virus Diseases

Although the use of a mild strain to protect Pilowsky 1975), Belgium (Vanderveken and the plants against more virulent strains has been Coutisse 1975), former USSR (Vlasov 1972), the suggested by Kunkel (1934), the technique has Netherlands (Rast 1972, 1975), Japan (Oshima not been widely adopted by tomato growers until et al. 1965;Gotoetal.1966; Nagai 1977;Oshima Rast (1972, 1975) from the Netherlands demon- 1981), New Zealand (Mossop and Procter 1975), strated the commercial benefits of inoculating German Democratic Republic (Schmelzer and crops with the avirulent TMV strain MII-16. Wolf 1975), the United States (Ali Ahoonmanesh Growers in the Isle of Wight (UK) were the first and Shalla 1981) and China (Tien and Chang to adopt the immunisation technique in 1964, 1983). although a mild strain of TMV has not been Most of these immunisation experiments were available and the normal fairly severe strain of attempted with Rast’s MII-16 strain. In Denmark, Tomato mosaic was used for inoculation in the Paludan (1975) worked with Danish attenuated seedling stage. The proportion of unsalable and TMV strain (K58-45-0) and found it not as en- poor quality fruit was reduced from about 30 to couraging as MII-16. The TMV strain K58-45-0 3% in 1965. These results have led to the idea that caused weak to moderate symptoms throughout the benefit gained from seedling infection would the growing period. The TMV-MII-16 strain gave be greater if a mild strain of TMV that would pro- higher protection than the TMV strain K58-45- tect the plants from severe strains could be used. 0. In Japan, Oshima et al. (1978) used LIIA, an In capsicum and tomatoes, TMV is both ex- attenuated tomato strain of TMV, and proved it ternally and internally seed transmitted. Exten- to be ineffective for the existing TMV-resistant sive attempts have been made to combat TMV tomato cultivars having the Tm-1 gene. This was infection in tomatoes through immunisation, in possibly because the strain multiplied very little which the plants are deliberately inoculated with in these cultivars and the plants were readily avirulent strain which will give protection against infected by virulent strains such as CH2. the severe strain of the same virus. In the Nether- In order to obtain another attenuated strain for lands, Rast (1972) isolated an almost symptom- protecting these cultivars, strain LllA 237 was less mutant using the nitrous acid mutagenic isolated by successive passage of LIIA through technique which gave sufficient protection to five TMV-resistant GCR 237 tomato bearing homozy- strains of TMV, when challenge inoculated. One gous TMV-resistant gene Tm-1. The new strain of the effects observed on tomato plants follow- also caused no symptoms in tomato and Samsun ing early infection with MII-16 was a temporary tobacco but differed from LllA in that it multi- check of growth which delayed flowering and plied much faster in the existing TMV-resistant fruit set, indicating the necessity to advance the cultivars (Tm-1/T). It sometimes caused necrotic sowing date by about a week to compensate for rings on Xanthi-nc tobacco in the environment this. He also found that plants inoculated at the in which LllA causes necrotic spots. In immu- seedling stage with MII-16 gave better yields than nisation tests, a resistant cultivar, LllA 237, in- those inoculated at the same growth stage with terfered more strongly with the multiplication of the parent strain. Upstone (1974) reported that in CH2 than did LllA. In glasshouse experiments, 27 trials in the UK, plants inoculated with Rast’s inoculation of resistant tomato Tanamo (Tm-1/T) MII-16 strain, on average, yielded 5% more than with LllA 237 gave good control of the disease the uninoculated plants. (Oshima et al. 1978;Oshima1981). In China, a The values of immunisation as a control symptomless TMV mutant, N14 was produced measure against TMV infection in tomatoes by using nitrous acid mutagenic technique and grown in glasshouse conditions have been well this mild strain under field inoculations increased established in different countries like Denmark tomato yield by 5Ð60% (Tien and Chang 1983). (Paludan 1975), France (Migliori et al. 1972), As this immunisation technology proved ben- the UK (Evans 1972; Fletcher and Rowe 1975; eficial to tomato growers in the Netherlands, Channon et al. 1978), Israel (Zimmerman and the UK, Japan, New Zealand, etc., techniques 8.10 Approved Seed Certification Standards 209 were developed for mass inoculation. Rast (1972) 2. The inoculated plants should induce milder reported a spray gun method of inoculation to be symptoms than isolates commonly encountered better than manual inoculation because of the risk in the fields and should not alter the yield and of contamination with the other strains. quality of the crop. It should also be mild in The MII-16 strain became commercially avail- all its cultivated hosts including those which able in the UK in 1973 and is sold in 5 ml quanti- are not targets for the cross protection. ties of concentrated virus which has to be diluted 3. Mild strain multiplication should be con- 1,000 times before use. In addition to facilitating ducted in highly protected under strict higher yields, this mild strain offers an advantage phytosanitary supervision in order to eliminate in seed production. Seedling inoculation with risk. this strain results in negligible TMV infection of 4. For mass multiplication, it is essential to pro- seeds. In contrast, infection is higher in plants vide to farmers and farm advisers an easy and inoculated with the virulent strains, where the efficient mild strain inoculation technique. virus is both exogenously and endogenously seed However, some apprehensions have been ex- transmitted. So the deliberate inoculation with the pressed about immunisation. First, the mild or mild strain could be used by seed growers and is protecting strain could mutate to a highly vir- an alternative to heat treatment. Steeply (1968) ulent form, leading to crop losses rather than found a lower incidence of seed infection with a protection. Second, the protecting virus could act heat-attenuated strain. synergistically with an unrelated virus to create a Prunus necrotic ring spot virus (PNRSV) and disease combination that is more damaging than Apple mosaic virus (ApMV) are seed transmit- either virus on its own. Third, the mild strain ted in some fruit crops; limited information is could be virulent in other crops, making it unwise available on the use of cross protection technique to spread it so extensively. Finally, the protecting to protect these crops. For example, Wood et al. strain generally causes a small but significant (1975) found that the yields of Jonathan apple reduction in yield. trees infected with ApMV almost doubled when Even with these limitations, plant immunisa- these trees were top worked with scionwood con- tion has the potential for being highly effective in taining a mild strain of ApMV and the symptoms modern agriculture. For the success of this phe- of virus were alleviated. However, the yields nomenon in different crops, the selection of the obtained were still substantially lower than yields mild strain is of paramount importance. An in- from mosaic-free trees, suggesting that replanting tensive approach is required to find a milder strain with healthy trees would be more economical in having desirable properties in terms of qualitative the long term. Mild strain protection of cherries and quantitative crop yields. The strain should be against Cherry rugose mosaic strain of PNRSV tested regularly for both avirulence and protective may be possible. Mink (1983) reported a strain of power. The methods of application and timing of PNRSV which produces no detectable effect on virus inoculation in relation to conditions of plant either tree vigour or fruit quality. Circumstantial growth need careful study. evidence suggests that this symptomless strain might provide protection against later infection of trees by severe strains of the virus. 8.10 Approved Seed Certification The following points are to be seriously taken, Standards while immunisation technique is followed under field condition in large-scale crop cultivation: Seeds are the foundation to crop production, 1. The mild strain of the virus should be fully and seed health is related to food production in systemic and invade all host tissues. Indeed, various ways. Seed being the foundation of suc- cross protection effectiveness depends on the cessful agriculture, the demand for quality seeds presence of the mild strain in all tissues to be of improved varieties/hybrids is growing fast and protected from severe strains. adoption of new technologies around the world 210 8 Methods of Combating Seed-Transmitted Virus Diseases by the farmers is happening at an amazing pace. multiplication. The agencies involved in the Therefore, production and supply of high-quality production of breeder seeds are agricultural seed of improved varieties and hybrids to the universities and the ICAR institutes in India. grower are a high priority in agricultural growth Foundation seed is the progeny of breeder and development. seed, and it is supervised and certified by the Seed Governments are strongly encouraged to Certification Agency. It should possess the min- implement a predictable, reliable, user-friendly imum genetic purity of 99.00%. Expert handling and affordable regulatory environment to ensure is needed for producing foundation seed, and they that farmers have access to high-quality seed are produced on seed farms where specialists and at a fair price. In particular, FAO member facilities are available. Agencies involved in the countries are participating in the internationally production of foundation seeds are the State De- harmonised systems of the Organization for partment of Agriculture, Horticulture, State Seed Economic Cooperation and Development Corporation and the State Farms Corporations of (OECD), the International Union for the their respective countries. Protection of New Varieties of Plants (UPOV), Certified seed is the progeny of foundation the International Treaty on Plant and Genetic seed and certified by the certification agency. The Resources for Food and Agriculture (ITPGRFA) agencies involved in the production of certified and the International Seed Testing Association seeds are the State Seed Corporation through (ISTA). Participation in these systems will their Registered Certified Seed Growers and the facilitate the availability of germplasm, new Private Seed Companies. Its genetic purity is plant varieties and high-quality seed for the 98.00% for varieties/composites/synthetics, and benefit of their farmers, without which their for cotton and castor hybrids, it is 85%. ability to respond to the challenges ahead will The seed should meet minimum standards of be substantially impaired. It is inevitable that germination and purity prescribed and labelled as governments need to develop and maintain per Seed Act 1966. Use of quality seed and plant- an enabling environment to encourage plant ing material which are free from seed-transmitted breeding and the production and distribution diseases including viruses contributes to the en- of high-quality seed. hancement in crop production by better reali- sation of its own productivity potential (to the extent of 20Ð25% depending on the crop) and 8.11 Stages of Seed also by contributing to enhance the utilisation Multiplication efficiency of other inputs to the extent of another 20Ð25%. Therefore, availability of good quality Genetically pure, disease-free seeds are the disease-free seeds of superior varieties/hybrids prerequisite for a healthy, vigorous and high- needs to be ensured for boosting the agricultural yielding crop. There are three important stages of production. The central seed certification board, seed multiplication. department of agricultural and cooperation, min- Nucleus seed, also known as original parental istry of agricultural, and Government of India, material, is the product of scientific breeding by New Delhi, have prescribed disease certification crop breeders who evolved the varieties/hybrids. standards in foundation, and certified seed crops Breeder seed is the first and is the product are provided in the table (Table 8.2). of scientific breeding by crop breeders who evolved the particular variety. Breeder seed (sexually/asexually propagating material) is directly controlled by the original plant breeder. 8.12 Inoculum Threshold The production is also personally supervised by the breeder. It should possess the maximum The level of the pathogen in seeds which gives genetic purity (99.5Ð100%). Breeder seed is rise to an unacceptable risk of disease is often made available in small quantities for further referred to as ‘inoculum threshold’, although the 8.12 Inoculum Threshold 211 early and late varieties hills and plains, respectively 1.01.0 At harvesting At harvesting ) 2000 0.5 0.75 1.0 Stage II Ð 60 and 70 days in 1.00.5 2.0 0.75 3.0 1.0 Stage I Ð 35 and 45 days in 1.00.05 2.0 At maturity prior to 0.10 harvest At flowering stage 0.100.100.10.10.10.10.1Ð 0.20 0.20 2.0 0.5 At flowering and fruiting 0.5 stage At flowering and fruiting 0.5 stage 0.5 At maturity prior to harvest At fruit maturity 3.0 Prior to harvesting Prior to harvesting Prior to harvesting At harvesting ) and Narayan Rishi ( 1988 Mild mosaic Severe mosaic Leaf roll Jute chlorosis Leaf roll Watermelon mosaic Severe mosaic Common mosaic Bean mosaic Cucumber mosaic Cucumber mosaic Tomato mosaic Lettuce mosaic Mosaic Mild mosaic Maximum per cent virus disease certification standards in foundation and certified seed crops Potato tuber Stage I seed Stage II seed Certified seed Table 8.2 CropCowpea French bean Jute Muskmelon Summer Virus squash Tomato Lettuce Sweet potato Potato true seed Foundation seed Certified seed Evaluation stage Based on Tunwar and Singh ( 212 8 Methods of Combating Seed-Transmitted Virus Diseases term ‘tolerance standard’ is perhaps less mislead- in parts of Eastern Europe, Russia, southern ing and to be preferred. The risk of a ‘significant’ Africa and Australia for green manure, forage epidemic developing is dependent on the rate of and medicinal purposes, and CMV affects crop transmission from seed to seedling and the rate yields and also virus is seed transmitted to of disease increase in the crop (both of which are the extent of 12Ð18% (Jones 1988, 2000)and highly dependent on environmental conditions). causes epidemics. An integrated virus disease The use of infected seed for sowing is an- management approach was used to minimise the other major factor contributing to disease inci- yield losses, and sowing lupin seed stocks with dence and eventual crop loss. For instance, in minimal virus contents provide a key control England, lettuce crops are grown from 5 com- measure within this strategy (Bwye et al. 1995, mercial seed lots with 2.2Ð5.3% infection of 1997, 1999; Jones 1988, 1991b, 2000, 2001; LMV, the plants became infected with mosaic Jones and Proudlove 1991; Thackray et al. 2004). were 25Ð96%, whereas 6 seed lots at <0.1% Since 1988, a commercial testing service for infection have produced crops with fewer than CMV in lupin seed samples has provided an 0.5% of infected plants at the end of the growing estimate of per cent CMV infection based on season (Tomlinson 1962). However, where aphid a 1,000-seed test. This helps farmers to avoid population was high, as in the case of Califor- sowing seed with infection levels likely to result nia (USA), lettuce seed at <0.1% infection has in serious yield losses. Bwye et al. (1994)have produced severe outbreaks (Grogan et al. 1952; reported that sowing 1% CMV infected lupin Zink et al. 1956). Even in the case of peanut, the seed resulted in significantly decreased yields incidence of PMV depends partly on the initial in 2 experiments, while 0.75 and 0.5% infected level of seed infection (Paguio and Kuhn 1974). seed caused significant losses in 1 experiment In irrigated plots, an average yield reduction (16Ð19% losses). For lupin-growing areas at among three barley cultivars of Betzes, Compana lower risk from CMV, a threshold of <0.5% and Vantage ranged from 24 to 35% when plants seed infection suffices to avoid serious yield were BSMV infected by mechanical inoculation losses in most years, but for high-risk areas, a (Carroll 1980). The reduction in barley grain threshold level of <0.1% is used for lupin grain yield, number of heads and seed weight due to crops, that is, a zero test result on a 1,000-seed BSMV was found to be linear in response to the sample (Bwye et al. 1995; Jones 2000, 2001). A level of seed infection (Nutter et al. 1984). model forecasting CMV epidemics in lupins was Attempts on inoculum threshold were also incorporated into a decision support system by made by Coutts et al. (2009) in pea infected with the Department of Agriculture and Food, Western Pea seed-borne mosaic virus (PSbMV). If the Australia, for use by farmers and agricultural primary virus inoculum through pea seed is 0.3Ð consultants in planning CMV management 6.5%, the final PSbMV incidence reached 98%, (Thackray et al. 2004) and is accessed via the and the yield losses were 18Ð25% (Fig. 8.2). departments’ websites. Neya et al. (2007) have studied epidemics de- From the four field experiments conducted velopment of Cowpea aphid-borne mosaic virus at Mysore (India), Udayashankar et al. (2010) in cowpea by using 0Ð5% initial seed infection. showed that sowing of cowpea seeds with 10, 5, Because of the high level of resistance in the 3, 2, 1, 0.75, 0.5 and 0.05% infection of Bean cowpea varieties used, the progress of the disease common mosaic virus (Blackeye cowpea mosaic was very low (<50%) in all treatments. Even strain) BCMVÐBICM results in an average cu- Puttaraju et al. (2002) have also noticed that mulative disease incidence of 90, 53, 37, 26, cowpea seeds with 5% Blackeye cowpea mosaic 12, 8, 6 and 1%, respectively. The seed yield virus have resulted in yield loss of 34Ð53%, while reduction was directly correlated to levels of seed 0.5% seed infection did not cause significant loss. infection. Seed infection of 10, 5 and 1% resulted Narrow-leafed lupin (Lupinus angustifolius) in 74, 54 and 18% seed yield loss per plant. Infec- is one of the important cool season grain legumes tion of 0.75 and 0.5% reduced the seed yield per 8.13 International Seed Testing Association (ISTA) 213

Fig. 8.2 Plots of field pea cv. Kaspa at Avondale in obvious depressions caused by widespread PSbMV infec- 2005 (experiment 1) originally sown with (a)6.5%and tion in (a) and vigorous growth with a uniform canopy in (b), 0.3% Pea seed-borne mosaic virus (PSbMV)-infected (b) (Source: Coutts et al. 2009; R.A.C Jones) seed. Note pale appearance and uneven canopy with plant by less than 1%. These field experiments in Prunus seedlings can be achieved and will demonstrated that sowing >1% BCMVÐBICM- be demanded in the future. There is requirement infected seed can lead to significant losses in of maintaining the inoculum threshold levels in grain yield, while the spread of BCMVÐBICM almost all certification schemes of fruit trees. infection resulting from sowing 1% infected seed More details on inoculum threshold level can be did not significantly decrease seed yield. had from the review articles (Jones 2000; Stace- In some of the Prunus species and other stone Smith and Hamilton 1988; Roberts 1999). fruit crops, which are propagated on root stocks also carry seed-transmitted virus diseases like Prunus necrotic ring spot virus and Prunus dwarf 8.13 International Seed Testing virus. Most of the stone fruit certification pro- Association (ISTA) grammes specify that Prunus seedling lot must contain fewer than 5% virus-infected plants. Be- The International Seed Testing Association cause the demand of certified seed is greater (ISTA) was founded in 1924 during the 4th than the supply available in most years, nurs- International Seed Testing Congress held in eries often purchase large amounts of Prunus Cambridge, United Kingdom. As on June 2012, seed of unknown virus contents, which exceeds its membership consists of about 201 member the 5% tolerance (Mink 1984). Actually even laboratories, 52 personal members and 42 5% tolerance level results in the production and associate members, from 79 countries around distribution of contaminated bud wood. By the the world. Nearly 120 of the ISTA member application of serological and molecular virus laboratories that are accredited by ISTA are diagnostic tests, there is every reason to believe entitled to issue ISTA international seed lot that a zero tolerance level for these two viruses analysis certificates. ISTA is independent and 214 8 Methods of Combating Seed-Transmitted Virus Diseases acts free from economic interest and political 8.13.2.1 Orange International Seed Lot influence, and it is unbiased, objective and fair. Certificate Furthermore, the hitherto unsurpassed expertise An Orange International Seed Lot Certificate is of ISTA is based on the non-profit, cooperation issued when both sampling from the lot and of the international community of approximately testing of the sample are carried out under the re- 400 experienced, competent and energetic seed sponsibility of an accredited laboratory, or when scientists and analysts. sampling from the lot and testing of the sample are carried out under the authority of different accredited laboratories. Where the accredited lab- oratory carrying out the sampling is different to 8.13.1 Objectives of ISTA the one carrying out the testing, this must be stated. The procedure followed links the Orange (a) The primary objective of the association is International Seed Lot Certificate with the seed to develop, adopt and publish standard pro- lot. The certificate is coloured orange. In the case cedures for sampling and testing seeds and of Orange International Seed Lot Certificates, the to promote uniform application of these pro- results reported refer strictly to the lot as a whole cedures for evaluation of seeds moving in at the time of sampling. international trade. (b) The secondary objective of the association 8.13.2.2 Blue International Seed is to promote research in all areas of seed Sample Certificate science and technology, including sampling, A Blue International Seed Sample Certificate is testing, storing, processing and distributing issued when sampling from the lot is not under seeds; to encourage variety (cultivar) cer- the responsibility of an accredited laboratory. The tification; to participate in conferences and accredited laboratory is responsible only for test- training courses aimed at furthering these ob- ing the sample as submitted. It is not responsible jectives and to establish and maintain liaison for the relationship between the sample and any with other organisations having common or seed lot from which it may have been derived. related interests in seed. The certificate is coloured blue. In the case of Seed quality determination, as established by Blue International Seed Sample Certificates, the ISTA, on seed to be supplied to farmers is an results reported refer strictly to the sample at the important measure for achieving successful agri- time of receipt. The Blue International Seed Sam- cultural production. The establishment or main- ple Certificate refers only to the sample submitted tenance of an appropriate infrastructure on the for testing. scientific as well as technical level in developed and developing countries is highly commendable. 8.13.2.3 Provisional Certificate A provisional certificate is an ISTA certificate issued before the completion of a test or tests. 8.13.2 ISTA Certificates It is marked PROVISIONAL and must include a statement under ‘other determinations’ that a Certificates like Orange International Seed Sam- final certificate will be issued upon completion. ple Certificate and Blue International Seed Sam- ple Certificates are issued by accredited member and laboratories of ISTA and must only be is- 8.13.3 Conditions for Issuance sued in accordance with the ISTA rules currently of ISTA Certificates in force after seed analysis. Well qualified and trained personnels are involved in ISTA labs and ISTA certificates must be issued only on forms international standards are maintained in seed obtained from the ISTA secretariat and approved quality testing. by the ISTA executive committee. There are two 8.14 Seed Certification Against Plant Virus Diseases 215

kinds of certificates: Orange International Seed (h) For an Orange International Seed Lot Lot Certificates and Blue International Seed Sam- Certificate, each container in the lot must ple Certificates, as defined earlier. be marked, labelled and sealed in accordance An ISTA certificates may be issued only by with ISTA (ISTA 2011). the seed testing laboratory which either carried (i) An Orange International Seed Lot Certificate out all the tests to be reported or subcontracted shall be valid until it is superseded by sampling and/or some of the tests to be reported, another valid Orange International Seed Lot and under the conditions listed below: Certificate subsequently issued on the same (a) The issuing laboratory must be currently au- lot. Not more than one Orange International thorised to do so by the executive committee. Seed Lot Certificate shall be valid for a lot at (b) The seed tested must be of species listed in one time for any particular test. the ISTA manual (ISTA 2011) according to (j) For an Orange International Seed Lot the ISTA rules and shall be considered to be Certificate, the submitted sample must be covered. tested by an accredited laboratory. The issuing Consequently, no certificates may be laboratory must ensure that sampling, sealing, issued for species not listed in the current identification, testing and issuance of the ISTA rules, nor for mixtures of species which certificate are in accordance with the ISTA are in the ISTA rules, as no procedures are rules, although subcontracting of sampling prescribed for mixtures. and/or testing to another accredited laboratory (c) The tests must be carried out in accordance is permissible. The laboratory which carries with the ISTA rules. However, additionally out sampling must provide all the information and on request, results of tests not covered that is necessary to complete the Orange by these rules may be reported on an ISTA International Seed Lot Certificate. certificate. Results of analyses not covered by the cur- rent ISTA rules may be included on a certifi- 8.13.4 Accredited Laboratory cate only if results of at least one test covered by the ISTA rules are also being reported. An accredited laboratory is an ISTA-accredited (d) For the result of determination of moisture member laboratory authorised by the ISTA ex- content to be reported on an ISTA certificate, ecutive committee under Article VII of the ISTA the sample must be submitted in an intact, constitution to sample and test seeds and to is- moisture-proof container from which as much sue ISTA certificates. Blank ISTA certificates for air as possible has been excluded. seed analysis are produced by ISTA and only (e) To report results of tests which are in provided to accredited laboratories for reporting the ISTA rules, the laboratory must be the results of tests. These completed certificates accredited for these tests, either directly or are the property of ISTA and may only be issued through subcontracting to another laboratory under the authority of ISTA. The list of accredited accredited for these tests. laboratories of the member countries and abstract (f) The assessment of any attribute reported on of ISTA member laboratories are provided ISTA a certificate must be calculated from tests website. carried out on one submitted sample. (g) In the case of Orange International Seed Lot Certificates: 8.14 Seed Certification Against Ð The seed lot must comply with the Plant Virus Diseases requirements prescribed in ISTA manual (ISTA 2011). Quality control of seed for viruses is not very Ð The submitted sample must be drawn and common due to the relative ‘weight’ of previ- dealt with in accordance with ISTA rules. ous available biological assays and to the high 216 8 Methods of Combating Seed-Transmitted Virus Diseases number of seeds to be tested for assessing low tested in number of hosts viz., BSMV/barley virus transmission rates. (Lister et al. 1981), LMV/lettuce (Ghabrial et al. The enzyme-linked immunosorbent assay 1982), PeMoV/peanut (Bharathan et al. 1984), (ELISA) which offers greater sensitivity of PSbMV/pea (Hamilton and Nichols 1978;Maury detection and enables a large number of samples et al. 1987b), SMV/soybean (Bossennec and to be analysed per day has become a technique of Maury 1978; Lister 1978; Maury et al. 1983) choice for large-scale testing of seed-transmitted and SqMV/cucurbits (Nolan and Campbell 1984) viruses (Maury and Khetarpal 1989). have shown a large variation in the concentration There is no doubt that the recent development of virus among infected embryos. The virus has of the polymerase chain reaction (PCR) been found in the axis and not in the cotyledons technique has resulted in a thousand times gain of about 20% of the infected embryos of soybean in sensitivity of detection of viruses in plant and pea (Maury et al. 1983; Masmoudi et al. tissues. Since 1992, using reverse transcription 1994b) and also in a proportion of peanut (Zettler (RT-PCR), certain RNA viruses have been et al. 1993) and bean embryos (Klein et al. 1992). detected in seeds, namely, PSbMV in pea seeds However, such low concentration is positively (Kohnen et al. 1992), CMV in lupin seeds detected by ELISA when extracting the whole (Wylie et al. 1993), BCMV in bean seeds soybean embryos at the 200 w/v dilution and (Saiz et al. 1994) and AMV, Bean yellow mosaic pea embryos at 500 w/v for detecting SMV virus (BYMV), Clover yellow vein potyvirus and PSbMV, respectively (Maury et al. 1983; (CYVV) and Subterranean clover mottle virus Masmoudi et al. 1994a). (SCMoV) in germplasm of subterranean clover Good correlations have been found between and medic seeds (Bariana et al. 1994). The use the percentage of ELISA-positive embryos of PCR for seed testing of viruses in routine in a given seed lot and the percentage of would require the step of extraction of RNA infected seedlings raised from the same seed from large number of seeds, which in fact is lot for SMV/soybean seed (Maury et al. 1984), laborious, time-consuming and inconvenient. PeMoV/peanut seed (Bharathan et al. 1984)and Moreover, nucleic acid extraction of a sample PSbMV/pea seed (Maury et al. 1987b). Johansen cannot be exploited for simultaneous detection of et al. (1994) considered that infectivity assays seed-transmitted bacterial and fungal pathogens often indicate an absence of intact virions in because the progress in the latter field comes cotyledons. If the virus is inactivated in cotyle- from bio-PCR, which detects the pathogen after dons, ELISA-positive embryos where the virus enrichment on a culture medium. In this case, is present in the cotyledons and not in the axis a variant of PCR, that is, immunocapture-PCR would be false-positive embryos. Such a distribu- (IC-RT-PCR) which has an inherent simplicity of tion of virus in embryos has been found rarely for amplifying sequences of viral RNA without prior SMV/soybean (Maury et al. 1985) and more fre- RNA extraction, was presumed to facilitate use of quently for BICMV/cowpea seed (Gillaspie et al. PCR in routine. However, recently, a lower level 1993). It was also established that the BICMV of sensitivity and lack of reliability of IC-PCR from cowpea cotyledons is not seed transmitted. has been observed for group testing of pea seed (b) Avoidance of interference of non-embryonic infected by PSbMV (Phan et al. 1997). Thus, tissues during extraction: Since the presence of this technique needs an adaptation to seed testing virus in the seed coat does not relate to virus before becoming popular for routine. transmission from seeds to seedlings, whole-seed serological assays are not suitable for estimating rates of seed transmission (Johansen et al. 1994). 8.14.1 The Quality Control by ELISA While preparing samples for determining the transmission rate in routine conditions on Studies of correlation between detection of virus large number of seeds, it is therefore necessary in infected embryos and seed transmission. Were that the viral antigen from non-embryonic 8.14 Seed Certification Against Plant Virus Diseases 217 tissues (i.e. the seed coat in general) be not 8.14.2.1 Lettuce simultaneously extracted in order to prevent A field applicable seed certification programme false-positive reactions. That introduces a of lettuce seed production has been developed potential complication as manual processing of against Lettuce mosaic virus was worked out by large number of seeds is poorly compatible with number of workers. Grogan et al. (1952)devel- the routine. oped a system in which lettuce seed was produced Surprisingly, a lack of viral antigen in testa has from virus-free plants grown under glasshouse been reported for mature peanut seed transmitting conditions. In isolated areas, only the healthy BCMV (PStV) (Demski and Warwick 1986;Xu plants were grown to maturity after roguing out et al. 1991) and PeMoV (Adams and Kuhn 1977) the infected plants. The tolerance levels of seed as well as for LMV/lettuce seed (Falk and Purci- transmission varied from place to place depend- full 1983). This could also be due to a failure in ing on the ecology and epidemiology of the virus extraction of the virus from testa as demonstrated disease. in case of SMV/soybean (Maury et al. 1985), or Researchers in California (Zink et al. 1956; a failure in detection as shown for PSbMV/pea Wisler and Duffus 2000) and Florida (Purcifull (Masmoudi et al. 1994a). In the latter case, it and Zitter 1971) have established that if seed was indeed observed that the PSbMV capsid is transmission exceeded 0.1%, control was not partially cleaved in testa of infected pea seeds, satisfactory because of large winter and spring during the maturation process. Antisera to the aphid population. In England, when lettuce seed cleaved polypeptide detected PSbMV specifically with 0.1% infection was sown, only 0.5% of the in the embryos but not in the testa, thus en- plants were infected (Tomlinson 1962). It was abling use of whole seed for testing without prior observed that the initial level of 1.6% Lettuce decortications. mosaic virus infection in lettuce seed reached to In other virus/seed systems, testae have to 29%, whereas zero starting infection reached be removed in order to avoid the false-positive only 3% (Zink et al. 1956). The number of reactions and to correctly determine the seed- infected lettuce seedlings emerging per acre at transmission rate of a seed lot. the 0.1% rate would be 200 per acre or 20,000 infected seedlings per 100 acres (Greathead 1966). This is based upon an estimated 400,000 8.14.2 Certification Schemes seeds/lb, planting 1 lb/acre and with 50% Against Crops emergence. Programmes in both the Salinas Valley and Imperial Valley of California have, Among all the methods of obtaining healthy seed however, stressed the use of seed which is material, seed certification is the most depend- double tested Ð both the seed companies and able and prudent measure which helps to main- by a committee of lettuce growers Ð to show tain reasonable health standards without affect- zero mosaic/30,000 seeds tested (Kimble et al. ing seed germination. Programmes against seed- 1975; Grogan 1980, 1983). This has reduced transmitted viruses must start at the basic level losses by an estimated 95Ð100%, resulting in of germplasm collection available to the plant considerable increase in yield and quality. At breeder and continue through the subsequent de- Florida since 1974, the use of double-indexed velopment of varieties and the increase of seed seed has eliminated Lettuce mosaic virus,amajor through breeder seed (pre-basic seed), founda- limiting factor in lettuce production (Zitter 1977). tion seed (basic seed), registered seed (certified However, during 1995, in Florida, due to non- seed, first generation) and certified seed (second compliance with the lettuce mosaic certification generation). Some of the details of certification rule 5B-38, an outbreak of LMV in commercial schemes against crops like lettuce, barley, beans, lettuce production was noticed (Raid and Nagata pea and cowpea are outlined below. 1996). 218 8 Methods of Combating Seed-Transmitted Virus Diseases

8.14.2.2 Barley seed determined by test to be BSMV-free was In barley, Barley stripe mosaic virus (BSMV) used to produce certified seed which was then which has no biological vector, seed transmission made available to growers. is the major factor facilitating disease spread. The It was reported that by 1971, for all practical simplest way of ameliorating the losses caused by purposes, BSMV was eliminated in North Dakota this virus is by planting uninfected seed. A seed (Timian 1971). Similarly at Montana, the two ma- certification programme to obtain barley seed free jor barley cultivars, namely, Compana and Uni- from BSMV was attempted at Kansas (Hampton ton, suffered losses of approximately 4 bushels et al. 1957), Montana and North Dakota in the per acre from 1953 to 1967, but by 1967Ð1970, USA (Carroll 1983). It began with the growing these declined to 2 bushels per acre. Since 1970, of samples of seed in glasshouses during late au- losses due to BSMV were reported to be about tumn and winter to select those which are virus- half a bushel per acre after using the certified seed free, for further propagation. The control pro- (Carroll 1980; Jackson and Lane 1981). gramme involved assaying seedlings for presence of BSMV by serology and roguing infected plants 8.14.2.3 Pea from certified seed plots which significantly re- Among the viruses infecting peas, PSbMV has duced the incidence of the virus. In 1972, a zero seed transmission up to 100%, and even 23% of tolerance for seed-transmitted BSMV was placed pea accessions are infected in USDA collection on all certified seeds sold in Montana, and as a (Hampton and Braverman 1979). Because result, losses due to BSMV declined dramatically. of high seed transmission and potent aphid The certification scheme against BSMV in vector, schemes to produce virus-free seed are Montana involved inspection of each barley field developed. In Canada, the mother plants in dry being considered for certification according to (field) pea breeding programmes are propagated the field standards published by the Montana under aphid-free conditions and screened by Seed Growers Association and Montana State visual inspection and a serological assay based University. When a field intended for the pro- on SDS-gel immunodiffusion (Hamilton and duction of registered or certified barley seed was Nichols 1978). Subsequent seed increase plots inspected for quality and purity, it was also care- are grown in isolation in areas of Manitoba and fully checked for plants exhibiting symptoms of Saskatchewan where populations of aphid vectors BSMV. One or two inspections of each field were are minimal. Before release to the commercial made by the secretary-manager of the Montana seed trade, the standing crop of a new variety is Seed Growers Association or the Agronomist inspected visually, and samples of harvested seed with the Montana Cooperative Extension Service, (200 seed/50 lb) are subjected to a growing-on Bozeman-Montana or a member of his staff. test. Seedlings are examined, and five randomly If infected plants were found in such a field, selected seedlings are assayed for PSbMV by that field was no longer eligible for certification. SDS-gel immunodiffusion. Virus-free lots are The barley harvested from an uncertifiable field then released to the Canadian Seed Growers was usually sold for feed purposes. The second Association or to SeCan, a federal government procedure of the certification programme is the agency for propagation in a seed certification inspection of seed from each potentially certifi- programme. However, no further testing for able barley field by employing serological tests. PSbMV is done. In the USA, in the primary seed- At Montana, because of the implementation of producing areas (Washington and Idaho), there is certification schemes, the percentage of infected a zero tolerance for PSbMV-induced symptoms seed lots denied appreciably from 1967 onwards. observed during field inspections (10 days post- At North Dakota, the certification scheme against emergence and 10 days prior to harvest). Visual BSMV resembled the Montana scheme, which inspection is being supplemented by direct assay involved both a procedure for inspection of seed of seed (200 seeds/seed lot irrespective of size of fields and seed lot examination. Only foundation seed lot), and there is increasing use of ELISA for 8.14 Seed Certification Against Plant Virus Diseases 219 the testing of breeder seed, seed from breeding monitoring remaining plants by ELISA. Seed lines and seed for commercial growers. Official from such lines has been exported to breeding certification (phytosanitary certificate) of testing programmes in other international institutes, but for PSbMV by ELISA is required for shipment the continued maintenance of virus-free nursery of pea or lentil seed from Washington and Idaho becomes the responsibility of the recipient breed- to Australia, New Zealand or South Africa. ing programmes (Hamilton 1983).

8.14.2.4 Beans 8.14.2.6 Cowpea BCMV in French beans is seed transmitted to the In cowpea, out of the 12 seed-transmitted viruses, tune of 83%, and economic yield losses are en- CpAMV, CMV and Cowpea mottle viruses are countered, whenever virus-infected seed is used. highly seed transmitted and are worldwide in Hence, in the USA and Canada, the certifica- distribution. In Nigeria, the International Institute tion programme of field and garden bean against of Tropical Agriculture (IITA) and the Institute BCMV is based only on field inspection of the of Agricultural Research and Training are the crop. These crops are not allowed to BCMV main centres for cowpea improvement, and it is diseased plants when grown for foundation seed, their policy to test cowpea lines before being 0.5% mosaic diseased plants for registered seed sent to other institutions for breeding purposes and 1% for certified seed (Anonymous 1971). In or for increase in other parts of Nigeria. Samples Brazil, bean lines are screened visually for major of 1,000 seeds are planted, and the resulting virus diseases, primarily BCMV, and apparently seedlings are examined visually for the presence virus-free lines are increased under insect-free of seed-transmitted viruses; differential hosts and conditions in greenhouses. That seed is multi- serological tests are used to facilitate further plied under field conditions in arid regions of identification. Seed lots showing 2% or more seed Brazil for two generations, with periodic visual transmission are not distributed; those with less inspection for virus symptoms before being sold than 2% seed transmission are so indicated and to certified seed growers. Yield increases of about the recipient is advised. Preliminary results indi- 30Ð100% have been reported following use of cate that the ELISA may be applicable in detect- such certified seed (Hamilton 1983). ing Cowpea mottle virus in seedlings (Hamilton 1983). 8.14.2.5 Soybean In soybean, SMV, CMV, TRSV, Soybean stunt, 8.14.2.7 Peanut and Tobacco streak viruses are seed transmit- In peanut crop, PStV and PMV are very ted at higher percentage (up to 100%) and are important, and seed transmission is recorded widespread in occurrence. Despite the fact that to the extent of 8.5Ð30% both for PMV and SMV is present and can cause serious dam- PStV. At Georgia (USA), a seed certification age wherever soybean is cultivated, there ap- programme against PMV was successfully pears to be no bona fide seed certification pro- implemented (Kuhn and Demski 1975). Even grammes against this or other seed-transmitted against PStV, a well-worked out certification viruses in this crop. Two major soybean improve- programme was framed in the USA. Inspection ment programmes (INTSOY at the University of of the standing crop is the method used Illinois, Urbana, and the Asian Vegetable Re- for detecting PStV-infected plants in peanut search and Development Center, Shanhua, Tai- seed fields in Virginia, a major producer of wan) are screening soybean lines for resistance peanut seed for commercial peanut growers to some strains, but neither of these agencies em- in Georgia, which demands zero tolerance for ploys formal certification schemes (Hashimoto PStV. Infected plants are rouged from seed 1986). International Soybean Program (INTSOY) fields. The effectiveness of the detection system has eliminated SMV from a number of tropical has been monitored by serological assays of soybean lines by roguing standing crops and suspicious plants; correlation between diagnosis 220 8 Methods of Combating Seed-Transmitted Virus Diseases and confirmation has been essentially 100% interference of non-embryonic tissues (which do (Demski and Lovell 1985). Even at Florida, not play a role in transmission of virus through Zettler et al. (1993) developed a scheme for seeds) in routine assessment of seed transmission production of peanut seed free of Peanut stripe rate and its role in seed certification programmes. and Peanut mottle viruses by using virus-indexed The germplasm samples are usually received greenhouse-grown seed. as a few seeds/sample, and thus, it is often not possible to do sampling because of the few seeds and also because of the fact that a part of the 8.15 Conclusion seed is also to be kept as voucher sample in the gene bank of any country apart from the disease- In seed certification schemes, precautions should free part that has to be released. Hence, extreme be taken by restricting the regions for raising precaution is needed to ensure that whatever is the seed lots along with the protective measures the result obtained in the tested part, it should as during the cropping period. If these precautions far as possible not to denote a false-positive or are not taken according to seed health certifi- a false-negative sample. The importers need to cate requirements, then (1) proper crop stand as be encouraged to get as much sample as possi- expected by seed analysis will not be achieved, ble to allow effective processing. More attention and (2) the disease will develop and lead to total needs to be given to non-destructive techniques devastation of the crop, resulting in deterioration wherever possible, and as in case of groundnut, in the quality of seeds and, consequently, their the whole seed is not used for ELISA testing for market value. If the diseased seed is distributed viruses, and only cotyledonary part of the seed is within the country or sent to other countries, analysed. these seed-transmitted virus diseases are further Manual processing of large number of seeds disseminated. The seed after harvest should be is not possible in a routine test (Maury and carefully analysed by some of the advanced virus Khetarpal 1997). Therefore, efforts should be diagnostic techniques described in Chap. 6 to made to develop techniques which could detect decide whether the seed samples in question virus (e.g. PSbMV) only in embryo, although the should be released for use or rejected. Review whole seed is used for extraction (Masmoudi, chapter of Maury et al. (1998) and Albrechtsen’s et al. 1994a, b). Moreover, if the number of textbook (2006) are additional sources for seed imports is more, it would be difficult to subject all certification. the samples for growing in post-entry quarantine (PEQ) greenhouses. The PEQ of bulk imports in India is being 8.16 Quality Control of Bulk carried out under the supervision of the inspec- Seed Lots tion authorities who are the senior level plant protection scientists of various universities and The size of consignment received is very critical institutes. However, these inspection authorities, in quarantine from processing point of view. Bulk in general, lack sufficient funds to carry out their seed samples of seed lots need to be tested by work effectively. This can be a major bottleneck drawing workable samples as per norms. The pre- in the functioning of this aspect of the quarantine scribed sampling procedures need to be followed operations (Khetarpal 2004)(Fig.8.3). strictly, and there is a need to develop and adapt protocols for batch testing, instead of individual seed analysis (Maury et al. 1985). Maury and 8.16.1 Determination of Seed Khetarpal (1989) discussed in depth the use of Transmission Rate ELISA for detecting viruses in single embryo, determination of seed transmission by coupling If the per cent seed transmission of a seed lot is it with group analysis, mode of eliminating the high, individually excised embryos can be tested 8.16 Quality Control of Bulk Seed Lots 221

Fig. 8.3 Growing-on test of legume germplasm in post-entry quarantine greenhouse at NBPGR, New Delhi (Source: NBPGR, R.K. Khetarpal; V.C. Chalam) by ELISA. However, if the per cent transmission bryos having the lowest titre of virus. Therefore, is assumed to be low, a large number of embryos with a given antiserum, a workable group size need to be tested that calls for use of a group- limit is determined, coherent with a probability testing method. Psl D l of detecting one infected embryo in such Due to the variation in virus titre in different a group of seeds (Maury et al. 1985; Maury et al. embryos, it is not possible to correlate, as previ- 1987b; Maury and Khetarpal 1989). ously suggested for BSMV (Lister et al. 1981), The systematic use of such quality control has an ELISA absorbance value for a sample of been efficiently protecting the lettuce crops in embryos with the number of infected embryos in California and Florida from LMV outbreaks. this sample. Therefore, the group-testing method The determination by this method of seed adopted for determining seed transmission rates transmission rates is also used by the seed in- consists of dividing a representative sample of the dustry for classifying seed lots in classes of qual- seed lot to be analysed into a number of groups ity, but the following approach mentioned in (b) of n seeds, each made at random. As compared would make it simpler. with single embryo tests, the number of tests According to the General Agreement on is divided by n. The percentage of transmission Tariffs and Trade (GATT), now known as World can be estimated as a function of the number Trade Organization (WTO), any phytosanitary of ELISA-negative groups, with a confidence measure should be based on a pest risk analysis. interval which determines the precision of the The pest risk analysis enables each country determination (Maury et al. 1985). For example, to define its appropriate level of protection. while testing 60 groups of 500 seeds of lettuce, The classification of seed lots according to if all the 60 groups are negative, the confidence seed transmission rates would much help in a interval is 0Ð0.012% at 95% level of probability. pragmatic determination of tolerance thresholds The number n of seeds per group has a limited if quality controls are now extended to other value depending on the dilution limit of the em- crops. 222 8 Methods of Combating Seed-Transmitted Virus Diseases

8.16.2 Infection Status of a Bulk Seed The first concern is a complete lack of stan- Lot with Respect dardised protocols. To solve this gap, it is neces- to a Tolerance Limit sary to control all the causes of variability which would induce result discrepancy when several Is the infection of a given seed lot under or above laboratories perform the same test on the same a non-tolerable level of infection (Int)? Answer- seed lot. An important cause is the variabil- ing this question does not require determining ity of the antiserum, due to the specificity, the the seed transmission rate. After testing k groups range of strains detected, the avidity and the of N seeds, a statistical approach which includes epitopes detected on the virus particle. Another two probabilities of errors leads to the following important difficulty is, as many laboratories from decision rule: ‘the seed lot is above the threshold developing countries will be diffcult to perform if at least one group of N seeds is found infected’ these tests if they they do not have the tech- (Geng et al. 1983; Masmoudi et al. 1994b). nology and advanced facilities. Finally, the lack Increasing each group size N reduces accord- of accurate working sheet leaves each labora- ingly the number k of groups to be tested if it tory functioning in isolation with its rudimentary is also taken into account a probability PsI of methodology. detecting one infected embryo in a group of N seeds. This probability depends on the avidity of the antiserum used. As an example, to determine 8.17 World Trade Organization with a good accuracy the status of a pea seed (WTO) Regime lot with respect to a threshold of 0.1%, it was and Its Implications sufficient to analyse 31 groups of 200 seeds each, that is, one ELISA microplate (Masmoudi et al. The World Trade Organization (WTO), estab- 1994b). It is important to underline the significant lished on 1 January 1995, is the legal and in- simplification in the number of ELISA samples to stitutional foundation of the multilateral trading be examined and the ease with which a seed lot system. The main purpose of the WTO is to can thus be classified by this approach in view of promote free trade, serves as a forum for trade local or international trade. negotiations and settles disputes based upon the principles of non-discrimination, equivalence and predictability. 8.16.3 Infection Status of a Bulk Seed The WTO agreement contains more than 60 Lot with Respect to a Virus agreements covered under some 29 individual Not Known in the Importing legal texts encompassing everything from ser- Country vices to government procurement, rules of origin and intellectual property (http://www.wto.org)of A zero tolerance should theoretically be applied these, and the following four agreements have a to the international exchange of commercial seed direct bearing on agriculture and related activi- lots when a destructive virus exists in the ex- ties: porting country, that is, the country where the ¥ Agreement on Agriculture is designed to en- seed lot is produced and/or when the same is not sure increased fairness in farm trade. known to occur in the importing country. Since ¥ Agreement on Trade-Related Intellectual it is not feasible to achieve zero tolerance while property rights is aimed at improving certifying bulk seed lots, it is in the larger interest conditions of competition where ideas and to assess the risk associated with the possible inventions are involved. introduction of a viral disease not known to occur ¥ Agreement on Technical Barriers to Trade is in the importing country (Kahn 1979). to ensure that technical regulations and certi- (a) The Actual Difficulties Preventing a Gener- fication procedures do not create obstacles to alised Use of Quality Control trade. 8.18 The Role of Plant Biosecurity in Preventing and Controlling... 223

¥ Agreement on the Application of Sanitary and top virus (BBTV), Banana streak mosaic virus, Phytosanitary (SPS) Measures is in fact the Peanut stripe virus, etc., and BBTV was probably one which is going to have major implications introduced into India from Sri Lanka in 1940 on seed/plant health in trade (Khetarpal and (Magee 1953) and has since spread widely in Gupta 2002; Khetarpal et al. 2003, 2005a, b; the country. The international spread of BBTV Khetarpal 2004). is primarily through infected planting material (Wardlaw 1961). In India, an annual loss of Rs.40 crores due to BBTV has been estimated 8.17.1 Examples of Introduced Plant in Kerala alone. These introductions highlighted Viruses Through Seed the fact that increased pace of international travel Exchange and trade had exposed countries to the danger of infiltration of exotic viruses harmful to the Among the various disease-inciting agents agriculture. of plants, viruses assume special importance Since both seeds and vegetative propagules are by virtue of being sub-light microscopic, efficient carriers of viruses, infection is progres- causing systemic infection in the host and not sively transmitted through generations. Further, being generally controlled by physical and if the in vitro cultures are raised from a virus- chemical methods. Plant viral diseases are infected mother plant, the plantlets are bound to known to cause serious yield losses. Probably carry the virus infection. The successful detection the greatest challenge is due to globalisation and control of viruses in seed and other planting and the international trade in agricultural and material depend upon the availability of rapid, horticultural produce, which is breaking down reliable, robust, specific and sensitive methods the traditional geographical barriers to the for detection and identification of viruses. Over movement of viruses. There are several instances the years, a great variety of methods have been of inadvertent introduction of pests along with developed that permit the detection and identi- introduced seed or planting material into a fication of plant viruses. Virus indexing is done country or region within a country. Trade and by deploying a combination of some of the well- exchange of germplasm at international level known virus detection techniques mentioned in play a key role in the long-distance dissemination Chap. 6 keeping in view the intended purpose. of a destructive virus or its virulent strain. The worldwide distribution of many economi- cally important viruses especially those attacking 8.18 The Role of Plant Biosecurity legumes, such as Bean common mosaic virus, in Preventing Soybean mosaic virus (Goodman and Oard and Controlling Emerging 1980), Pea seed-borne mosaic virus (Hampton Plant Virus Disease and Braverman 1979)andPeanut mottle virus, Epidemics is attributed to the unrestricted exchange of seed lots. Peanut stripe virus (later identified as a The emergence of a global community and an strain of BCMV) was introduced into the USA increase in plant virus discovery in the last in the 1980s through groundnut seed germplasm 15 years have increased the requirement for imported from China (Demski et al. 1984b). countries and regions to protect their farming The same pathogen was intercepted in Australia systems from these exotic pests. In a recent (Persley et al. 2001) from imported groundnut report, plant viruses were identified as the cause seed held in post-entry quarantine. of 47% of the emerging infectious diseases Like in other countries, a number of exotic of plants that were recorded on the PubMed plant viruses have been introduced into India database during the period of 1996Ð2002, and a along with imported planting material causing se- further 4% were attributed to phytoplasmas, and rious crop losses. These included Banana bunchy it is likely that this trend will continue. 224 8 Methods of Combating Seed-Transmitted Virus Diseases

Plant biosecurity can be defined as a set agencies to detect known and unknown plant of measures designed to protect crops from viral agents. Pre-emptive breeding strategies have emergency plant pests (EPPs) at national, also been initiated to protect plant industries if regional and individual farm levels (Anonymous and when key exotic viruses become established 2005). Countries are required to comply with in local communities. With the emergence of international obligations as defined by the free trade agreements between trading partners, World Trade Organisation (WTO) Agreement on there is a requirement for quality assurance mea- the Application of Sanitary and Phytosanitary sures of pathogens, including viruses, which are Measures (WTO 1995). More specifically, present in both the exporting and importing coun- international guidelines and standards are tries. These measures are required to ensure mar- outlined under the International Plant Protection ket access for the exporting country and also min- Convention to demonstrate pest-free areas (FAO imise the risk of the establishment of a disease 1995) that stipulate that a pest/pathogen is epidemic in the importing country (Khetarpal and ‘known not to occur’ in a given geographical Gupta 2007; Rodoni 2009). region (van Halteren 2000). This edict is A number of research strategies have been significantly different from the previous wording initiated over the last decade to enhance plant of ‘not known to occur’ and has resulted in biosecurity capacity at the pre-border, border and additional requirements for surveillance of post-border frontiers. In preparation for emerg- EPPs in countries to demonstrate area freedom. ing plant virus epidemics, diagnostic manuals Countries that import plant products can demand for economically important plant viruses that from exporting countries or regions evidence for threaten local industries have been developed the absence of pests which do not occur in that and validated under local conditions. Contin- importing country. Importing countries also have gency plans have also been prepared that provide an obligation to provide more justification for guidelines to stakeholders on diagnostics, surveil- their quarantine measures and not conceal or fail lance, survey strategies, epidemiology and pest to look for the presence of pests in their territory risk analysis. Reference collections containing (van Halteren 2000). validated positive virus controls have been ex- In this context, a number of research strategies panded to support a wide range of biosecurity sci- have been initiated over the last decade to en- ences. Research has been conducted to introduce hance our biosecurity capacity at the pre-border, high-throughput diagnostic capabilities and the border and post-border frontiers. In preparation design and development of advanced molecular for emerging plant virus disease epidemics, diag- techniques to detect virus genera. These diagnos- nostic manuals for economically important plant tic tools can be used by post-entry quarantine viruses that threaten local industries (e.g. Plum agencies to detect known and unknown plant pox virus) have been developed and validated viral agents. Pre-emptive breeding strategies have under local conditions. Contingency plans have also been initiated to protect plant industries also been documented that provide guidelines to if and when key exotic viruses become estab- stakeholders on diagnostics, surveillance, survey lished in localised areas. With the emergence strategies, disease epidemiology and pest risk of free trade agreements between trading part- analysis. Reference collections containing vali- ners there is a requirement for quality assurance dated positive controls have been expanded to measures for pathogens, including viruses, which support a wide range of biosecurity sciences. may occur in both the exporting and importing Research has been conducted to introduce high- countries. These measures are required to en- throughput diagnostic capabilities and the design sure market access for the exporting country and and development of advanced molecular tech- also to minimise the risk of the establishment niques to detect virus families. These diagnos- of a damaging virus epidemic in the importing tic tools can be used by post-entry quarantine country. 8.19 Pest Risk Analysis (PRA) 225

major pests and diseases is to be studied. The en- 8.19 Pest Risk Analysis (PRA) try status refers to the entire range of decisions or policies or regulations that serve as guidelines for Pest categorisation is the key component of pest rules and regulations of that government whether risk assessment. This is not just an identification or not a potential carriers like plants, plant prod- of the pest species but an analysis of its potential ucts, cargo, baggage, mail, common carriers, etc., danger as a pest. A pest of quarantine significance are enterable and if enterable, under what safe- refers to a pest of potential economical impor- guards, into one geographical region to another. tance to the area endangered, but not yet present Pest and pathogen risk is based on an evaluation there, or present but not yet widely distributed of biological variables. Examples of such vari- and being officially controlled. Pest categorisa- ables are the (1) ecological range of a hazardous tion includes the following major elements: organism compared to the ecological range of its ¥ Identification of the pest hosts in the importing country, (2) hitchhiking ¥ Definition of the PRA area activity of the pest and (3) ease of colourisation of ¥ Distribution and official control programmes the pest. Attitudes towards entry status may range within the PRA area from ‘conservative’ to ‘liberal’, but ‘liberal’ in ¥ Potential of the pest for establishment and this context does not imply ‘lax’. The most con- spread in the PRA area servative attitude is that the plant material is ¥ Potential economic impact potential in the excluded without any exceptions, whereas the PRA area most liberal attitude is that the plant material ¥ Endangered areas may enter freely without agricultural regulatory The other major components of PRA include restrictions. When the points of valid matchings Economical Impact Assessment (basically a thor- are plotted and connected, a biological curve may ough examination of the economic risk associated be drawn. By illustrating the pathogen and pest with the process) and the Probability of Intro- risk analysis diagrammatically as a curve on a duction, which looks at the prospects of both graph, one can effectively communicate genera- entry and establishment of the pest. Essentially, tion philosophy, principles, policies and decisions pest risk management is the process of deciding not only to quarantine officers but to the scien- how to react to a perceived risk, deciding whether tific, commercial or growers. It is also required action should be taken to minimise the risk, and, to explain a quarantine decision or activity that if so, what action should be chosen. These steps is against the interest of the importer. This curve should provide a clear scientific basis for pest is also useful in domestic quarantine matter in risk assessment, risk management and quarantine making understand the interaction of biological, decisions, and prevent these from being used in economic and political factors to the trainees. It an inconsistent manner and as barriers to interna- also helps the quarantine officers in diagnosing tional trade. priorities or discussion budgetary matters.

8.19.1 Pest and Pathogen Risk Analysis 8.19.2 Pest Risk Analysis for Viral Diseases of Tropical Fruits Availability of accurate and reliable information on the occurrence of the pests and diseases and Virus and virus-like diseases have been causing the damage they cause is one of the essential serious damage to fruit production in tropical requirements for the effective operation of the areas. They are not usually transmitted through quarantine services. Unless such information is seeds, but are often carried across national available, the plant quarantine or plant protection boundaries by infected bud wood, vegetatively officer has no basis on which to act in regulating propagated seedlings and transmission by insect the importation of plants. The entry status for the vectors. 226 8 Methods of Combating Seed-Transmitted Virus Diseases

With regard to disease risk analysis, one has also. When costs are entered into the pest risk to have sound knowledge about the major virus analysis, plant quarantine officers consider the diseases of economically important cereals, veg- costs of adequate safeguards like virus indexing, etables, and fruit crops, in terms of the aetiology heat therapy, meristem tissue culture, etc. and epidemiology of the diseases, current diagno- The pest and the pathogen risk analysis curve sis methods, their geographical distribution and can be exemplified with the little cherry disease, their economic impact. The various strains of present in Washington. This little cherry disease important viruses infecting fruit crops are to be is a phytoplasma disease spreads due to the sup- critically identified. For framing suitable man- ply of infected plant material by nursery men agement measures in recent years tissue culture and secondary spread in the field by the vectors techniques have gained importance in producing and is found in the dooryard cherries in a 40- virus-free planting material and advantages and square-mile area in the Washington state. Studies disadvantages of this technique are to be studied on the risk analysis curve to illustrate specific depending on the virus and the crop. Plantlets problems in plant quarantine due to little cherry propagated by tissue culture have been found to US agriculture and various quarantine actions to be more susceptible to Banana mosaic virus taken are (1) destruction of all species of Prunus than seedlings of sucker origin. Since tissue cul- in a given geographical area; (2) destruction of P. ture is widely used to produce disease-free ba- avium, P.cerasus and all following cherry species nana seedlings, this suggests that intensive vector like P. serrulata, P. subhirtella and C. yedoensis control is essential in banana plantations where in a given geographical area; (3) destruction of all plantlets are raised by tissue culture are being sweet (P. a v iu m) and sour cherries (C. cerasus)in grown. the area; (4) destruction of only sweet and sour Greening is one of the most serious citrus dis- cherries showing diagnostic symptoms (either eases which is now identified to be Liberibacter with or without confirmatory laboratory staining asiaticus C gram-negative bacterium. It affects results); (5) survey for symptoms to determine most citrus species and is vectored by psyllids in the distribution of the agent in P. a v iu m and P. a persistent matter. Most natural spread probably cerasus in the Western States or the entire United occurs when young shoots are sprouting, at a time States and (6) no regulatory action (Kahn 1989; when succulent growth is present on donor and Kahn and Mathur 1999). receptor plants, and psyllid populations are high. The problem presented by the little cherry Recent work by an FFTC survey team, using new agent in the dooryard cherry in Washington is to methods of diagnosis and indexing, had found determine which one or more of the quarantine greening to have a much wider distribution in the actions shown on the ‘Y’ axis should be imple- Pacific than previously recognised. Some strains mented so that the quarantine action and pest are virulent to pomelo, which had been thought risk interact somewhere on the pest risk analysis resistant to the disease. This illustrates the im- curve. Another good example of pathogen risk portance of using advanced scientific procedures analysis is the Cadang-cadang disease,ofthe when certifying planting materials, particularly coconut palm, whose aetiology is proved to be imported ones, as virus-free. of viroid in nature and the incubation period is The pest and pathogen risk is based on two long and uncertain. Similarly, there are risks in- general precepts Ð (1) the benefits must exceed volved in sending carnation and chrysanthemum the risk, and (2) the benefits must exceed the cuttings to warmer climates to avoid the growing costs. In international transfer of genetic stocks, conditions of the northern European winter. At the benefits usually consist of the opportunity to present, cuttings are sent from the United King- introduce new crops or new varieties of old crops dom to overwinter in places such as Malta, Sar- or to introduce new genes to improve the old dinia, the Canary isles, Kenya and South Africa. varieties. Such improvement may consist of in- The cuttings are grown as mother plants in the creased yields through increased pest or pathogen warmer climate until large enough to provide resistance coupled with increased nutritive values cuttings, which are then returned to Britain or 8.20 Biosafety 227 other European countries. In these warmer coun- the composition of the local ecosystem. There- tries, viroid diseases like chrysanthemum stunt, fore, in most countries, environmental studies are chrysanthemum chlorotic mottle, cucumber pale required prior to the approval of a GM plant fruit, citrus exocortis and potato spindle tuber for commercial purposes, and a monitoring plan are highly hazardous and multiply much more must be presented to identify potential effects rapidly and reach greater concentrations in plants which have not been anticipated prior to the at temperatures in the range of 30Ð35ıC. Under approval. these conditions, each of them is able to infect the principal host plants of the other viroids. This factor should not be overlooked, for example, 8.20.1 Biosafety Regulations where chrysanthemums send to the Canary isles may be growing near to potato or cucurbit crops, Application of GM technology for commercial so that infections might be transferred in either crop production is faced with a number of issues direction (Hollings and Stone 1973). and challenges the most important being safety Pest risk analysis is generally accepted as the of environment, and human and animal health principal strategy to make sure that plant quaran- (Grumet and Gifford 1998;Khetarpal2002; tine standards are transparent and justified on a Philippe 2007). These concerns are based on sound scientific basis. However, its application to the argument that recombinant DNA-based GM individual pests will require great deal of further technology differs from traditional breeding in discussion and further detailed work. Once this is that totally new genes using potentially risky done, PRA can be expected to remove unneces- technology are transferred between widely sary barriers to trade. unrelated organisms, and the location of these There is an urgent need for all countries to genes on the recipient genome is random, reach an equal level in plant quarantine, in terms unlike when gene transfer takes place through of technology and equipment. An international conventional breeding. These differences demand network on quarantine pest monitoring is also that adequate laboratory safeguards are used needed, to meet the growing danger of exotic and plants developed by GM technology are pest invasion as a result of growing international rigorously assessed for their performance as also tourism and trade, and the long-distance migra- for the likely risks they pose to the environment tion of insect pests. and human health and a few other concerns Disease risk analysis has to be followed by related to the application of biotechnology in control of the diseases. In general, these vector- agriculture. borne systemic plant virus diseases can be effec- tively controlled by integrated control measures, including production and cultivation of virus-free 8.20.2 History of Biosafety Protocol seedlings, elimination of inoculum sources and and Regulations prevention of reinfection through IPM of vector insects and cross protection with mild strains. Because of the risk associated with genetically The establishment of pathogen-free nursery sys- modified organisms, there were concerns world- tems is the most important way of preventing wide to control it. It is also important to protect these diseases from spreading. the biological diversity of the nature while re- leasing or accidental escape of GMO to the envi- ronment. This leads to the formation of biosafety 8.20 Biosafety protocols dealing with genetically modified or- ganisms. The origins of the biosafety protocol Genetically modified plants can spread the trans- were found in the UN Convention on Biological gene to other plants or Ð theoretically Ð even to Diversity, which was signed by over 150 govern- bacteria. Depending on the transgene, this may ments at the Rio ‘Earth Summit’ in 1992, and pose a threat to the environment by changing which came into force in December 1993. In 228 8 Methods of Combating Seed-Transmitted Virus Diseases the Convention on Biological Diversity (CBD), The protocol was adopted on 29 January 2000. it was acknowledged that release of GMOs (re- The protocol has been signed by 103 countries ferred to in the CBD as ‘living modified organ- (except the USA). The Cabinet (GOI) approved isms’ or LMOs) may have adverse effects on the proposal, and India signed the biosafety pro- the conservation and sustainable use of biological tocol on 23 January 2001. Subsequent to the diversity. All countries that signed up to the CBD Cabinet approval on 5 September 2002, India has were expected to: acceded to the biosafety protocol on 17 January (a) ‘Establish or maintain means to regulate, 2003. So far, 43 countries have ratified the pro- manage or control the risks associated with tocol. The protocol will come into force on the the use and release of living modified organ- 90th day after the date of deposit of the fiftieth isms resulting from biotechnology which are instrument for ratification by countries that are likely to have adverse environmental impacts, parties to the convention. taking also into account the risks to human health’. (b) ‘Consider the need for and modalities of a 8.20.3 Biosafety Regulations protocol setting out appropriate procedures of Asia-Pacific Countries in the field of the safe transfer, handling and use of any living modified organism resulting Beginning the late 1980s, Asia-Pacific countries from biotechnology that may have adverse initiated legislative measures to manage the effect on the conservation and sustainable use potential risks associated with GM technology. of biological diversity’. In 1986, India enacted ‘Environment Protection In accordance with the precautionary Act’ and published ‘The Environment (Protec- approach contained in Principle 15 of the Rio tion) Rules’ to regulate environmental pollution Declaration on Environment and Development, by managing hazardous substances, including the objective of the protocol is to contribute hazardous microorganisms and GMOs. In 1990, to ensuring an adequate level of protection in ‘Philippines Presidential Order’ established a the field of the safe transfer, handling and use national biosafety committee, and during the of living modified organisms resulting from early 1990s, India and Thailand published the modern biotechnology that may have adverse first guidelines on research and environmental effects on the conservation and sustainable use aspects of GMOs. of biological diversity, taking also into account The biosafety regulatory systems vary across risks to human health, and specifically focusing countries; some developing an entirely new on trans boundary movements. biosafety specific system while others making The Cartagena Protocol on biosafety, the first modifications in the existing regulatory systems international regulatory framework for safe trans- to address biosafety issues. The legal instruments fer, handling and use of living modified organ- used for the purpose have been new or modified isms (LMOs), was negotiated under the aegis laws, acts, decrees, guidelines, rules, etc. of the Convention on Biological Diversity. The Alongside, administrative systems have been protocol contains reference to a precautionary developed to operationalise the legal instruments. approach and reaffirms the precaution language Following one or the other approach, a number in Principle 15 of the Rio Declaration on En- of countries have currently in place regulations vironment and Development. The protocol also on development, contained use, environmental establishes a Biosafety Clearing-House to facil- release, commercialisation and import of GM itate the exchange of information on LMOs and crops and products. Several other countries have to assist countries in the implementation of the their biosafety regulations either in drafting or protocol. implementation phase. 8.22 Quarantines 229

8.21 Risks Associated 8.22 Quarantines with Genetically Modified Crops The term quarantine is derived from Latin word, quarantum, meaning forty. The objective of plant Genetically modified crops deal with the growth quarantines is to protect the agriculture, by pre- of plants. Transgenic plants exposed to the open venting the introduction and spread of important environment, and the interaction takes place with pests and diseases by legislative restrictions on other organisms in the field. Many of them con- the movement of plants and plant products. In tain toxic gene and antibiotic resistance marker other words, quarantines are important disease with them. Therefore, genetic modification in control measures based on avoidance and ex- agriculture has become a more sensitive issue clusion technique. Migrations, military conquests than those experiences with recombinant bio- and occupations, voyages of discovery, religious therapeutics. Transgenic plants need special at- expeditions and germplasm exchange have in tention because they are exposed to environ- many ways contributed to the spread of plant ment. Until today, there is no major risk concern material. A number of diseases that were not associated with the marketed transgenic crops present earlier in certain countries have been such as cotton, tomato, corn and soybean. Un- introduced into new areas either on or in the fortunately, the public debate over the hazards plant materials which in turn had spread and of transgenic plant or transgenic food suffers caused disastrous crop losses. For increase of the from misinformation and misunderstanding of food production by breeding techniques, there is the basis of genetic manipulation in plant sys- a constant exchange of germplasm throughout tem. These GM foods carry a label and have the globe, and hence, a great impact is noticed been to extensive field trials for safety and en- in agricultural outputs. Most of our major crop vironmental impact before they are approved for plants now grown in areas where they did not commercialisation. With the continuing accumu- evolve, but have been disseminated by man, are lation of evidence of safety and efficiency and introductions. The phenomenal increase in ad- no harm to public or the environment, more vanced transport techniques during the recent and more transgenic plants and food are getting years has increased the movement of the plant appreciated and used by the people. Neverthe- material by man from place to place irrespective less, thorough assessment of the risk and safety of geographical barriers. While air travelling, a associated with GM crops and food needs com- small bud wood stick or cutting can be carried in a plete evaluation before they are released to the viable condition without any protective measures. environment. In the case of ship travel involving prolonged The major safety concerns associated with duration of time, much propagative material will GM crops and GM food are as follows: be lost during the passage, and the inspection 1. The effect of GM crops on environment and can be done leisurely at the port of entry. It biodiversity is also established from Table 1.1 that nearly 2. Gene pollution (escape of gene through 231 viruses are seed transmitted, while on the pollen) contrary, the propagules derived from the virus- 3. Toxicity of the GM plant due to altered infected vegetative material are mostly infected. metabolism The dissemination of seed-transmitted viruses is 4. Safety, toxicity and allergenicity of the GM also occurring due to the massive movement of food seed, particularly under ‘green revolution’. In the 5. Insect- and herbicide-resistant varieties of developing countries, import of large quantities plant of food grains may result in certain inadvertent 6. Undesirable effect of transgenic plant on non- disease introduction, and in certain times, farmers targeted or beneficial insect or plant in the use the same in smaller amounts as planting environment material. 230 8 Methods of Combating Seed-Transmitted Virus Diseases

All countries are involved in the export and 8.22.1 Plant Quarantine import of agricultural products and are depending heavily on agriculture for their export earnings. Plant quarantine promulgated by a government The key concept in the new free trade situation or group of governments to restrict the entry of is pest risk analysis, an objective assessment of plants, plant products, soil, cultures of living or- the dangers of invasion by a particular known ganisms, packing materials and commodities, as and unknown pests and diseases including plant well as their containers and means of conveyance virus and virus-like diseases which may damage to protect agriculture and the environment from agriculture production more drastically if they avoidable damage by hazardous organisms. They were accidentally introduced. exclude dangerous organisms while permitting Scientific community is also introducing the plants and plant products to enter (Kahn 1988). infected genetic stocks for research investigations The term exclusion conveys this objective more unaware of national regulations or due to clearly than plant quarantine. Exclusion means sheer ignorance. On several occasions, seeds the practice of keeping out any materials or ob- are sent across the world by post enclosing jects that are contaminated with pathogens or them in envelopes or are brought by tourists. disease plants and preventing them from entering For scientific purposes, there is no embargo the production system for which quarantine rules exchange of germplasm when safeguards are and regulations are to be implemented. adequate and quarantine regulations of every country are playing major role in this regard. Plant quarantine is a government endeavour 8.22.2 Plant Quarantine Measures enforced through legislative measures to regulate the introduction of planting materials, A number of quarantine measures are available, plant products, soil, living organisms, etc., and may be used, either separately or collectively, in order to prevent inadvertent introduction in an attempt to prevent the establishment of a of pests (including fungi, bacteria, viruses, new plant pest or disease in an area previously nematodes, insects and weeds) harmful to the free of it. The governments enact regulations agriculture of a country/state/region and, if in- and stipulate safeguards in accordance with the troduced, prevent their establishment and further risk presented by the importation of the plant spread. materials from areas where hazardous pests and It is evident from the third chapter that the pathogens are known to occur. The quarantines virus and virus-like diseases are potentially dan- operate between different countries or between gerous in causing heavy toll of the crops. Some- adjacent states in one country. The regulations times, the catastrophic consequences suffered by are formulated and implemented by the federal agriculture through the introduction of exotic or state government or both. Persons desiring diseases compelled every nation or a group of detailed information about the quarantine regu- government to impose legal restrictions on inter- lations of foreign countries should request their national trade of plant materials which sometimes own country’s plant protection and quarantine unnoticingly carry pests and diseases. The entry service. In each country, the government regu- and further spread of these dangerous pests and lations issued by ministries or Departments of diseases in each country are controlled by quar- Agriculture have been received and abstracted. antines. Khetarpal et al. (2004, 2005a, 2006a) In general, these regulations are as follows: (1) and Khetarpal and Gupta (2007) have reviewed Specify requirements of import permits, (2) re- various methods for the safe movement of plant quire phytosanitary certificates, (3) require cer- germplasm. tificates of origin, (4) stipulate inspection upon 8.22 Quarantines 231 arrival, (5) prescribe treatment upon arrival to Asian greening and its vectors, once established eliminate a risk, and (6) prescribe quarantine or in an area, have the potential of totally destroying post-entry quarantine depending on the nature of citrus as an economic crop (Fraser et al. 1966; the risk. Salibe and Cortez 1966). There are new strains Some of the quarantine measures comprise of Citrus tristeza virus which can spread very the following: embargoes, inspection at the port rapidly (Bar-Joseph and Loebenstein 1973)and of entry, inspection at the port of dispatch, field can affect many citrus root stocks and scions inspection during the growing season (preclear- once considered tolerant to the virus. Even the ance), controlled entry (i.e. disinfection, treat- introduction of exocortis viroid into a country ments) and post-entry quarantine stations. Some such as Japan or Uruguay, where the predominant details of these quarantine measures are men- root stock is P. trifoliata, is a distinct hazard tioned in the subsequent pages. because exocortis can be spread by unindexed bud wood or very effectively by cutting tools and is very damaging to Poncirus root stocks. Some 8.22.3 Functions of Plant Quarantine of the important virus and virus-like diseases introduced in different countries are discussed The prime functions of plant quarantine are as under separate heading. follows: (1) enforcing the measures for the pre- For the international exchange of germplasm, vention of foreign species of plants with pests the involved scientists or commercial nurseries and diseases from overseas and it is defined as whether importers or exporters should be cog- international quarantines; (2) quick identification, nizant of the quarantine regulations of the im- localisation and eradication of exotic pests and porting country. The quarantine regulations of diseases which are invaded within a country itself the importing country will determine the enter- and is called as domestic quarantine and (3) pro- ability of such germplasm. Even the quarantine viding to the country with as many new varieties service of the exporting country is involved as a as possible at the same time without allowing source of information about the requirements of entry of new diseases. the importing country and also in the issuance International Quarantine: This will prohibit or of phytosanitary certificates, if required by the regulate the entry of imports from other counties’ importing country. necessitation international cooperation on Domestic Quarantines: In spite of the best phytopathological and entomological problems. precautions taken and stringent measures of quar- For long periods, plants and the organisms antine applied, even the most advanced countries pathogenic to them in many instances coexisted have not been able, on several occasions, to in balanced equilibrium. Interference by man prevent the entry of the diseases. In such cases, invariably disturbs this balance, and the man has taken recourse to domestic quarantine, pathogens of minor significance in one situation which will help in the confinement of the diseases may cause epiphytotics when transferred to new and pests to particular area only, due to the environment/places. It may be because of plants restriction in the movement of the plant material developing in the absence of the pathogen, have between the states. They will primarily deal with no opportunity to select resistant factors specific the new diseases and pests which are already against an introduced pathogen, as a consequence entered in a country and will be made to elim- of which when they are grown in a place where inate them before they become established. The they have been imported become extremely most effective quarantine action is that which is vulnerable to disease attack. In certain cases, applied at the source of spread. The problems of the introduced diseases may also mutate to more domestic quarantine are basically similar to those destructive forms and cause epidemics. of international quarantine. In practice, federal There are a number of examples, where the and state governments jointly enforce regula- introduced plant viruses into new areas have tions to prevent intrastate as well as interstate resulted into heavy crop losses. For example, the spread. 232 8 Methods of Combating Seed-Transmitted Virus Diseases

In each country, some legislations are also In Sudan, legislation is passed for pulling of passed against certain devastating virus diseases; the volunteer cotton plants and the regrowth to for example, in California, an ordinance No. 1053 be prevented for the control of Cotton leaf curl of the county of Monterey was implemented virus. Growing of okra before 15th September is to prevent losses of lettuce crop due to mosaic strictly prohibited. Any programme of regulation disease and reads in part as follows: and free registration can succeed only if it is supported by the growers and nursery men. ‘Mosaic is a virus disease which infects lettuce crops and is a direct cause of crop failures. It is In California, they have provided outstanding a disease which is seed transmitted and carried by support and cooperation of various regulatory aphids and, if not prevented, spreads from field and quarantine programmes. Similarly, in South to field affecting large areas of production and Africa, a cooperative programme between causing great losses to growers ::: The disease threatens to seriously affect the general economy growers and the government regulated in if prompt and effective measures are not taken at legislation passed in 1927 providing for the once to eradicate this menace. eradication of all psorosis-infected citrus trees It shall be unlawful for any firm or corporation to showing bark lesions (Doidge 1939). This plant any lettuce seed in the unincorporated area programme carried out between 1930 and 1950, of the country of Monterey which has not been effectively reduced psorosis in South Africa mosaic Ðindexed. from one of the most destructive diseases to Any violation of this ordinance constitutes a mis- one that is now rare. In the Philippines during demeanour punishable by a fine of not more than 1962Ð1974, the area of mandarin, sweet orange $500.00 or by punishment in the county jail for a and pomelo cultivation was greatly reduced in period not exceeding six (6) months’. certain parts up to 60% due to leaf mottling This ordinance mentioned above has been ef- (greening). In 1969, a quarantine measure was fective in reducing losses in lettuce production for issued through an administrative order by the nearly 20 years. Similarly, Western celery mosaic Secretary of the Department of Agriculture to virus is effectively controlled by the establish- prohibit entry of citrus planting materials into ment of a legally defined period in which no cel- noninfected areas. All citrus for planting will be ery can be grown in a given district was described required to certification papers from the point by Milbrath (1948). Due to the implementation of of origin for verification in inspection centres this legislation in the VeniceÐSawtelle region of and quarantine stations throughout the citrus- California, celery yields were increased by 50% growing areas of the Philippines (Altamirano in the first 2 years following passage of the law et al. 1976). Similarly, in Florida, legislations and increased by another 50% in the subsequent were passed for certifying the peach nursery years. stocks. Prior to June 30th, of every year, Phony In most of the countries, the legislations were peach trees should be eradicated which are passed for the nursery men for the supply of found are mile within the environs, of the disease-free plants. The nurseries were inspected nursery. Ultimately, the success of quarantine while growing, by the quarantine staff so that is dependent on a substantial degree of public only certified planting stocks were sold which support, but if regulations can be ignored with meets certain prescribed standards. In most of impunity by a few, they soon lose the support of the developed countries, seed potatoes are certi- many. fied. In India, the Directorate of Plant Protection, Quarantine and Storage takes necessary steps to regulate the interstate movements of plants and 8.22.4 Pathways of Spread of Pests plant materials to prevent the further spread of and Pathogens destructive pests and diseases. Some of the successful test control aspects by Pests and pathogens may move along natural or legislations in different countries are as follows: man-made pathways. Whether a pest or pathogen 8.22 Quarantines 233 becomes established at the end of the pathway in some, but not all, foreign countries, but the depends on the synchronisation of three factors: importation originates from a country where sufficient viable inoculum (or population thresh- such risks have not been reported. Post-entry old), the presence of susceptible hosts and a quarantine is usually reserved for government favourable environment. At the end of the path- services, in situations and qualified individuals way, if any one of the factors is not operative, whose facilities meet post-entry quarantine an organism, although it may have gained entry, requirements. Safeguards consist of inspection does not become established. In theory, countries upon arrival and during a special post-entry are bombarded by exotic organisms entering on period, usually for at least two growing seasons, natural pathways: Yet, there is usually no estab- at the premises of the importer. The fourth and lishment if the three factors are not operating last category, restricted entry, is usually assigned when the pest or pathogen arrives (Kahn 1988). to only restricted plants that are received by the general public, and they were subjected to inspection and treatment upon arrival at a port of 8.22.5 Quarantine Status of Plant entry or inspection station. Importations

A number of precautions are to be undertaken 8.22.6 Types of Materials Received to minimise pest/disease risk. They include the regulations themselves, phytosanitary The genetic stocks may be in the form of certificates, with or without added declarations, ‘vegetative propagule’ or ‘seed’ type. Vegetative permits, inspection upon arrival, treatments, propagative material may be bud wood, scion etc. Based on their quarantine status, when material or unrooted cuttings, all of which, even a plant or seed material is received at although dangerous, carry less risk than does the inspection station or port of entry, they are rooted vegetative propagating material and can placed in one of the four categories mentioned be transported quickly by air. The vegetative here: (1) absolute prohibition, (2) quarantine, (3) propagative material also includes plant materials post-entry quarantine and (4) restricted entry. like rooted cuttings or bulbs, tubers, corms Whenever the risk of introducing pests and or rhizomes. The plant material can also be pathogens is very high, then the importation exchanged in the form of ‘seed’. In the earlier of that particular plant material is completely chapter, the seed-transmitted viruses and the prohibited, even for government services and methods to make them virus-free were discussed. such material will be included under absolute The introduced plant material should be tested at prohibition category. This is invoked by the the quarantine stations and which involve some importing country when there are no adequate time and labour. The imported plant material safeguards and when isolation from commercial should be tested by germination, indexing, crop production is not possible. For example, an serology, electron microscopy, etc., which were absolute prohibition against coconut from eight earlier described in this chapter. The seeds are specified geographical areas was recommended less likely to carry virus diseases than is the by the South West Pacific Commission, because vegetative propagative material. The propagules of the diseases like cadang-cadang (Hanold and derived from virus-infected mother plants will Randles 1991). The second category, quarantine, be mostly infected. The import of seed is covers the plant materials on which the pests not considered to involve a risk of the same and diseases have already been reported from magnitude as that presented by living plant the country. Admission of plants is usually material, except that in some cases, viruses are reserved for government services and not for carried through seed. Since no case of seed- private agencies. In the third category, post- transmitted disease transmitted by whiteflies entry quarantine, the genera are placed on has been definitely established, the required which pests and pathogens have been reported germplasm can be introduced through seeds. 234 8 Methods of Combating Seed-Transmitted Virus Diseases

With the discovery of Potato spindle tuber existing knowledge in all disciplines relating to viroid which is transmitted through true seed of the phytosanitary safety of germplasm transfer. tuber bearing species of Solanum can no longer This has prompted FAO and IBPGR to launch be regarded as freely interchangeable among a collaborative programme for the safe and ex- breeders and collectors. peditious movement of germplasm reflecting the FAOÐIBPGR have recommended the fol- complementarity of their mandates with regard to lowing steps for majority of the ‘seed’-type the safe movement of germplasm. germplasm movement for majority of the crops, Frison et al. (1990) (Frison EA, Bos L, and with little modifications, one can plan Hamilton RI, Mathur SB, Taylor JW) under the depending on the size of the consignment. At instructions of FAO/IBPGR have brought out International Institute of Tropical Agriculture technical guidelines for the safe movement of (IITA), Nigeria, the in vitro techniques have legume germplasm in which they have furnished been applied to the conservation and exchange the information on virus and virus-like diseases of cassava germplasm to collaborators outside of legume crops and provided the guidelines Nigeria, and plantlets regenerated from meristem for international crop improvement programmes, culture were transplanted and indexed for African collecting, conservation and utilisation of plant cassava mosaic virus (ACMV). Plantlets regen- genetic resources and their global distribution. erated from nodal cuttings obtained from their In this preparation, the experienced crop virus-tested plant were used for distribution upon experts, namely, Albrechtsen SE, Johnstone GR, request. NARS has distributed these certified Makkouk KM, Feliu E, McDonald D, Mink GI, virus-tested plantlets for evaluation in more than Morales FJ, Reddy DVR, Vermeulen H, Rossel 40 countries in Africa (Ng and Ng 1997). HW and Zhang Zheng, have provided the details The transfer of germplasm should be care- of virus and virus-like diseases of leguminous fully planned in consultation with quarantine au- crops and participated in the preparation of the thorities and should be in amounts that allow guidelines. adequate handling and examination. The mate- The movement of germplasm involves a risk rial should be accompanied with the necessary of accidentally introducing plant quarantine pests documentation. along with the host plant material; in particular, pathogens that are often symptomless, such as viruses, pose a special risk. To minimise this risk, 8.23 Role of FAO/IBPGR effective testing (indexing) procedures are re- in Germplasm Exchange quired to ensure that distributed material is free of pests that are of quarantine concern. General rec- FAO has a long-standing mandate to assist its ommendations on how best to move germplasm member governments to strengthen their Plant of the crop concerned and mention available Quarantine Services, while IBPGR’s mandate Ð intermediate quarantine facilities when relevant inter alia Ð is to further the collecting, conser- are provided. vation and use of the genetic diversity of useful plants for the benefit of people throughout the world. The aim of the joint FAO/IBPGR pro- 8.23.1 Conceptual Guidelines for gramme is to generate a series of crop-specific Exchange of Legume technical guidelines that provide relevant infor- Germplasm, Breeding Lines mation on disease indexing and other procedures and Commercial Seed Lots as that will help to ensure phytosanitary safety when Follows germplasm is moved internationally. The ever increasing volume of germplasm (a) Germplasm exchanged internationally, coupled with recent, ¥ All legume germplasm collections should rapid advances in biotechnology, has created a be maintained free of known seed- pressing need for crop-specific overviews of the associated pests (seed borne or seed 8.23 Role of FAO/IBPGR in Germplasm Exchange 235

transmitted in the case of fungi and ¥ Unless specified otherwise, seeds should bacteria; seed transmitted in the case be surface disinfected (with sodium of viruses). Descriptor data should be hypochlorite or a similar product) before obtained from pest-free germplasm. being given appropriate fungicide and ¥ Only seed lots certified to be free of such insecticide treatments. pests should be distributed. ¥ Seed lots suspected to contain insects ¥ In recipient countries, seed lots should should be fumigated with an appropriate be established and maintained for pesticide. one generation under conditions of ¥ Parcels containing seeds should be un- isolation (temporal and/or spatial) or packed in a closed packing material and containment, with periodic inspection, should be incinerated or autoclaved. testing and roguing. (b) Breeding Lines ¥ Legume seed lots to be exchanged among 8.23.3 Movement of Germplasm breeding programmes should be produced under conditions of isolation (with ap- (a) Introduction of Germplasm propriate chemical protection) or contain- ¥ Introduction of new germplasm entries ment, with periodic inspection and rogu- should satisfy local regulatory require- ing to eliminate seed-associated pests. ments. ¥ Seed lots should be tested for seed- ¥ Each new introduction should be grown associated pests and certified by the under containment or isolation. appropriate regulatory agency before ¥ Plants should be observed periodically. distribution. Plants suspected to be affected with seed- (c) Commercial Seed Lots associated pests should be destroyed. ¥ Commercial seed lots should continue ¥ All symptomless plants should be tested to be subject to current regulatory for latent infections by viruses known to procedures. occur in the place of origin of the ma- terial and in the country of maintenance. Ideally this testing should be carried out 8.23.2 The Technical Guidelines for at this stage or, if not possible, it should Exchange of Germplasm be carried out before the germplasm is and Breeding Lines distributed (see ‘International Distribution of Germplasm’). Infected plants should be (a) General Recommendations destroyed. ¥ Vegetative material of legume species ¥ Seed should be collected from healthy should go through intermediate or post- plants only. entry quarantine and should be tested for (b) Further Multiplication of New Introductions absence of viruses. or Rejuvenation of Germplasm Accessions ¥ Legume seed should not be moved inter- ¥ Seed should be sown under containment nationally in pods. or isolation with appropriate chemical ¥ Seed should be harvested at optimal time protection. for the crop and care taken to ensure effec- ¥ Plants should be observed periodically. tive drying. Plants affected by seed-associated pests ¥ Seed samples should be cleaned to elimi- should be removed and destroyed. nate all soil, plant debris, seeds of noxious ¥ Seed should be collected from healthy weeds and phanerogamic parasites. plants only. 236 8 Methods of Combating Seed-Transmitted Virus Diseases

(c) International Distribution of Germplasm ¥ Germplasm accessions that have been in- 8.24 Steps in Technical troduced and multiplied according to the Recommendations for Seed procedures described above can be certi- Germplasm Exchange in the fied and distributed internationally. Country of Origin ¥ Germplasm accessions which are not yet or Destination in a pest-free state should be handled ac- cording to the same procedures as de- ¥ Seed production should be carried out in areas scribed for new introductions. which are free from diseases of quarantine significance whenever possible. ¥ Movement of germplasm should comply ¥ Fruits should be harvested from healthy with regulatory requirements of the im- looking plants. porting country. ¥ In addition to the phytosanitary certificate, ¥ Seeds of normal size should be selected from a ‘germplasm health statement’, indicat- healthy looking fruits. ing which tests have been performed to ¥ Seeds should be treated according to the following recommendations, either in the assess the health status of the material, country of origin or in the country of should accompany the germplasm destination: accession. (d) Movement of Breeding Material Ð Immerse the seeds in water and discard any ¥ Seeds used for the multiplication of breed- floating seeds. ing material should be pest-free. Ð Treat the seeds immersed in water in a mi- crowave oven at full power until the water ¥ Breeding material under multiplication temperature reaches 73ıC and pour off the should be grown under containment water immediately after the treatment. or isolation with appropriate chemical Ð If a microwave oven is not available, treat protection. ı ¥ Plants should be inspected soon after the seeds with dry heat for 2 weeks at 60 C. emergence and periodically thereafter. Ð Dry the seeds and treat them with thiram dust. Plants infected with seed-associated pests Ð Pack the seeds in a paper bag. should be destroyed. For field-grown Ð After arrival in the country of destination, plants, suitable precautions should be taken to prevent soil spread from infected the seeds should be inspected for the pres- plants and introduction of possible seed- ence of insect pests. If found to be infected, associated pests from local sources of they should be fumigated or destroyed (if fumigation is not possible). infection. Ð Seeds should be sown under containment or ¥ Seeds should be harvested only from in isolation and kept under observation until symptomless plants. ¥ Seed samples of appropriate size should the plants are well established and normal be tested for seed-associated pests. healthy leaves are produced. ¥ When non-destructive seed health tests If bud wood or scion material is to be ex- ported, prior information is required to the sta- are available, all seeds should be tested tions which enable the officers involved to raise accordingly. the test plants. Healthy stock seedlings should ¥ Movement of germplasm should comply with regulatory requirements of the im- also be grown in advance, so that they will be porting country. suitable for grafting when the importation ar- ¥ In addition to the phytosanitary certificate, rives. Any material received with roots, there are chances of carrying nematode or fungal vectors. a germplasm health statement, indicating Therefore, it should be first grown in sterile soil, which tests have been performed to assess if possible under glass. Cuttings then can be taken the health status of the material, should accompany the breeding material. from the shoots of plants whose tops have been 8.25 Part of the Planting Material to Be Tested and Post-Entry Quarantine 237 found to be healthy, and these can be rooted or grafted to healthy root stocks. If it is sap 8.25 Part of the Planting Material transmitted, it should be tested on susceptible to Be Tested and Post-Entry hosts. The original roots must be destroyed. Quarantine In the UK, the Carnation necrotic fleck virus Methods available for detecting viruses include (CNFV) had been detected in the imported carnation cuttings from the Netherlands (Stone testing of seedlings using a combination of tech- and Hollings 1976). During March and April niques after subjecting the seeds for post-entry 1977, a series of consignments of Portuguese quarantine (PEQ) growing as per the technical guidelines developed by FAO/Bioversity Inter- glasshouse carnation cuttings severely affected national (formerly International Plant Genetic by CNFV were intercepted and destroyed at Resources Institute, IPGRI) for safe movement of the UK. Similarly, daphne mosaic plants were found in a lot of 250 Daphne mezereum plants germplasm (http://www.bioversityinternational. to the USA from Holland and were promptly org/scientific information/themes/germplasm destroyed under the supervision of state nursery health/). However, this includes growing for a season which requires good environment- inspector. When the unrooted cuttings were controlled PEQ greenhouses involving funds for received, they should be treated with some constructing greenhouses or a field in isolation, insecticide and rooted in an appropriate medium, and transplanted to richer compost when rooted which is not easily available. and indexed by all possible methods. Detection of viruses in embryo indicates In the USA between 1957 and 1967, a that the viruses are seed transmitted. Since the presence of virus in the seed coat does not relate total number of 1,277 vegetatively propagated to virus transmission from seeds to seedlings, plant materials of citrus, ipomoea, prunus, whole-seed serological assays sometimes are solanum and vitis were received from various countries, and 62% of the plant material was not suitable for determining seed transmission infected with different viruses (Kahn et al. (Maury and Khetarpal 1989; Johansen et al. 1967). In the next decade, that is, from 1968 1994). While preparing samples (i.e. extracting embryos) for determining the transmission rate to 1978, the percentage of imported infected on large number of seeds, it is therefore necessary plant material was only 50%, out of the 1,913 that viral antigen from non-embryonic tissues vegetative propagules of Saccharam, Theobroma, Vitis, Solanum, Ipomoea, fruit trees, grasses (i.e. seed coat) is not simultaneously extracted, and other plants (Kahn 1989). Some of the because only the virus in the embryo leads to virus diseases detected in ornamental plant seed transmission and extraction of virus from non-embryonic tissue may lead to false-positive importations from different countries to the USA reactions. Moreover, manual processing of large during 1968Ð1978 have also been reported by number of seeds is not possible in a routine test Kahn (1989) and Kahn (1988). For breeding programmes, the wild species are exchanged (Maury and Khetarpal 1997). Therefore, efforts from one country to another, and viruses are should be made to develop techniques which carried even in these hosts. Kahn and Monroe could detect virus (e.g. PSbMV) only in embryo, although the whole seed is used for extraction (1970) recorded 39% virus incidence in the (Masmoudi et al. 1994a, b). Moreover, if the wild Solanum introductions collected as tubers. number of imports is more, it would be difficult Similarly, Kahn and Sowell (1970) and Kahn (1988, 1989) reported 11% of the 46 wild Arachis to subject all the samples for growing in PEQ species collected from Uruguay, Argentina and greenhouses. Brazil were virus infected. The above examples The PEQ of bulk imports in India is being carried out under the supervision of the inspec- clearly indicate the need for effective measures tion authorities who are the senior level plant to be taken while introducing any type of plant protection scientists of various universities and material. 238 8 Methods of Combating Seed-Transmitted Virus Diseases institutes. However, these inspection authorities, SPS measures are defined as any measure ap- in general, lack sufficient funds to carry out their plied within the territory of the member state: work effectively. This can be a major bottleneck (a) To protect animal or plant life or health from in the functioning of this aspect of the quarantine risks arising from the entry, establishment operations (Khetarpal 2004). or spread of pests, diseases and disease- carrying/disease-causing organisms (b) To protect human or animal life or health 8.26 Exclusion of Exotic Plant from risks arising from additives, contami- Viruses Through Quarantine nants, toxins or disease-causing organisms in food, beverages or foodstuffs 8.26.1 International Scenario (c) To protect human life or health from risks arising from diseases carried by animals, The recent trade-related developments in interna- plants or their products, or from the entry, tional activities and the thrust of the WTO agree- establishment/spread of pests ments imply that countries need to update their (d) To prevent or limit other damage from the quarantine or plant health services to facilitate entry, establishment or spread of pests pest-free import/export. The SPS agreement explicitly refers to The establishment of the WTO in 1995 three standard-setting international organisations has provided unlimited opportunities for commonly called as the ‘three sisters’ whose international trade of agricultural products. activities are considered to be particularly History has witnessed the devastating effects relevant to its objectives: International Plant resulting from diseases and pests introduced Protection Convention (IPPC) of Food and along with the international movement of Agriculture Organization (FAO) of the United planting material, agricultural produce and Nations, World Organization for Animal Health products. It is only recently, however, that legal (OIE) and Codex Alimentarius Commission standards have come up in the form of Sanitary of Joint FAO/WHO. The IPPC develops and Phytosanitary (SPS) Measures for regulating the International Standards for Phytosanitary the international trade. The WTO agreement on Measures (ISPMs) which provide guidelines on the application of SPS measures concerns the pest prevention, detection and eradication. To application of food safety and animal and plant date, 34 standards (https://www.ippc.int/index. health regulations. It recognises government’s php?id=13399&type=publication&subtype=& rights to take SPS measures but stipulates that category id=&tx publication pi1[pointer]=0& they must be based on science, should be applied showAll=1#publication)asgivenbelowhave to the extent necessary to protect human, animal been developed, and several others are at different or plant life or health and should not unjustifiably stages of development: discriminate between members where identical 1. ISPM 1: Principles of plant quarantine as or similar conditions prevail (http://www.wto. related to international trade org). 2. ISPM 2: Guidelines for pest risk analysis The SPS agreement aims to overcome health- 3. ISPM 3: Code of conduct for the import and related impediments of plants and animals to release of exotic biological control agents market access by encouraging the ‘establishment, 4. ISPM 4: Requirements for the establishment recognition and application of common SPS mea- of pest-free areas sures by different members’. The primary incen- 5. ISPM 5: Glossary of phytosanitary terms tive for the use of common international norms is 6. ISPM 6: Guidelines for surveillance that these provide the necessary health protection 7. ISPM 7: Export certification system based on scientific evidence and improve trade 8. ISPM 8: Determination of pest status in an flow at the same time. area 8.26 Exclusion of Exotic Plant Viruses Through Quarantine 239

9. ISPM 9: Guidelines for pest eradication pro- 33. ISPM 33: Pest-free potato (Solanum spp.) grammes micropropagative material and minitubers 10. ISPM 10: Requirements for the establish- for international trade ment of pest-free places of production and 34. ISPM 34: Design and operation of post-entry pest-free production site quarantine stations for plants 11. ISPM 11: Pest risk analysis for quarantine Prior to the establishment of WTO, govern- pests including analysis of environmental ments on a voluntary basis could adopt interna- risks and living modified organisms tional standards, guidelines, recommendations 12. ISPM 12: Guidelines for phytosanitary cer- and other advisory texts. Although these norms tificates shall remain voluntary, a new status has been 13. ISPM 13: Guidelines for the notification of conferred upon them by the SPS agreement. non-compliance and emergency action A WTO member adopting such norms is 14. ISPM 14: The use of integrated measure in a presumed to be in full compliance with the SPS systems approach for pest risk management agreement. 15. ISPM 15: Guidelines for regulating wood packaging material in international trade 16. ISPM 16: Regulated non-quarantine pests: 8.26.2 National Scenario concept and application 17. ISPM 17: Pest reporting Plant quarantine is a government endeavour 18. ISPM 18: Guidelines for the use of irradia- enforced through legislative measures to regulate tion as a phytosanitary measure the introduction of planting material, plant 19. ISPM 19: Guidelines on list of regulated products, soil, living organisms, etc., in order pests to prevent inadvertent introduction of pests and 20. ISPM 20: Guidelines for phytosanitary im- pathogens harmful to the agriculture of a region port regulatory system and, if introduced, prevent their establishment 21. ISPM 21: Pest risk analysis for regulated and further spread. non-quarantine pests As early as in 1914, the Government of India 22. ISPM 22: Requirements for the establish- passed a comprehensive Act, known as Destruc- ment of areas of low pest prevalence tive Insects and Pests (DIP) Act, to regulate or 23. ISPM 23: Guidelines for inspection prohibit the import of any article into India likely 24. ISPM 24: Guidelines for the determination to carry any pest that may be destructive to any and recognition of equivalence of phytosani- crop, or from one state to another. The DIP Act tary measures has since undergone several amendments. In Oc- 25. ISPM 25: Consignments in transit tober 1988, new policy on seed development was 26. ISPM 26: Establishment of pest-free areas announced, liberalising the import of seeds and for fruit flies (Tephritidae) other planting material. In view of this, plants, 27. ISPM 27: Diagnostic protocols for regulated fruits and seeds (regulation of import into India) pests order (PFS order) first promulgated in 1984 was 28. ISPM 28: Phytosanitary treatments for regu- revised in 1989. The PFS order was further re- lated pests vised in light of the WTO agreements, and the 29. ISPM 29: Recognition of pest-free areas and plant quarantine (regulation of import into India) areas of low pest prevalence Order 2003 [hereafter referred to as PQ Order] 30. ISPM 30: Establishment of areas of low pest came into force on 1 January 2004 to comply prevalence for fruit flies (Tephritidae) with the sanitary and phytosanitary agreement 31. ISPM 31: Methodologies for sampling of (Khetarpal et al. 2006b). Until 12 June 2010, 14 consignments amendments of the PQ Order were notified, and 32. ISPM 32: Categorisation of commodities ac- ten draft amendments were prepared, revising cording to their pest risk definitions, clarifying specific queries raised by 240 8 Methods of Combating Seed-Transmitted Virus Diseases quarantine authorities of various countries, with both public and private sectors. NBPGR has revised lists of crops under the Schedules VI, developed well-equipped laboratories and post- VII and quarantine weed species under Schedule entry quarantine greenhouse complex. Keeping VIII. The revised list under Schedules VI and VII in view the biosafety requirements, National now includes 729 and 286 crops/commodities, Containment Facility of level-4 (CL-4) has been respectively, and Schedule VIII now includes 31 established at NBPGR to ensure that no viable quarantine weed species. The PQ Order ensures biological material/pollen/pathogen enters or the incorporation of ‘Additional/Special Declara- leaves the facility during quarantine processing tions’ for import commodities free from quaran- of transgenics. At NBPGR, adopting a workable tine pests, on the basis of pest risk analysis (PRA) strategy, a number of viruses of great economic following international norms, particularly for and quarantine importance have been intercepted seed/planting material (http://www.agricoop.nic. on exotic material, many of which are yet not in/gazette.htm). reported from India, namely, Barley stripe mosaic The Directorate of Plant Protection, Quaran- virus in barley, Broad bean stain virus in broad tine and Storage (DPPQS) under the Ministry bean, Cherry leaf roll virus on French bean of Agriculture is responsible for enforcing quar- and soybean, Cowpea mottle virus in cowpea antine regulations and for quarantine inspection and Bambara groundnut, Raspberry ring spot and disinfestation of agricultural commodities. virus and Tomato ring spot virus on soybean, The quarantine processing of bulk consignments etc. (Khetarpal et al. 2001, 2006a, b;Chalam of grain/pulses, etc., for consumption and et al. 2005a, 2008, 2009a, b, c; Chalam and seed/planting material for sowing are undertaken Khetarpal 2008). In addition, many interceptions by the 35 plant quarantine stations located in have also been made of viruses not reported on different parts of the country, and many pests the host on which it is intercepted. In India, the were intercepted in imported consignments seeds of cowpea, mung bean, soybean and broad (http://www.plantquarantineindia.org/docfiles/ bean received from Nigeria, Taiwan, Columbia, appendix-8.htm). Import of bulk material for Brazil, Myanmar, Australia, Hungary, Spain and sowing/planting purposes is authorised only Bulgaria are screened at NBPGR, and about through five plant quarantine stations. There eight viruses which are seed transmitted are are 41 inspection authorities who inspect the intercepted (Khetarpal et al. 2001; Parakh et al. consignment being grown in isolation in different 2008; Chalam et al. 2008, 2009a, b, c). Until parts of the country. Besides, DPPQS has to date, 8,700 samples of transgenic crops developed 21 standards on various phytosanitary comprising Arabidopsis thaliana, Brassica spp., issues such as on PRA, pest-free areas for fruit chickpea, corn, cotton, potato, rice, soybean, flies and stone weevils, certification of facilities tobacco, tomato and wheat with different traits for treatment of wood packaging material and imported into India for research purposes were methyl bromide fumigation. Also, two standard processed for quarantine clearance, wherein they operating procedures have been notified on are tested for associated exotic pests, if any, and export inspection and phytosanitary certification also for ensuring the absence of terminator gene of plants/plant products and other regulated technology (embryogenesis deactivator gene) articles and post-entry quarantine inspection which are mandatory legislative requirements. (www.Plantquarantineindia.org/standards.htm). Barley stripe mosaic virus and Wheat streak The National Bureau of Plant Genetic mosaic virus which are not reported from India Resources (NBPGR), the nodal institution for were intercepted in wheat. Also, Maize dwarf exchange of plant genetic resources (PGR), mosaic virus not reported on wheat in India was has been empowered under the PQ Order to intercepted (Chalam et al. 2009a). handle quarantine processing of germplasm All the plants infected by the viruses were including transgenic planting material imported uprooted and incinerated. The harvest from virus- for research purposes into the country by free plants was released to the indenters. If not 8.28 Quarantine for Germplasm and Breeding Material 241 intercepted, some of the above quarantine viruses The process requires detailed information on could have been introduced into our agricul- pest scenario in both countries importing and tural fields and caused havoc to our productions. exporting the commodity. Efforts to develop Thus, apart from eliminating the introduction a database for endemic pests have been made of exotic viruses from our crop improvement by the plant quarantine station in Chennai, programmes, the harvest obtained from virus-free and NBPGR has compiled pests of quarantine plants ensured conservation of virus-free exotic significance for cereals (Dev et al. 2005)anda germplasm in the National Genebank. checklist for viruses in grain legumes (Kumar et al. 1994). Compilation of pests of quarantine significance for grain legumes by Chalam 8.27 Challenges in Diagnosis and Khetarpal (2008) and the Crop Protection of Pests in Quarantine Compendium of CAB International, UK, are some of the useful assets to scan for global pest The issues related to quarantine methodology and data (CAB International 2007). challenges in exclusion of plant viruses during As one faces challenges to crops from transboundary movement of seeds were anal- intentional or non-intentional introductions of ysed/reviewed recently (Khetarpal 2004;Cha- viruses, speed and accuracy of detection becomes lam and Khetarpal 2008). The challenge prior paramount. The size of consignment received is to import is preparedness for pest risk analysis very critical in quarantine from processing point (PRA). PRA is now mandatory for import of new of view. Bulk seed samples of seed lots need commodities into India. The import permit will to be tested by drawing workable samples as not be issued for the commodities not covered per norms. The prescribed sampling procedures under the Schedules V, VI and VII under the PQ need to be followed strictly, and there is a need Order. Hence, for import of new commodities to develop/adapt protocols for batch testing, in bulk for sowing/planting, the importer should instead of individual seed analysis (Maury et al. apply to the Plant Protection Adviser to the Gov- 1985; Maury and Khetarpal 1989). On the other ernment of India for conducting PRA. In case of hand, germplasm samples are usually received germplasm, import permit shall be issued by the as a few seeds/sample, and thus, it is often not Director, NBPGR, after conducting PRA based possible to do sampling because of few seeds on international standards (http://agricoop.nic.in/ and also because of the fact that a part of the Gazette/Psss2007.pdf). seed is also to be kept as voucher sample in the The ISPM-2, ISPM-11 and ISPM-21 deal with National Genebank in India apart from the pest- the guidelines for conducting PRA for quarantine free part that has to be released. Hence, extreme pests and regulated non-quarantine pests (http:// precaution is needed to ensure that the result www.ippc.int/ipp/en/standards.htm). There are obtained in the test was not denoted a false- two kinds of PRA, that is, pathway based and positive or a false-negative sample (Khetarpal pest based. It consists of three stages: initiating 2004; Chalam and Khetarpal 2008). the process for analysing risk, assessing pest risk and managing pest risk. Initiating the process involves identification of pests or pathways for 8.28 Quarantine for Germplasm which the PRA is needed. Pest risk assessment and Breeding Material determines whether each pest identified as such, or associated with a pathway, is a quarantine Scientists, hobbyists and growers worldwide have pest, characterised in terms of likelihood of entry, contributed to horticultural and silvicultural well- establishment, spread and economic importance. being. There have been a vast number of ex- Pest risk management involves developing, otic plant introductions in agriculture. This plant evaluating, comparing and selecting options for movement was somewhat haphazard and uncon- reducing the risk. trolled until the end of the nineteenth century. 242 8 Methods of Combating Seed-Transmitted Virus Diseases

In 1898, the US Federal Government began con- (b) Germplasm Exchange and Quarantine: The ducting plant explorations to introduce new and increase in exchange of seeds of germplasm unusual plants and began maintaining system- accessions that are likely to be contaminated atic and detailed records on plant introductions. by viruses has necessitated the need to certify Most germplasm enters the United States as a them to be free from viruses through stringent result of government-sponsored explorations or quarantine measures, keeping in mind the as a result of requests by nurserymen, botan- possibility of introducing unknown viruses or ical gardens, hobbyists and various scientists. virulent strains into a country. SMV, PSbMV Germplasm from original or secondary gene cen- and BCMV (PStV) are the classical examples tres is now in great demand. With the high pri- of economically significant, seed-transmitted ority awarded to resistance, introduction often viruses of soybean, pea and peanut, respec- comes from developing countries with very lim- tively, which are known to have spread to ited quarantine resources and limited information different countries through seeds of infected on endemic diseases. germplasm (Demski et al. 1984a, b; Good- In case of germplasm accessions and breeding man and Oard 1980; Hampton and Braver- lines, a zero tolerance is advocated. Plant genetic man 1979). resources are actually the key elements in any Seeds of germplasm material are usually crop improvement program. Germplasm which exchanged in small quantities. As quarantine represents a rich variability in plant genotype is processing of only a portion of the seed lot collected from diverse agro-climatic zones. The- does not indicate the status of the untested oretically in those geographical regions where portion, it is necessary to plant seed of an sources of resistance in wild genotypes are found, imported accession in isolation. Plant expressing there is a strong possibility of the emergence suspicious symptoms should be rogued, and of virulent isolates of the virus because of se- only seeds from plants shown to be virus-free lective inoculum pressure. In the case of seed- by appropriate tests should be harvested and transmitted viruses, there is also the possibility released to breeders for use in crop improvement of perpetuating such pathotypes through infected programmes (Jones 1987; Khetarpal et al. 1991). seeds of susceptible germplasm lines. By adopting such a procedure, a number Therefore, it is essential to certify that seeds of of important seed-transmitted viruses, namely, genetic resources and breeding lines at different CABMV, SBMV, SMV and many others, were stages of utilisation and exchange are free from intercepted in accessions of legumes imported viruses (Hampton et al. 1993). into Australia (Jones 1987). Similarly, SMV, (a) Germplasm Evaluation and Trials: Germplasm CPMoV, CABMV, BYMV, BBSV, BCMV materials are generally multiplied and and PSbMV were intercepted in seeds of evaluated in the field. During the course legume germplasm and breeding lines imported of multiplication, seed-transmitted virus(es) from different countries into India (Khetarpal serve as primary source(s) of inocula and, in et al. 1993, 1994; Chalam et al. 2004, 2008, the presence of insect vectors, are transmitted 2009a). The importance of interception can be to neighbouring susceptible lines. Hence, highlighted by the fact that the economically the virus is finally spread to different lines damaging virus of cowpea, CPMoV, is still of the collection in which it becomes seed not known to occur in India where cowpea transmitted. A very large percentage of is an important source of protein for a the germplasm collections from different predominantly vegetarian population. Moreover, regions are thus found to be infected. Since other intercepted viruses are known to possess germplasm is collected from diverse regions different virulent strains. and is often exchanged internationally, Production of virus-free seeds for conserva- considerable viral strain variation can be tion can be achieved simply by identifying virus- expected in these collections. free plants from which harvested seeds are to be 8.29 Role of IPGRI and NBPGR in Germplasm Maintenance and Exchange 243 used for conservation. The more rapid alternative Programme and (3) the International Network which consists in testing a representative sample for the Improvement of Banana and Plantain of the seed lots for presence of viruses before (INIBAP). The international status of IPGRI is being conserved would not be foolproof. conferred under an Establishment Agreement Seeds of germplasm stored in a gene bank which, by January 2003, had been signed must also be periodically multiplied in the field by the Governments of Algeria, Australia, for either replenishing the depleting stock or to Belgium, Benin, Bolivia, Brazil, Burkina circumvent the problems of seed viability. Such Faso, Cameroon, Chile, China, Congo, Costa multiplication needs to be protected from vec- Rica, Coteˆ d’Ivoire, Cyprus, Czech Republic, tors for ensuring freedom from seed-transmitted Denmark, Ecuador, Egypt, Greece, Guinea, viruses. Hungary, India, Indonesia, Iran, Israel, Italy, Technical guidelines jointly issued by FAO Jordan, Kenya, Malaysia, Mauritania, Morocco, and IPGRI (Frison et al. 1990) and the checklist Norway, Pakistan, Panama, Peru, Poland, of seed-transmitted viruses (Kumar et al. 1994) Portugal, Romania, Russia, Senegal, Slovakia, are useful references for ensuring safe movement Sudan, Switzerland, Syria, Tunisia, Turkey, of germplasm material. Uganda and Ukraine. For IPGRI’s research, financial support is provided by more than 150 donors, including governments, private 8.29 Role of IPGRI and NBPGR foundations and international organisations. in Germplasm Maintenance For details of donors and research activities, and Exchange please see IPGRI’s Annual Reports, which are available in printed form on request from ipgri- IPGRI: The International Plant Genetic [email protected] or from IPGRI’s website Resources Institute (IPGRI) is an independent (www.ipgri.cgiar.org). international scientific organisation that promotes the conservation and use of plant genetic diversity for the well-being of present and future 8.29.1 Objectives of NBPGR generations. It is one of 15 centres supported by the Consultative Group on International NBPGR (National Beauro of Plant Genetic Re- Agricultural Research (CGIAR), an association sources) is one of the ICR institutions established of public and private members who support by GOI at New Delhi (India). The objective of efforts to mobilise cutting-edge science to reduce this institution are: hunger and poverty, improve human nutrition ¥ To plan, organise, conduct and coordinate and health and protect the environment. More exploration and collection of indigenous and than 90% of respondents felt that agricultural exotic plant genetic resources biodiversity could help to meet these challenges ¥ To undertake introduction, exchange and quar- and that it could make a ‘major’ contribution to antine of plant genetic resources food security and environmental conservation. ¥ To characterise, evaluate, document and con- IPGRI has its headquarters in Macanese, near serve crop genetic resources and promote their Rome, Italy, with offices in more than 20 other use, in collaboration with other national organ- countries worldwide. It has five regional groups, isations with collective global coverage. IPGRI’s main ¥ To develop information network on plant ge- focus is on the developing countries, but the netic resources regional groups also establish contacts with ¥ To conduct research, undertake teaching and institutions in the more developed countries. training, develop guidelines and create public The institute operates through three programmes: awareness on plant genetic resources (1) the Plant Genetic Resources Programme, NBPGR has a separate Division of Plant (2) the CGIAR Genetic Resources Support Quarantine to meet the quarantine requirements 244 8 Methods of Combating Seed-Transmitted Virus Diseases in respect of the germplasm materials being The data presented in the table includes the exchanged through it. The division has trained seed-transmitted viruses intercepted in crops like scientific and technical staff representing the cowpea, mung bean, beans, soybean and broad disciplines of entomology, nematology and bean (Khetarpal et al. 2001; Chalam et al. 2004, plant pathology, well-equipped laboratories, 2008, Chalam et al. 2009a, b, c; Parakh et al. greenhouses and post-entry isolation growing 2008). field facilities to discharge its quarantine respon- National Bureau of Plant Genetic Resources sibilities efficiently. In case of certain crops, after (NBPGR) is the nodal agency for quarantine laboratory examination at NBPGR, the exotic processing of exotic germplasm and hence under- material is passed on to the specific crop-based taken the quarantine processing of all germplasm institutes for post-entry isolation growing, before including transgenic planting material under ex- it is released to the indenters. These institutes change for research purposes. Kumar et al. (1994) have established adequate post-entry isolation and Khetarpal et al. (2001) have summarised the growing facilities, and required expertise is viruses intercepted in leguminous crops during also available with them. These are Central 1991Ð2000. Similar type of information is avail- Potato Research Institute, Shimla; Central Tuber able on the list of intercepted plant viruses in Crops Research Institute, Trivandrum; Central almost all tropical countries. NBPGR also deals Tobacco Research Institute, Rajahmundry; with testing for absence of terminator technology Sugarcane Breeding Institute, Coimbatore; and which is mandatory as per national legislation. Central Plantation Crops Research Institute, This authorisation was vested upon NBPGR for Kasaragod. NBPGR has established a regional germplasm vide Article 6 of PQ Order 2003 and plant quarantine station at Hyderabad to fulfil for transgenic planting material vide Government the quarantine requirements of the International of India Notification No. GSR 1067(E) dated Crops Research Institute for the Semi-Arid 05.12.1989. Tropics (ICRISAT), Directorate of Rice Research and other research organisations in the region. It is also proposed to establish quarantine facilities 8.29.2 Methods of Testing for temperate fruit crops at NBPGR’s Regional at Quarantine Stations Station, Bhowali, and an offshore Quarantine Station at Port Blair in the Andaman Group The testing of the plant material received is done of Islands for vegetatively propagated tropical by indexing on indicator hosts either by mechan- crops. During the last 12 years or so, a large ical/insect transmission and also by grafting. The number of exotic insects and mites, plant parasitic symptoms are expressed within a few days or nematodes, plant pathogens and weeds have weeks when herbaceous test plants are used, but been intercepted from the imported germplasm may require months or years when woody plants materials, many of which are of major quarantine are used in which case the entry of the genetic significance and are not yet known to occur in stocks will be delayed. The various methods the country. While processing the germplasm for involved in detecting the viruses in seeds and seed quarantine clearance, all out efforts are made stocks are described in the Chap. 6. Indexing the to salvage the infested/infected materials so viruses infecting fruit crops is difficult than the that valuable exotic germplasm could be made other crops. available in a healthy state for exploitation in For example, for testing cocoa planting crop improvement programmes in the country. material, Amelonado plant (West African Cocoa In this direction, Chakrabarty et al. (2004)have Research Institute Selection C14) is the best listed the different pests intercepted in oil seed known indicator plant. The surface-sterilised germplasm imported during 1986Ð2003. The bud wood sent from the donor country is Table 8.3 shows the list of intercepted plant budded onto decapitated Amelonado plants under viruses in India. glasshouse conditions. The health of scion and 8.29 Role of IPGRI and NBPGR in Germplasm Maintenance and Exchange 245

Table 8.3 Virus and virus-like diseases intercepted in exotic germplasm at NBPGR and quarantine stations in India Virus Sl. no Crop intercepted Source of country Reference 1 Beans TBRV CIAT, Columbia Chalam et al. (2004) 2 Broad bean AMV ICARDA-Syria, Eritrea, Iraq, Chalam et al. (2009c) Spain 3 Broad bean BYMV, PSbMV Spain, Syria Chalam et al. (2009c) 4 Broad bean BYMV, BBSV, Bulgaria Khetarpal et al. (2001) PSbMV 5 Cowpea AMV, CMV, IITA, Nigeria Chalam et al. (2008) TBRV 6 Cowpea CABMV Eritrea Chalam et al. (2008) 7 Cowpea CABMV USA Khetarpal et al. (2001) 8 Mung bean CMV China Chalam et al. (2008) 9 Mung bean USA Chalam et al. (2008) 10 Mung bean CABMV AVRDC, Taiwan Chalam et al. (2008) 11 Peanut PStV Myanmar Prasada Rao et al. (1990) 12 Soybean AMV AVRDC-Taiwan, IITA-Nigeria, Parakh et al. (2008) Brazil, Myanmar, USA 13 Soybean BCMV AVRDC-Taiwan, IITA-Nigeria, Parakh et al. (2008) USA 14 Soybean CABMV AVRDC-Taiwan, IITA-Nigeria, Parakh et al. (2008) Myanmar, USA 15 Soybean CMV AVRDC-Taiwan, IITA-Nigeria, Parakh et al. (2008) Brazil, Myanmar, USA 16 Soybean SBMV AVRDC-Taiwan, IITA-Nigeria, Baleshwar Singh et al. Australia, Brazil, Hungary, (2003) and Parakh et al. Thailand, USA (1994, 2008) 17 Soybean TRSV IITA-Nigeria, Myanmar Parakh et al. (2008) the indicator shoots from the root stock are or symptomless, and this has been demonstrated checked weekly for virus symptoms. For safety, in certain fruit trees. For example, Kunkel et al. the scion should be top worked later with an (1951) and Manns et al. (1951) have reported for Amelonado bud. If free from symptoms after peach yellows and little peach Myrobalan plum three or four leaf flushes, bud wood is sent to (Prunus cerasifera) as a symptomless carrier, and the recipient country. In certain plant materials, several varieties of the Japanese plum (Prunus some of the latent viruses cannot be detected salicina) are symptomless carriers of little peach. during the routine inspection, as they do not The Grape vine corky bark virus is latent in exhibit well-marked symptoms. Some virusÐ Vitis vinifera cv. ‘Emerald Riesling’, whereas host combinations have long incubation periods in ‘Pinot Noir’ and ‘Almeria’, it causes severe which vary from few weeks to several years. For disease (Hewitt 1975). In many grape varieties, example, certain of the citrus viruses have 3Ð8- viruses-like fleck (marbrure), vein mosaic and year incubation period. Inspection and treatment vein necrosis are latent. They can be detected by at ports of entry may not be adequate safeguards indexing on Vitis ruperstris St. George, Riparia to prevent the entry of these latently infected gloire and Berlandieri x Rupestris (Hewitt et al. plant materials. Plant experts and plant breeders 1972; Legin and Vuittenez 1973). also introduce the wild/cultivated plants which Even certain diseases are latent in some orna- were not showing the symptoms, considering it mentals like chrysanthemum, dahlias, gladiolus, as resistant/tolerant source (Kahn 1976). Some carnations, etc. Sometimes, the latent virus may times more than one virus may become latent not damage the host plant, and it proves to be 246 8 Methods of Combating Seed-Transmitted Virus Diseases very economically important to other crops. For reduced the cost and permitted virus testing at example, virus YN strain which is symptomless locations where facilities are limited or even or causes only very mild symptoms in pota- absent. Immunochromatographic assay (ICA) is toes will spread rapidly by several aphid species another ELISA variant which added speed to and can completely destroy the tobacco crop in virus identification, where results can be obtained which it causes severe necrosis. Tomato spotted within 10Ð15 min, as compared to 2Ð3 h for wilt virus in arums, begonias, chrysanthemums TBIA. However, ICA is more expensive than and dahlias often cause no visible symptoms, TBIA. The development of nucleic acid-based but if it is introduced, it causes a great loss tools was another new dimension of virus detec- to the crops like tomato, tobacco, lettuce, peas, tion. The most common among these techniques beans, pineapple and other economic crops. Dod- are cDNA hybridisation and polymerase chain der (Cusanta californica) is reported by Bennett reaction (PCR). In addition, PCR can be used as (1944) as a symptomless carrier of a virus causing a confirmatory test for TBIA, where processed damage to sugar beets, cantaloupes, tomatoes and blots can be cut individually and tested by PCR. several other crops. Even swollen shoot of cocoa This proved to work well with both DNA and and sunblotch of avocado are symptomless in RNA plant viruses. Furthermore, unprocessed some varieties. In such cases, plant materials are plant tissue blots on nitrocellulose membrane sent to special quarantine, where they are grown represent a good sample for PCR amplification. for one or two seasons under special greenhouses. PCR products can also be used for cloning and Besides the indicator hosts, the other tests subsequent sequencing which is extremely use- comprising histopathological, serological and ful for identification of new viruses or virus electron microscopic techniques are also enabling strains. for the quick and authentic identification of the virus and virus-like diseases of the imported plant materials of certain fruit crops. Based on these 8.30 Important Cases laboratory tests, the quarantine staff will decide of Introduction the further measures to be taken against the materials under inspection (James et al. 2001). Lack of proper quarantine measures for the in- Only healthy plant material those guaranteed free troduced plant materials has resulted in the in- from infection will be released. The details of troduction of some very highly devastative plant the inspection techniques can be obtained from pathogens from one country to another and has USDA Manual 1971 and also from the handbook proved to be catastrophic. This has been resulted of phytosanitary inspectors in Africa (Caresche into increased cost of food material. Evidence et al. 1969). about the entry of disease into new areas is, Since testing through indicator hosts is time- however, often circumstantial, and it is rarely consuming in almost all quarantine stations and possible to say with certainly about how a disease research organisations are following molecular- has been introduced. A good example is tristeza based diagnostic tests for virus detection. Proper virus of citrus, originated probably in China, virus identification is always the key in devel- and introduced into South Africa by 1900 and oping appropriate practical solutions to manage probably at times into the USA, but its presence plant virus diseases. Recent advances in biotech- was marked due to the use of tolerant varieties nology and molecular biology have played a sig- and the absence of vector (Broadbent 1964). nificant role in the development of rapid, specific Prior to 1920s, the citrus industry of Argentina and sensitive diagnostic tests. The use of ELISA, and Brazil flourished despite the use of tristeza by employing either polyclonal or monoclonal susceptible root stock, but 20 years after tristeza- antibodies, was a significant step in adding sensi- infected nursery stock was imported from South tivity and precision to virus detection. The devel- Africa and Australia, 20 million trees had per- opment of the tissue-blot immunoassay (TBIA), ished due to the spread of the virus by an abun- as a variant of ELISA, greatly simplified testing, dant local vector Aphis citricida (Costa 1956; 8.30 Important Cases of Introduction 247

Ducharme et al. 1951). In North America and in 30 years. Experts convened by EPPO in 1963, the Mediterranean region, no disease state existed made surveys to estimate the situations of viruses for decades. A few varieties infected with tristeza of deciduous tree fruits and small fruits, which were introduced into North America at the end were already widespread or locally established in of the nineteenth century (Olson 1955b). In the Europe and those not known to be present within Mediterranean region, tristeza was introduced in the area (Granhall 1964). There is circumstantial the 1930s (Reichert and Bental 1960), but natural evidence, too, that important virus diseases of spread was encountered in Spain only in 1960s raspberry and the strawberry might have been and in Israel in 1970 (Bar-Joseph et al. 1974). introduced into the USA when tolerant varieties Another example is of Star Ruby isolate of citrus (perhaps infected with the disease) were brought Ring spot virus(CRSV-SR) discovered in a ‘Star in around 1920. Banana bunchy top,whichis Ruby’ grape fruit tree (Citrus paradisi)andwas threatening the banana industry in India, has brought into Florida by a private grower from been introduced from Ceylon through the in- commercial sources in Texas, without authorisa- fected suckers during 1940 (Vasudeva 1959). The tion from the State Department of Agriculture. Fiji disease of sugarcane is quite serious in Fiji is- CRSV-SV has not been found in ‘Star Ruby’ lands, and from there, this disease is introduced to trees imported officially for testing and release Madagascar and to other countries (Kahn 1989; (Garnsey et al. 1976). The other serious diseases Kahn and Mathur 1999). Potato spindle tuber vi- of citrus like psorosis, exocortis, xylopsorosis, roid has its home in North America and was intro- greening and stubborn are also proved to be duced into Russia and Poland from N. America quite dangerous as they were introduced through and barred on to Rhodesia. The infected plants infected bud wood and nursery stock (Klotz et al. of Abutilon striatum Dickson var. thompsonii 1982; Knorr 1965). with Abutilon infectious variegation virus show Plum pox virus (Sharka disease) was intro- attractive leaf variegation and hence were brought duced into England during 1965, through the in- into Europe from Brazil more than 100 years fected root stocks from Germany (Cropley 1968), ago (Hollings and Stone 1979). Circumstantial and adequate precautions are to be taken in other evidence suggests that Tomato aspermy virus parts of the world against introducing this disease in chrysanthemum may have been introduced in plum, peach, apricot and a variety of suscep- into the USA with chrysanthemum imports from tible ornamental Prunus species. The imported Japan (Brierley 1958) or from Europe in the early cherry material from European and Asian sources 1950s. Certain new cultivars of pelargonium have to Canada exhibited virus symptoms, when in- been imported into the United Kingdom from dexed on commercial cherry, apricot, prunus and the USA in recent years, and a number of them peach varieties (Welsh and James 1965). Stevens have been infected with Tomato ring spot virus (1931) states an available evidence which indi- (Hollings and Stone 1979). According to Leppik cates that cranberry false blossom first appeared (1964), Squash mosaic virus was introduced into in Wisconsin, probably as early as 1885, and the USA by the seed from Iran and dissemi- spread from there to Massachusetts, New Jersey nated in Iowa by cucumber beetles. After several and Oregon in shipments of infected vines. Early years of intensive work, this disease has been spread of the disease was largely by diseased eradicated. From the USA, the same virus has plants, but as the disease became established, been introduced into New Zealand through the its spread from plant to plant by the natural seed of Honey dew rock melon plants (Thomas vector, Euscelis striatulus, increased in the east- 1973). In Great Britain, Barley stripe mosaic ern states. The disease has apparently been of virus has been isolated only twice in barley seed little importance in the Pacific Northwest, pos- imported from France (Kassanis and Slykhuis sibly largely due to scarcity or absence of vec- 1959;Watson1959) and from Hordeum zeocriton tors. There has been little or no extension of seed imported from Denmark (Catherall 1972). infected area in the United States in the past 25Ð Many more examples can be cited where interna- 248 8 Methods of Combating Seed-Transmitted Virus Diseases tional movement of planting material has helped seeds (Konate et al. 2001). In citrus some of the in distributing pathogens from one part of the virus diseases which are emerging are at present world to the other. confined to one or two countries, and some of Besides the infected plant material, in some the diseases mentioned below are infectious and cases, the insects which are acting as vectors might spread, if introduced into new areas. They were also reported with the early free movement are bud-union crease in Argentina, abnormal bud- of plant material and also which in due course union problems in trees on rough lemon in South were established and became potent vectors. Africa, brittle twig yellows in Iran, decline of For example, Aphis citricola (Dspiricola) has rangpur lime in Brazil, Dweet mottle and yellow recently been introduced to the Mediterranean vein in California, gum pocket disease of trees on region and may also have come from Spanish Poncirus trifoliata in Argentina and South Africa, and Portuguese overseas territories, although gummy bark in Egypt, gummy pitting of P. tri- now widespread (Reichert 1959). Macrosiphum foliate in New South Wales, infectious mottling euphorbiae was apparently not present in Britain on navel orange in Japan, leaf curl in Brazil, leaf before 1917 and may have been introduced to variegation in Spain and Greece, marchitamiento Southern Europe from America some time before repentino or sudden wilt in Uruguay, the mis- that could have been a direct introduction from siones disease in Argentina, multiple sprouting in North America on new varieties of potatoes. It South Africa and Rhodesia, narrow leaf in Sar- is now thought that the disease and the vector, dinia, small fruit and stunting in Argentina and the leaf hopper Circulifer tenellus may have been young tree decline in Florida. There should be introduced together to California on live plant thorough indexing against these diseases, when- material used as a animal fodder on ships sailing ever the plant material is dispatched from these round Cape Horn during the ‘Gold Rush’ era countries. The need for strictly enforced quar- (Bennett and Tanrisever 1957). antines in every citrus-producing country is self The interception of known insect vectors of evident. plant viruses that depend solely on specific in- Plum pox (Sharka disease) is at the moment sects for spread may prevent the establishment of confined to Europe, and adequate precautions the viruses, provided no indigenous species are are taken in other parts of the world against capable of serving in a vector capacity. The best introducing this disease in plum, peach, apricot way of restricting the vector entry is by defoli- and a variety of susceptible ornamental Prunus ating the plants to be imported and treating with species (Matheys 1975; Capote et al. 2006). some systemic insecticide, before its dispatch. Cocao swollen shoot virus and Cocao necrosis virus are restricted to West Africa Ghana and Nigeria. The plants belonging to the families 8.31 Important Diseases Sterculiaceae, Bombacaceae,andTiliaceae are Restricted to Some Countries potential carriers of swollen shoot, and their ex- port should be strictly controlled. Abaca mosaic Many diseases have limited distribution and have virus of banana is restricted to the Philippines. been reported from only one or two countries. Yellow dwarf of potato is known only from North US plant quarantine regulations (1977) prohibit America. Cadang-cadang and coconut root wilt importation of plants and plant parts from speci- diseases are restricted to the Philippines and In- fied countries because of the occurrence in these dia, respectively. Another serious disease of co- countries of pests and pathogens of quarantine conut, lethal yellowing (Kaincope), is limited to significance to the United States. Rice yellow Jamaica, Cuba, Cayman Islands, Bahamas, Haiti, mottle virus is confined to some of the African West Africa and Florida. countries only and the virus was found in the The movement of cassava vegetative mate- fresh harvested seeds, but the seed transmission rial from Africa to American continent should of the virus was not noticed in the dried rice be banned to avoid the introduction of Cassava 8.32 Effective Methods of Plant Importations 249 mosaic in the western hemisphere. An additional certificate has international standard in testifying risk would be the introduction of whitefly vector the quality of the seed for trade transactions (B. tabaci) races better adopted to breed on cas- which is issued after examining the seed lots sava than those existing in America. according to the prescriptions laid down in the international rules of seed testing (ISTA 1966), whereas the Blue certificate is issued for samples 8.32 Effective Methods of Plant which have not been drawn officially. The Rome Importations certificate has the international standard, set up in accordance with FAO model phytosanitary 8.32.1 Phytosanitary Certificates certificate. These phytosanitary certificates are issued The risk of introducing the pathogen can be to the plant material which does not show checked by phytosanitary certificates for export- any external symptoms and also tested by ing or importing the plant materials, and permits mechanical/graft transmission to the indicator in the form of phytosanitary certificates issued by hosts. It is also confirmed by using the other the quarantines in relation to its health are highly techniques like histopathology, serology and useful in restricting the entry of the diseased ma- electron microscopy. In some of the virusÐhost terial into new area. It is understood that when the combinations, visual observations and indexing certificate is issued with the importing country do not help in complete restriction of diseased policies and regulations, the plant materials are material. Generally, quarantine inspectors depend to be healthy. These phytosanitary certificates, the on symptoms to detect the presence of viruses. imported material may be liable to be destroyed However, symptoms alone are totally unreliable at the port of entry of the importing country, as if they are not diagnostic and if the plant is per the international agreement. infected only recently and incubation period is ‘Certified’ refers that the plant material is incomplete. In some cases, the diseases will free from virus and virus-like diseases and also be latent. Since three decades the molecular from other pests and diseases. In some of the diagnostic tests are followed for screening the advanced countries, there are approved certified plant materials at the quarantine stations. nurseries. For example, Canada and United States accept certified Prunus, Cydonia and Malus nurs- ery stock from the certified nurseries in the UK, 8.32.2 Closed Quarantines France, Belgium, the Netherlands and West Ger- many. Similarly, the East African Plant Quaran- Because of latent or symptomless behaviour of tine Station at Kenya accepts the certification of the virus in a particular consignment, certain chrysanthemums by the Nuclear Stock Associa- serious diseases have escaped inspection in tion in the UK and the certification of grapevine the country of origin. All these imported plant by the Foundation Plant Materials Service of the materials should be grown in secured glasshouses University of California. These certificates are in a controlled atmosphere to avoid external con- issued by the central and state governments after tamination and tested for virus. This is known as careful examination of the material. Although the closed quarantines. From the small consignment variety of phytosanitary certificates varies from of plant material accepted for closed quarantine, country to country, depending on their compe- a few should be grown, and all plants should tence of their regulatory agency, their use has be inspected daily. If necessary, propagations reduced the movement of plant diseases between derived from the indexed mother plants or seeds countries. from the second crop should be released. If For evaluating the seed health, there are some suspicious symptoms are observed and the casual international certificates like orange certificate, virus is determined to be one not yet established blue certificate, Rome certificate, etc. The Orange within the country, the safest procedure is the 250 8 Methods of Combating Seed-Transmitted Virus Diseases destruction of the entire consignments in the 8.32.4 Open Quarantine station incinerator. If the crop is very important, by means of thermotherapy and tissue culture, This is the quarantine of plants without the plants can be raised, and the healthy plant using such physical confinement structures as material can be handed over to consignee, and glasshouses or screenhouses. This can be used the details are discussed in this chapter. While to reduce or eliminate the risk of spread of working in glasshouses, the precautions like the pests by adhering to a quarantine protocol. The treatment of the floor of the entrance cubicle with technique has been used successfully in East and a disinfectant, using gumboots and laboratory Central Africa to exchange germplasm resistant coats within the glasshouse unit, using sterilised to EACMV-UG. Open quarantine has facilitated soil for raising plants, washing hands and safe introduction of large quantities of cassava instruments with detergents, maintaining distance germplasm, which would not have been possible between plants and use of partition screens through other means. The method was cheaper to avoid contact transmission, etc. are to be than micropropagation, and plant mortality was followed. The principles of the closed quarantine also low. This method can be used in germplasm procedures have been described by Sheffield exchange programmes where the climate and (1968). This type of plant quarantine stations pest species are more or less similar. are at, Maguga, Kenya, the Post-entry Plant Quarantine Station Ibadan, Nigeria and US Plant Introduction Station, Glenn Dale, Maryland. 8.32.5 Examination of Exportable Crops During Active Growth

8.32.3 Quantity of Plant Materials Examination of the plants before harvest under field conditions will help in detecting and The risk of introducing the pathogen and pest assessing the incidence of some of the virus can be minimised by reduced quantity of a given diseases but, once again, depends upon the variety or clone. For vegetative propagative ma- virus, virus combinations and environment. terials, small quantities are to be considered like For example, with annual imports of flower 5Ð10 roots, tubers or corms; 50 buds, 20 unrooted bulbs from Holland, US quarantine inspectors, cuttings or 10 rooted cuttings. In case of seeds, following previous agreement between the parties quantity required for sowing a 10-m row will involved, visit and inspect the flower fields in do the purpose. Initially, these small quantities Holland. If they find the fields to be disease- of planting materials should be maintained, and free, they issue inspection certificate allowing it should be free from all destructive pests and the import of such bulbs into the USA without diseases. After confirmation as healthy material, further tests. it can be tested in the field. The vegetative prop- The USA annually imports large quantities of agative materials should be defoliated, which will vegetables from Mexico, and arrangements were avoid the entry of vectors, which are colonised on made for the USA and Mexican personnel to con- leaves. The plant material should be examined for duct field surveys in the vegetable production ar- the eggs and different stages of vector and should eas of Mexico. Such cooperative field inspections be dusted or sprayed with the insecticide before during growth season at points of origin provide packing for exporting. The majority of vectors greater protection than checking at ports of entry. can be controlled by methyl bromide fumigation. The growing season inspection is generally more It should be sent in unused cloth or paper bags sensitive than pre-export inspection of planting or envelopes. The importers or the exporters of material or produce. This method will be very germplasm should ascertain well in advance the effective wherever the vegetatively propagated requirements of the importing country for avoid- materials are exported. Stover (1997) described ing unnecessary delay at quarantine stations. an effective method of exporting bananas for 8.32 Effective Methods of Plant Importations 251 commercial or research purposes. The rhizomes this type of safeguard is that it advocates the should be taken from a disease-free area, at least passage through country C only of genera that for 1 year. They should be pealed free of all root pose no threat to country C because (1) the stubs, until only white tissue remains. Later, they crop is not grown in country C, (2) the harm- should be submerged in hot water at 54ıCfor ful organisms of that crop have narrow host 10 min. The cartons having the suckers should be ranges so they will not attack other crops of shipped air freight with the required phytosani- country C, (3) the harmful organisms that may tary documents at temperature above 14ıC. After gain entry to country C would not become es- receiving the rhizomes at quarantine stations, the tablished because susceptible hosts are absent or plants should be raised for nearly 1 year, and later, the climate is unfavourable. The third-country they can be released. quarantine locations include the plant quarantine facility at Glenn Dale, Md; the US Sub-tropical Horticultural Research Unit, Miami; Kew Gar- 8.32.6 The Intermediate Quarantine dens, UK; and Royal Imperial Institute Wagenin- gen and IRAT at Nogent-Sur-Marne, France. For The genes for resistance to virus and virus-like example, the export of cacao propagating ma- diseases are likely to be found in regions where terial primarily from West Africa to the other the plant in question originated (primary gene cacao-growing countries is first quarantined at an centres) or has subsequently been grown (sec- intermediate quarantine station of third-country ondary and tertiary gene centres). It is in such quarantine. Facilities may exist within the trop- places that long association between pathogens ics reasonably far from growing cacao as at and plants has occurred with consequent elim- Mayaguez in Puerto Rico or Salvador in Bahia, ination of susceptible plant genotypes through but safety usually required intermediate quaran- natural selection. Hence, resistance material is tine in a temperate country, for instance, at Kew, generally searched for in what are thought to be Glenn Dale; Miami or Wageningen. Similarly, as the gene centres of cultivated plants. While sugarcane sets entering East Africa are first put collecting the sources of resistance from the gene into quarantine at the East African Agriculture centres, one should not inadvertently introduce and Forestry Research Organisation (EAAFRO) genes for virulence by collecting the new forms at Maguga to see if they carry viruses. of the horizontal and vertical pathotypes. As dis- cussed earlier, many virus diseases occur latent, without expressing any visual external symp- 8.32.7 Aseptic Plantlet Culture toms. The risk of introducing the new pathogens, either from the gene centres or from exporting Another safeguard in the international exchange country, can be reduced by transferring a ge- of germplasm is to import only plantlets estab- netic stock to a third country instead of send- lished as aseptic cultures. The required plant ma- ing directly, where that crop is not grown and terials which are proved virus-free after indexing the pathogen would not establish there. Third- can be developed by tissue culture technique and country or intermediate quarantine is an inter- can be sent to the other country without any national cooperative effort to lower the risk to hazards. As the size of the consignment will be country B associated with transferring genetic small and in aseptic conditions, there will not be stocks from country A by passing these stocks any chance of reinfection with other pathogens through isolation or quarantine in country C. like fungi, bacteria and also with other pests like The plants are maintained in country C to test insects, mites and nematodes. In this technique, for obscure pathogens, or they are detained to an imported variety or clone may be represented allow incipient infections to surface or to per- by meristem tips or exercised buds or embryos mit treatment at a weak point in the life cycle instead of several cuttings, scions, tubers, seeds, of hazardous organisms. The salient feature of etc. The tissue culture technique in combination 252 8 Methods of Combating Seed-Transmitted Virus Diseases with thermotherapy or chemotherapy or both has crops like ginger, chrysanthemum, sweet potato been used in the production of virus or virus- and banana and used for exchanging virus-free like disease-free planting material of number of genetic stocks between countries. This technique horticultural crops. During 2009,Wangetal.have is also useful to the germplasm explorers, where developed cryotherapy technique and produced they can collect the required perishable plant virus-free plants of citrus, grapes, Prunus spp. materials right in the field. They have to carry and certain tuber crops like potato and sweet tubes having tissue culture media, and after potato. thorough disinfection of the plant material, they Prior to the establishment of tissue cultures, can be transferred to the medium, with a higher the mother plants were indexed and tested risk contamination. serologically for viruses. The use of this technique was first reported by Kahn (1976) for Asparagus officinalis L. In 1972, asparagus 8.32.8 Embryo Culture clones from France were indexed for viruses at Glenn Dale. From the plants that indexed As some of the viruses are internally seed trans- negatively for viruses, aseptic plantlets were mitted, countries which have a zero tolerance developed on agar medium in screw cap against the seed-transmitted pathogens have pro- bottles and were shipped to Kenya. Roca hibited the entry of the seed from other coun- et al. (1978) shipped 340 aseptic cultures of tries where these specified organisms are known Solanum germplasm from the International to exist. For commercial purposes, the imports Potato Center (CIP) in Lima, Peru, to the are strictly prohibited; however, a very small countries like Australia, Bolivia, Brazil, Canada, quantities are permitted for scientific purposes. Colombia, Costa Rica, India, Indonesia, Kenya, Kahn (1979) developed during 1970Ð1972 an Mexico, the Philippines, Turkey, the United embryo culture technique by using tissue cul- Kingdom and the United States. The clones were ture methodology. In this technique, instead of successfully established in 12 of the 14 countries. complete seed, only embryo axes were exercised At CIP, multiple shoots in tissue culture were and used for culturing, and it can be mailed produced in shake cultures. Plantlets of potato without any risk. This technique is also useful regenerated from nodal cuttings of multi- for germplasm explorers. Guzman and Manuel meristem shoots were then shipped in culture (1975) used this technique for coconut plant ma- tubes from CIP to corresponding countries terial. Embryos collected in large numbers in (Roca et al. 1979). While developing the aseptic diseased area can be maintained in laboratory cultures, heat treatment is also employed for culture for 16 weeks and grown in screened plant materials in which viruses were not glasshouses for some months before they need easily eliminated. Waterworth and Kahn (1978) to be planted. If no virus, phytoplasma, viroid developed sugarcane plantlets of three varieties and other pathogens are found, then adequate that indexed negatively for Sugarcane mosaic samples were screened by standard methods, it virus (SCMV) by hot water treatment and aseptic could be considered safe to plant embryo cultured bud culture. The hot water treatment was three seedlings of coconut. Braverman (1975) modified sequential exposures of cuttings at 24-h intervals this technique so as to include one microbio- for 20 min at 52, 57 and 57ıC. The SCMV- logical test and virus indexing. As a safeguard infected canes were sent from the East African against the breaking of agar in the glass bottles Plant Quarantine Station, Kenya, to Glenn Dale or tubes during transit, it is desirable to add where they were subjected to heat therapy warm sterile agar before shipping, until the con- followed by bud culture. The aseptic plantlets tainer is almost full. The plantlets are submerged were returned to Kenya in screw cap bottles for in agar, but they ship well and usually arrive transplanting and indexing (Kahn 1976, 1977). in excellent condition with the agar remaining Aseptic cultures were also developed against undisturbed. 8.33 General Principles for the Overall Effectiveness of Quarantines 253

8.32.9 Use of Shoot Tip Grafting 4. Consignments of vegetatively propagated or Micrografting material should be small; that is, each variety or species should be presented by a few Micrografting is another technique which tubers, scions or cuttings. provides a means whereby plants can be 5. A stock plant should not be reused for prop- transported from one country to another. The agation if a foreign bud or scion failed to culturing of lateral buds in vitro to induce survive on the stock. Bud or graft union multiple shoots may have potentially important failures may be caused by pathogens such as applications. This method is proposed by Navarro viruses transmitted from the introduction to et al. (1975). Bud wood could be fumigated and the stock. shipped in test tubes or in a sealed container, 6. It should never be assumed that all vegetative and the lateral buds excised in the receiving propagations of a given species or variety country and cultured in vitro for production of were derived from the same mother plant. multiple flushes, whose shoot tips then be used 7. Each scion, cutting or tuber of a clonal intro- for grafting. The method involves holding the duction should be considered as a sub-clone. previously trimmed shoot with tweezers under 8. When pest or pathogen detection tests in- the microscope, and the very tip end of the dicate that a particular sub-clone is eligible growing bud (0.88 mm) is removed with a razor for release from quarantine, propagations for knife blade and quickly transferred to the cut release should come only from the sub-clone edge of the inverted ‘T’ on the seedlings. The that was tested and not from other sub-clones resulting graft plants after 3Ð5 weeks could then that were not tested, even though these sub- be indexed for a broad spectrum of pathogens clones constitute part of the original acces- in a special quarantine facility and destroyed sion. if pathogens were found. This should pose no 9. If introductions are received as roots, such as serious problems with quarantine regulations to sweet potato, cuttings derived from the roots the country of origin or the country processing the should be released rather than the original bud wood, since all the bud wood, bud or explants root itself, which should be destroyed. would be under continuous cover. This method 10. Visual observation is not satisfactory for di- also helps in exchanging of superior cultivars. agnosing virus diseases because neither the presence nor absence of virus-like symptoms is necessarily indicative of the presence or 8.33 General Principles for absence of virus. the Overall Effectiveness Besides the above ten principles, the follow- of Quarantines ing four types of quarantine actions will mostly restrict the entry of the diseases: Here details of quarantine regulations are not in- 1. The consignment should be followed by em- cluded as they vary from one country to another, bargo and airport permit. but a general set of principles contains the basic 2. Inspection (field inspection, laboratory tests, guidelines: etc.) in the exporting country before shipment 1. Seed rather than vegetative material should of the consignment and to be on the safe be introduced unless clonal propagation is side the material must be given chemo- and necessary. thermotherapy. 2. For clonal propagations, non-rooted prop- 3. Post-entry growth inspection of the importing agative material such as scions or cuttings country in closed quarantine. should take precedence over rooted plants. 4. Certification Ð phytosanitary certificate 3. Woody plant introductions should not be attested for freedom from disease and more than 2 years old. pests. 254 8 Methods of Combating Seed-Transmitted Virus Diseases

with monoclonal antibodies provided by, and 8.34 Quarantine Facilities financial support from, the International Devel- opment Research Centre (IDRC), Canada (Thot- The type of quarantine facilities depends on the tappilly et al. 1993). Many existing networks are climate at the introduction station, the crops and crop based and are often divisive from the point its temperature requirements, pest and pathogen of view of the virology community because the risks and duration of the quarantine period as it networks rarely link together. The networks such affects plant size. Features that can be incorpo- as the Cassava Biotechnology Network (CBN), rated into a greenhouse to improve phytosanita- ProMusa, East Africa Root Crops Research Net- tion and thus facilitate quarantine include a series work (EARRNET) and the Southern Africa Root of small glasshouses, air conditioning, filtered air, Crops Research Network (SARRNET) take a big humidity control, concrete floor with drains, par- step in the right direction. However, until capacity tition screens, soil sterilisation, fumigation cham- and confidence are increased, many of the virol- bers, etc. Other features are pathogen-free water ogists from developing countries are unable to supply, heat therapy unit, tissue culture rooms, participate fully and gain maximum benefit from hot water treatment facilities, black-light insect the interactions. traps, shoe disinfectants and fungicide and insec- The organisation of working group meetings ticide spray programmes. Big and well-equipped at regular intervals, either at the regional or sub- quarantine stations are started whenever a coun- regional level, is an ideal way to promote the try is both agriculturally and scientifically well networking concept. These meetings would in- advanced, whereas less advanced countries will volve scientists from all over the world who have small stations of nonexistent. A sufficient are working on the plant virology problems of number of adequately trained and experienced their country. It would enable priority constraints inspectors are required for the effective exam- to be identified and research needs of national ination of incoming plant material at airports, programmes to be determined. A framework for railway stations, seaports and frontier parts. collaborative research programmes in partner- ship with IARCs and research institutes in ad- vanced countries must be developed and sustain- 8.35 Need for Networking for able funding obtained. Sustainability of funding the Developing Countries must be considered a key issue that must be addressed. Working groups on crop-specific virus Networking within and between the subregions, disease problems have already been developed, and with institutes in developed countries and in- for example, the International Working Group ternational agricultural research centres (IARCs), on Groundnut Viruses in Africa and the Virol- is critical to strengthen plant virus research in the ogy Working Group of ProMusa.However,a world. There are numerous advantages of such formal network must be developed for the up- international networking (Plucknett and Smith coming countries which these collaborative ini- 1984). Networking is a rapidly growing mecha- tiatives and other virus-specific subgroups can nism, as funding becomes increasingly scarce, to interact. This will ensure better coordination of optimise resource utilisation and to facilitate the efforts and synergy between different partners efficient transfer of technologies to developing and stakeholders in addressing a broad range of countries. It is in developing countries where plant virology needs of the national programmes. the impact of these technologies can be felt al- However, it is vital that any initiative that is taken most immediately if the resources are available regarding networks and collaboration is assured to utilise them. A good example of such an effort of funding for long enough for the activities to was the collaborative project on identification be self-sustaining. Any plant virology network of cowpea viruses coordinated by the Interna- must be endowed with sufficient funds to have tional Institute of Tropical Agriculture (IITA) a dynamic coordinator and secretariat with au- 8.36 Perspectives 255 thority (perhaps under the auspices of the steering Phytosanitary Measures of WTO, wherein the committee of the network) to initiate appropriate consignments can be justifiably rejected, if the research and donor contacts. Regional and sub- exporting country does not follow the importing regional organisations should take an active role country’s requirements. in encouraging and supporting such initiatives. Strict regulatory measures together with More information can be also obtained from the growing new introductions under containment or chapters in edited books and also review arti- isolation and collection of seeds from only virus- cles on plant quarantines (Chiarappa 1981;Kahn free plants must be followed to eliminate the 1989; Ebbels 2003; Khetarpal et al. 2004). risk of introducing seed-transmitted viruses/their strains if any. Conservation of virus-free seeds of a crop in gene banks will minimise the spread 8.36 Perspectives of ‘germplasm-borne’ viruses as these materials are multiplied and exchanged worldwide. The Detection and diagnosis of viruses are crucial for countries should take note of a series of trade and for exchange of germplasm. But the technical guidelines for the safe movement level of confidence, knowledge and accuracy on of germplasm prepared by FAO/Bioversity the part of the workers needs to be improved International (formerly IPGRI), Rome (Frison for precise detection and diagnosis. Training is and Diekmann 1998). required for quarantine officials, germplasm cura- Database on all seed-transmitted viruses, in- tors and scientists who are involved in assessing cluding information on host range, geographical the conformity to the international standards es- distribution and strains, should be made available pecially in developing and least developed coun- for its use as a ready reckoner by the quarantine tries. There is also a need to seek technical personnel. There is a need to have antisera for assistance of international agencies in the area all the seed-transmitted viruses in the quarantine of human resource development. Compared to laboratories to facilitate the interception of exotic the other pest detection techniques, virus index- viruses and their strains. Information access and ing requires technically more advanced equip- exchange on diagnostics of plant viruses and ment, reagents, etc. Accreditation of few well- quarantine are crucial for effective working. Es- established laboratories would offer a reasonable tablishment of a National Diagnostic Network for solution for reduction of costs. India has limited Plant Viruses including antisera bank, database laboratories, which are well equipped and have of primers, seeds of indicator hosts, national trained experts to deal with diagnosis of plant DNA bank of plant viruses, lateral flow strips/dip viruses. Such laboratories need to be accredited sticks which can detect multiple viruses, mi- on priority to enhance their credibility at interna- croarray technology, DNA barcoding and, ulti- tional level. mately, a national biosecurity chip for diagnosis At NBPGR, the legume germplasm was of all current threats to crop plants would be found to be infected by 29 viruses when the backbone for strengthening the programme tested by ELISA and, in some cases, by on plant quarantine. The National Diagnostic electron microscopy and RT-PCR. Jones (1987) Network for Plant Viruses, if established, can emphasises the need for disease-free seed stocks be a storehouse of information on biology of in germplasm collections to enable countries plant viruses, diagnostic procedures and policies, with less sophisticated quarantine systems to international standards and related issues. Also import disease-free seeds, intended for improving Regional Working Groups of Experts for De- crop production and not for introducing new tection and Identification of Plant Viruses thus pathological problems. However, this is not need to be formed to explore future cooperation a problem in case of bulk consignments in terms of sharing of expertise and facilities, with the trade being under the gamut of for example, in South Asia where the borders Agreement on Application of Sanitary and are contiguous. This would help in avoiding the 256 8 Methods of Combating Seed-Transmitted Virus Diseases introduction of plant viruses not known in the the danger of bringing in infected or infested region and also the movement of plant viruses plant materials from abroad through advertising within the region. The importance of quarantine leaflets, posters and broadcasting on radio. has increased manifold in the WTO regime and Unless quarantine regulations are scientifically adopting not only the appropriate technique but sound and administratively feasible, they cannot also the right strategy for plant virus detection be successful and may cause serious political and diagnosis would go a long way in ensuring and economic problems. On the other hand, there virus-free trade and exchange of germplasm, and must be adequate legal authority and appropriate is considered the best strategy for preventing enforcement in prosecuting wilful violators. The transboundary movement of plant viruses. enforcement of quarantines requires considerable expenditure of money, much interference in trade travel and other normal activities 8.37 Conclusion of man. The international cooperation and strengthening regional organisations are of The foregoing discussion clearly stresses the paramount importance in attaining the objectives need for effective quarantines. The expenses of plant quarantine. incurred in establishing and maintaining the quarantines are but a fraction of the economic losses that would be suffered, if plant diseases 8.38 Biotechnology gain freely entry into the country. The success and Virus-Derived of the plant quarantine measures mainly depends Resistance on the proper composition of the state service of plant quarantine, the scientific background Engineered resistance to viruses includes virus- and the qualifications of the inspectors and the derived resistance, reviewed by Beachy (1997), specialists and the availability of necessary and several other approaches which are being equipments at the quarantine stations and explored (Robaglia and Tepfer 1996; Varma et al. laboratories. The post-entry quarantine service 2002). for all imported seed must be established, so that In some of the crops, partial success has been it can produce and distribute pathogen-free seed achieved in dealing the aspects of virus-derived derived from the imported infected material. It resistance which have already been applied or will also be useful if world distribution of seed- are going to be applied for controlling seed- transmitted pathogens is mapped. The safest transmitted viruses. The concept postulates that source of the healthy materials should be from the expression of a viral gene in a host can inter- a country with efficient quarantine services fere with hostÐvirus interactions, thus rendering which has talented staff who can diagnose the the host resistant. Virus-derived resistance can in- indigenous pests and diseases. There is a vital clude a variety of different viral gene sequences, need for public awareness of the importance of most of which interfere at different points in the quarantine regulations. It is well recognised by replication cycle. Two dates are important in the quarantine officials that success of a quarantine history of this approach: programme is dependent on cooperation between ¥ The first capsid protein-mediated resistance government agencies and the public. Generally, was described in 1986: tobacco TMV. the public will co-understood. Quarantine action ¥ The first commercial sale of virus-resistant is better received and followed if it is based on transgenic crop in the USA in 1995: squash cooperation between concerned groups rather WMV2 C ZYMV sold by Asgrow Seed than on a unilateral compulsion. In this age Company. of rapid and greatly expanded movement of Strategies for virus-derived resistance are people and cargoes by air, it is extremely divided into those that require the production important to alert the public by all means to of proteins and those that require only the 8.39 Molecular Approaches 257 accumulation of viral nucleic acid sequences. In general, the former confers resistance to a 8.39 Molecular Approaches broader range of virus strains and viruses, and Molecular approaches have high level of sen- the latter provides very high levels of resistance sitivity and discrimination over classical tech- to a specific virus strain. The future challenge for niques. The nucleic acid-based methods do not scientists is to develop strategies that broaden the effect and increase the degree of resistance. depend on the metabolic state of the virus or the state of gene expression as both coding and non- coding regions of the genome (Torrance 1992; 8.38.1 Capsid Protein-Mediated Matthews 1993;Hull2004; Webster et al. 2004). Resistance The applications of molecular tools to detect the seed-transmitted virus diseases are relatively Capsid protein-mediated resistance can provide recent, and this method is useful for detection and either broad or narrow protection. The CP of strain determination of plant viruses and virus- TMV provided effective levels of resistance to like diseases. High sensitivity and specificity are closely related strains of TMV and decreasing the major goals of virus detection, and it is used levels to tobamoviruses that share less CP se- in quarantine programmes. Rapid advances in the quence similarity. Apparently CP interferes with techniques of molecular biology have resulted in the disassembly of TMV, thereby preventing in- the cloning and sequence analysis of the genomic fection. Certain mutants of TMV CP can confer components of a number of plant viruses. Basic much greater levels of resistance than wild-type molecular approaches are used for detection and CP. Other viral proteins can similarly confer re- diagnosis of plant virus type and molecular sizes sistance, for example, replicase interrupted by an of the virion-associated nucleic acids, cleavage insertion sequence element produced high levels pattern of viral DNA or cDNA, hybridisation of resistance in tobacco plants to a wide range of between nucleic acids and polymerase chain re- tobamoviruses (Donson et al. 1993). action (Hull 2002). For potyviruses, wild-type CP was not Recent progress in biotechnology and the efficient, while CP deleted of its N-terminal part availability of novel techniques in molecular conferred capsid protein-mediated resistance. biology and genetic engineering now makes it However, heterologous protection to another possible to consider new approaches to plant potyvirus has also been observed (Dinant et al. virus disease management. It will involve genetic 1993). engineering techniques to introduce specific Nucleic Acid-Mediated Resistance: The most genes into plants or genetic codes, not only from widely studied example of nucleic acid-mediated plants but also from the viral genome itself. resistance is referred to as RNA suppression, a phenomenon that describes the targeted post- transcriptional destruction of RNA sequences 8.39.1 Molecular Interactions (Baulcombe 1996). of Seed-Transmitted Viruses A high correlation exists between RNA- mediated resistance and multiple copies of gene Molecular mechanism of few viruses has inserts. The cell detects abnormally elevated illustrated full length, and infectious cDNA levels of RNA sequences and activates a clones are available. Viral genes or products destruction mechanism that involves nuclease influence transmission mainly by regulating the destruction of the gene transcript and of viral regulation capacity and their movement in the RNA. Levels of RNA-mediated resistance may reproductive tissues of infected hosts, though be high; it is effective only against genomes that participation of host factors cannot be ruled out. contain sequences that are similar or identical to Several host determinants participate in seed the transgene. transmission. Pseudorecombinants of few bi- or 258 8 Methods of Combating Seed-Transmitted Virus Diseases multipartite RNA viruses have been studied. (2009). Some of the approaches that are in RNA 1 component of both CMV with a tripartite use include antisense RNA, satellite RNA, coat genome and some of the nepoviruses have been protein genes and specific resistance genes to shown the determinant of seed transmissibility. antiviral activities in plants, etc. Though pseudo-recombinants have lower seed The genomic sequences of all the important transmissibility, other minor determinants are seed-transmitted viruses mentioned here are now also suspected in reducing the rate of virus established. The use of viral 2 sequences as trans- transmission. Helper component protease (Hc- genes depends now on advance in the technology Pro), a non-structural gene in potyviruses of transformation of each species. is involved in aphid-mediated transmission, The academic studies on plant transforma- replication and long-distance translocation of tion have concentrated first on model systems virus. Hc-Pro regulates replication and movement such as members of Solanaceae, tobacco and of PSbMV in pea, thus influencing invasion of the tomato. Other plants have been more recalci- embryo. Pea early browning virus, a tobravirus trant to in vitro manipulation and particularly (PEBV), has 12 k gene resembles to the Hc-Pro the legume species: Only soybean and peanut of a potyvirus and RNA ” gene of a hordeivirus. which are rich in proteins and in oil have received 12 k gene influences the virus transmission to the much attention in terms of improvement through gametes of the pea cultivars. BSMV contains biotechnology. RNA’,RNA“ and RNA”, and major seed determinants are located in 50 UTR and a 369-nt 8.39.2.1 Tomato/TMV and ToMV repeat present in the ”aand”b genes. ”b genes of Engineered protection to TMV and ToMV has hordeiviruses share cystein-rich domains that reg- already been achieved, improving elite tomato ulate transmission, replication and movement of varieties without altering their desirable char- the virus in the embryo (Khan and Dijkstra 2007). acteristics. In the first field trial ever done of plants engineered for virus resistance, Nelson et al. (1988) evaluated resistance conferred by 8.39.2 Transgenic Approach transformation with the CP gene of TMV. Transgenic plants displayed nearly complete Transgenic technique is one of the approaches resistance to mechanical infections by TMV, focused on the resistance of transgenic plant and only 5% had symptoms at the end of the against viral infection and suggested this trial, as compared with 99% of the control. Fruit as ‘pathogen-derived resistance (PDR)’. The yield was identical for inoculated transgenic and advantages and setbacks of genetic engineering uninoculated control plants. for crop improvement are clearly exemplified by Sanders et al. (1992) extended the field char- virus resistances in transgenic plants. Introducing acterisation of these TMV-resistant tomato plants more than one or two transgenes is usually and found they had a limited ability to protect problematic because of ‘transgenic silencing’. against ToMV since 56Ð89% of them were in- Hence, at present, state-of-the-art genetic fected with ToMV causing 11Ð25% yield loss. engineering is short of causing a remedy to all Tomato plants expressing the CP of ToMV were the pathogens of a given crop. It is not surprising developed to introduce ToMV resistance. that genetic transformation for virus resistance Transgenic line 4174 showed substantial resis- became one of the first approaches of its kind tance to both tobamoviruses. to protect plants against pathogens. Protection The evaluation in Japan of the impact of crops against viral pathogens by genetic of release of transgenic tomatoes on the engineering was reviewed by Lomonossoff environment concluded that released transgenic (1995), Beachy (1995), Galun and Breiman plants could be cultivated in the fields and (1997), Dudhare and Deshmukh (2007), Varma that resistance was maintained throughout and Praveen Shelly (2007) and Reddy et al. generations (Asakawa et al. 1993). 8.39 Molecular Approaches 259

Tomato was also successfully transformed 8.39.2.4 Pepper/PMMoV, TMV with the well-known resistance gene N from and ToMV Nicotiana glutinosa which confers in Nicotiana Transformation of pepper appears as more dif- durable resistance by hypersensitivity to both ficult than transformation of tomato. In view of TMV and ToMV (Whitham et al. 1996). This its transformation for resistance to PMMoV, a is a single example of an isolated resistance preliminary study of the engineered resistance gene introduced by bioengineering and the was studied in Nicotiana benthamiana (Tenllado demonstration that it is effective in a new et al. 1995). biological context. The gene N would also be a candidate to be introduced, although the only known strain Ob 8.39.2.2 Lettuce/LMV virulent on N genotype was selected from pepper. Lettuce transformation has been achieved by Agrobacterium-mediated transfer or by proto- 8.39.2.5 Soybean/SMC plast electroporation in France, Japan and the Soybean genetic engineering is the most ad- USA. Transgenic lettuce expressing the CP gene vanced among all grain legumes, and this reflects of LMV has been obtained (Dinant et al. 1997). the economic importance of the crop in the Constitutive expression of the CP gene was industrialised world. Since SMV is economically demonstrated by ELISA. In 60 plants of the R2 important seed-transmitted disease, coat protein Ð progeny, different phenotypes of protection were mediated resistance has been developed by using observed: 8 plants were completely resistant, a non-aphid transmissible isolate of SMV to whereas 51 plants recovered after a first phase reduce the risk of spreading new pathogenic of active multiplication of LMV. A breakdown strains by recombination (Reverse et al. 1996). of recovery was observed in greenhouse growth Furutani et al. (2007) have reported the virus conditions, leading to a late progression of viral resistance in transgenic soybean was caused by infection in a significant number of plants. post transcriptional gene silencing. Intensive Interestingly, both types of resistance were researches on transgenics are carried out in effective towards all strains including the virulent majority of the soybean growing countries. ones. This lack of strain specificity could be due to the high degree of CP similarity between 8.39.2.6 Peanut/BCMV (PStV), PeMoV strains. and PCV Two methods have been used successfully for 8.39.2.3 Squash/SqMV the Agrobacterium-mediated transformation of Development of the transgenic squash is a peanut (McKently et al. 1995) and particle significant breakthrough for squash improvement bombardment (Ozias-Akins et al. 1993). considering the economic importance of ZYMV Particularly, by bombardment of shoot meristems and WMV-2 and the difficulties in developing of mature embryonic axes, the nucleocapsid resistant cvs by traditional breeding (Fuchs protein of Tomato spotted wilt virus was and Gonsalves 1995). Moreover, multiple introduced into peanut (Brar et al. 1994). CP genes enable controlling several aphid- Resistance to seed-transmitted viruses is now transmitted viruses: Transgenic squash lines a realistic target. expressing the CP genes of ZYMV, WMV-2 and CMV have been developed (Tricoli et al. 8.39.2.7 Bean 1995), and field tests have demonstrated the Biotechnology programmes relating to bean im- potential of such transgenic squash in controlling provement and utilisation have been ongoing in mixed infections by these three viruses. This Mexico, Colombia and other countries. For bean strategy could thus be extended to the control too, the particle bombardment appears as the of SqMV. most promising method of gene delivery (Russell 260 8 Methods of Combating Seed-Transmitted Virus Diseases et al. 1993; Kim and Minamikawa 1996; Christou should thus progressively help in controlling 1997). Transgenic beans expressing the coat pro- many viruses. Particularly, for seed-transmitted tein of a non-seed-transmitted virus, Bean golden viruses, even an incomplete or a delayed mosaic begomovirus, have been obtained (Russell resistance could prevent the transmission of virus et al. 1993). This first success makes the control through seed that would solve the agricultural of BCMV/BCMNV a realistic target. problem of the primary inoculum. It is important to mention that, after release 8.39.2.8 Pea/PSbMV of transgenic soybean, a patent issued by the US Production of transgenic pea plants was obtained patent office and by the European patent office with difficulty, by Agrobacterium-mediated to a US Plant Biotechnology Company provided transfer (Puonti-Kaerlas et al. 1990; Bean et al. broad patent protection for all transgenic soybean 1997). Significant progress has been reported in (Lehrman 1994). Such type of right of intellectual the last 4 years in pea transformation. However, property should be revised because it is going efficiencies are still low, and methods are variety to prevent the achievement of programmes, the dependent, an inherent difficulty with Agrobac- objectives of which are to satisfy the basic needs terium-mediated transfer. Transformation with of a major part of world population. PSbMV-derived constructs is under study in Genetic modification of plants with viral CP Europe to complement the multiresistance genes is an example of pathogen-derived resis- conferred by three recessive sbm genes. tance that has been used successfully to produce Effective genetic engineering has started, and viral-resistant plants (Lomonossoff 1995; Beachy certain transgenic varieties of crops have been 1997). CP-mediated resistance in peas against launched on the market. For each virus/plant Pea enation mosaic virus (Chowrira et al. 1998), system, there is a possibility of controlling the Pea seed-borne mosaic virus (Jones et al. 1998) virus or even several viruses (as shown by the and Alfalfa mosaic virus (Timmerman-Vaughan example of squash). et al. 2001). Transgenic squash resistant to Cu- Major agrochemical and seed companies are cumber mosaic virus, Zucchini yellow mosaic taking the leadership in the race to develop new virus and Watermelon mosaic virus was released products for crops of higher commercial values. during 1996 (Tricoli et al. 1995). They often have branches in those developing Resistance to viral infections in plants has countries which express a rapid economic growth been exploited in two ways Ð first one is resis- (China, Taiwan, India, Singapore, etc.). tance gene-dependent responses and second one However, a major part of the populations is pathogen-derived resistance. Out of these two of the world is excluded from this market. approaches, the pathogen-derived resistance is Moreover, these populations often prefer more appropriate; thus, the creation of crop plants local susceptible varieties already adapted with broad resistance to more number of viruses to the culinary uses and also to the agro- is a potential area of future research. climatic conditions. Therefore, some developing countries, as exemplified by India, invest in 8.39.2.9 Coat Protein Genes an intensive programme of development of The coat protein of virus normally has a its own biotechnological capacity. In other protective function, insulating the virus nucleic developing countries, biotechnology penetration acid (RNA or DNA) from environmental assaults, is via the international centres CGIAR, some such as inactivation by ultraviolet light or institutes like the French Center of International digestion by host cell enzymes. In some cases, Cooperation in Agricultural Research for the protein has a role in host recognition in Development (CIRAD), the US Agency for early stages of infection. The recent approach is International Development (USAID) and other introduction of coat protein genes which will be organisations which promote the technology expressed in genetically engineered (transgenic) transfer. Transformation for resistance to viruses plants in order to produce plants protected 8.39 Molecular Approaches 261 against virus infection (Hamilton 1980;Galun genomes. Examples of these methods include and Breiman 1997). This strategy is based on directed recombination and mutation analysis of the concept of pathogen-derived resistance where both DNA and RNA (Ishikawa et al. 1986)viral the introduction of viral sequence into plants genomes. could interfere with the viral life cycle, leading to The available evidence indicates that at least resistance against the virus (Sanford and Johnston for TMV, the CP confers protection, and the 1985).Itmayalsohelpinthetransformationof mRNA and 30 untranslated sequences do not con- plants to enhance their virus resistance. This tribute to the observed virus resistance (Powell approach, which is an attempt to simulate natural et al. 1990). CP plays a major role in vector trans- cross protection between virus strains, seems mission, and CPMR confers additional advantage applicable to a wide range of viruses. The coat of resistance to vector inoculation in several cases protein strategy results primarily in symptom (e.g. PVX, PVY, CMV). TSWV CP transgene retardation and is sensitive to the strength of the tomato plants were resistant to thrips and trans- viral inoculum and to the amount of coat protein genic rice expressing high level of Rice stripe expressed (Powell Abel et al. 1986), and this is virus CP gene expressed resistance to virus in- known coat-protein-mediated resistance (CPMR) oculation by plant hopper. A remarkable increase (Beachy et al. 1990). in the yield of the several crops is established Single-stranded RNA plant viruses are geneti- by CPMR technology (Arif and Hassan 2000; cally simple, and most of them have genomes less Dasgupta et al. 2003; Sreenivasulu and Subba than 10,000 nucleotides. The cistron encoding Reddy 2006; Reddy et al. 2009). the coat protein is often located at 30 end of Wheat streak mosaic virus (WSMV) was the encoding RNA. CP coding region of various found widely dispersed in and around Australian plant viruses has been cloned and sequenced, continent and was shown to be seed-transmitted readily accessible technology exists to construct in wheat. CP gene of nine WSMV isolates suitable vectors with a chimeric form of the was sequenced and compared. Three sequenced gene for transfer and expression in plant system CP genes were closely related to the Pacific (Gasser and Fraley 1989). Peanut mottle virus Northwest sequences and differ by 0.76% resistance gene RPv1 in Williams isoline did only. The sequence of CP gene of third not give any protection against mottling caused seed-transmitted isolate originally from the by SMV. There was significance in virus, line same source differed from the other two and virus-line interaction for seed coat mottling. seed-transmitted isolates by two nucleotides, Non-seed-coat mottling gene (Im) in Williams indicating that the immigrant WSMV population isoline provided limited, if any, protection against may have been variable (Dwyer et al. 2007). mottling caused by SMV and none against Bean In the first report of stable transgenic resis- pod mottle virus (BPMV) (Wang et al. 2003). tance against mosaic disease of soybean, CP- Since TMV is one of the best characterised mediated resistance has been developed by using plant viruses, the gene coding for its coat pro- a non-aphid transmissible isolate of SMV to re- tein is introduced into tobacco. The transgenic duce the risk of spreading new pathogenic strains tobacco plants expressed the coat protein cod- by recombination (Revers et al. 1996). ing sequences of TMV RNA and exhibited the high degree of protection against TMV infection 8.39.2.10 Movement Protein Genes (PowellAbeletal.1986). Turner et al. (1987) (MP Genes) have also transferred the AMV coat protein into Movement proteins (MPs) are essential for tobacco and tomato plants which induced resis- plant viruses to move from one cell to another tance to AMV. In the future, it may be possible cell Ð called as cell-to-cell movement. MPs have to extend the range of viral sequences that can be been shown to involve in the gating function used in this way by applying information derived of plasmodesmata of the cell. They allow the from new methods of analysis of plant viral virus particles or their nucleoprotein derivatives 262 8 Methods of Combating Seed-Transmitted Virus Diseases to spread or to move to the adjacent cells. high levels of resistance to closely related strains Transgenic plants expressing viral movement of virus. This replicase-mediated resistance is protein are resistant to the virus from which achieved both through suppression of viral repli- the sequence was derived as well as to other cation and inhibition of systemic viral movement. viruses, a phenomenon termed as movement High level of resistance to closely related strains protein-mediated resistance (MPMR), and this and the inhibition of replication point to an RNA- was first used to engineer resistance against TMV mediated homology-dependent mechanism of re- in tobacco (Cooper et al. 1995). Single MP gene sistance. The suppression of viral spread hints in tobamoviruses is mediated by a set of three at the involvement of protein. PVY and AMV overlapping genes. Expression of the modified have been engineered, and the mechanism of re- 12-kDa TGB gene of PVX has shown to confer sistance was shown to be protein based since only MP-derived resistance in potato to potexvirus mutant replicase proteins conferred resistance. PVX and Potato virus M and Potato virus S. Viral replicase genes are effective in conferring The resistance depends upon the interaction of resistance to infection by both RNA and virions. the viral-derived and transgenic-derived MPs. Expression of various replicase sequences can TMV MP plants were resistant not only to TMV confer a very high level of resistance, and this will and other tobamoviruses Ð Tobacco mild green be highly useful for engineering plants for virus mosaic virus and Sunn-hemp mosaic virus Ðbut resistance. also to unrelated viruses Ð TRV, AMV, CMV, Peanut chlorotic streak virus and Tomato ring 8.39.2.12 Satellite RNA spot virus (TRSV). This broad resistance informs Certain groups of biological entities which are that viral MP interacts with plasmodesmata in naturally able to modify plant viral diseases are similar ways. Transgenic plants carrying mutant the satellite RNAs. These at RNAs have poten- MP may be one approach to generate broad viral tial in controlling viral disease since several of resistance of crop plants. However, MPMR is not these at agents result in amelioration of virus always that broad. Transgenic plants expressing symptoms. Some of the RNAs attenuate symp- one of the MPs of White clover mosaic virus, toms induced by the helper virus (e.g. CARNA a potexvirus, were resistance only to other 5 of CMV). Satellite RNA (sRNA) strategy is potexviruses. being exploited for certain viruses to make virus- free plants. The experiments with satellite RNA 8.39.2.11 Replicase Protein Genes (RP illustrate the potential of transformation strate- Genes) gies for engineering resistance to viral infection Viral specific transcriptase or replicase within (Baulcombe et al. 1987). The successful exam- the virus nucleocapsid is essential because no ples, involving satellite RNA, have used the prin- host cell contains any enzyme capable of de- ciple of turning the virus against itself, via the coding and copying the genome. This replicase expression of viral sequence in the transformed gene can be manipulated and used in transgenic plant (transgenic plant). condition because the replicase protein expres- Satellite RNAs are species of RNA associated sion is key to the resistance phenotype. This with some strains of certain plant RNA viruses, mechanism is known as replicase-mediated re- but not necessary for virus replication. Satellites sistance (Rep-MR). Rep-MR was first described are replicated in cells infected with the particular in tobacco plants carrying a 54-kDa fragment virus they depend on, and because they are of the TMV-U1 replicase. This technique has packaged with viral genomic RNAs in the virus been exploited in PVX, PVY, Cymbidium ring particle, they can accompany viruses released spot virus (Cy RSV), CMV, Pea early brown- from diseased plant tissue to new infection ing virus (PEBV), PLRV, Cowpea mosaic virus sites. Although they are completely dependent (CPMV) and TRV. The expression of a truncated on the virus for replication and transmission, CMV 2a replicase gene in tobacco plants gave their nucleotide sequence seems to be essen- 8.39 Molecular Approaches 263 tially unrelated to that of the viral genome. ter understood, it may also be possible to ex- Certain satellites reduce, sometimes markedly, tend the range using artificial satellite sequences the severity of disease symptoms resulting constructed in vitro to explore with other plant from virus infections where they are present viruses. (Courtice 1987). The principle involved in this approach is the 8.39.2.13 Antisense RNA introduction of a DNA sequence coding for satel- Antisense RNA expression is an another strategy lite RNA into the genome of transformed plants tried for producing virus-resistant plants against so that satellite RNA would be produced consti- plant viral CP gene sequences. The mechanism is tutively in those plants. If the transcribed satellite the antisense constructs conferring resistance RNA has the biological properties of naturally may be the same as homology-dependent occurring satellite RNA, the transformed plants degradation mediated by RNA. The virus would show attenuated symptoms when inocu- replication may be inhibited if the plant is lated with CMV. This phenomenon of satellite- induced to make antisense RNA stretches of RNA mediated protection has been exploited agronom- complementary to crucial sequences of the plant ically in China against CMV in pepper plants virus genomic RNA. By forming base pairs to the (Tien et al. 1987). genomic RNA, such wrecking stretches of RNA Satellite RNA is a less widespread feature may prevent expression of the virus genome. of plant viruses than coat protein. However, it Plants that showed some resistance to low has been possible to produce virus resistance inoculum concentrations have been produced for in transgenic plants by expression of symptom- CMV, PVX and TMV. RNA molecules that bind attenuating satellite RNA from TRSV (Gerlach to RNA transcripts of specific genes and prevent et al. 1987) as well as from CMV (Harrison their translation are called antisense or mic-RNA et al. 1987; Baulcombe et al. 1987). Some of (messenger-RNA-interfering complementary the strains of CMV encapsidate satellite RNA in RNA). Good resistance was reported in addition to the tripartite messenger sense ssRNA antisense transformants against BYMV, sense genome. CMV sRNA depends on its helper and antisense TRSV CP transformants were virus (HV) CMV for replication, movement resistant to the virus, antisense mRNA of within the plant, assembly and transmission. rep protein of TGMV and TYLCV and of Transgenic tobacco plants expressing multiple or CP of ToMoV. partial copies of CMV sRNA showed attenuated Antisense RNA, regulatory RNA that can in- symptoms when challenged with CMV. Tobacco hibit gene expression, is complementary to a plants transformed with antisense sRNA showed given messenger RNA (mRNA) species, with delayed symptom development with the cognate which it pairs, forming double-stranded RNA. virus. Tobacco plants transformed with CMV Antisense RNA is a natural mechanism for gene sRNA were resistant to satellite-free CMV and regulation in bacteria but has also been used produced mild symptoms, but sRNA replication experimentally. It has been shown to inhibit gene occurred while that of CMV was reduced. The expression in a variety of prokaryotic and eukary- result was the same with plants raised from the otic cells (Kim and Wold 1985; Weintraub et al. seed of the transgenic tobacco (Harrison et al. 1985; Green et al. 1986) including plants (Ecker 1987). and Davis 1986). The mechanisms by which anti- The advantage of satellite RNA over the coat sense RNA inhibits gene expression are not well protein phenomenon is that the protection is per- understood, and different modes of inhibition manent, independent of the strength of the viral have been described. In eukaryotic systems, the inoculum and insensitive to the concentration formation of double-stranded RNA in the nucleus of satellite transcript in the transformed plants may block mRNA transport to the cytoplasm (Harrison et al. 1987). In the future, when the (Kim and Wold 1985), or the RNAÐRNA hybrids principles of satellite-based protection are bet- formed in the nucleus may be rapidly degraded 264 8 Methods of Combating Seed-Transmitted Virus Diseases

(Melton 1985). Antisense RNA (mic-RNA) may that antisense inhibition will be a useful antiviral inhibit in mRNA translation bound to polysomes strategy in agriculture. (Melton 1985; Rosenberg et al. 1985). Ribozymes are catalytic RNAs those cleave The genomes of plus strand ssRNA viruses at specific sites on the target cRNAs, thus have structural similarities to mRNA and func- complementary to the target sequence and is an tion as mRNA during virus replication (Strauss antisense RNA. The constructs of PPV containing and Strauss 1983), and the antisense RNA in- hammerhead ribozyme given the strongest hibits virus replication (Sanford and Johnston resistance than normal antisense RNA construct. 1985). Antisense transgenic squash plant seeds were There is limited information on the use of tested for resistance against SqMV strains antisense RNA against a plant virus since this (SqMV-22, SqMV-127). Transgenic squash approach is attempted in recent years. Loesch- plants inoculated at 17 days after germination Fries et al. (1987) reported the synthesis of an- were not resistant, while those inoculated at tisense RNA of AMV in transgenic plants but 45 days after germination showed high level did not report any antiviral activity. It was also resistance on the virus strain (8% resistance with established through different regions of Tobacco strain SqMV-22 and 92% resistance with strain rattle virus (TRV) genomes were expressed as SqMV-127) (Jan et al. 2000b). antisense RNA in tobacco, and the transformed Antisense-mediated gene silencing (ASGS) plants did not show resistance to infection by with sense transgenics is remarkable, and TRV (Baulcombe et al. 1987), possibly because the mechanistic terms are similar with post- the encapsidated viral RNA is not accessible to transcriptional gene silencing (PTGS). In ASGC, the antisense RNA molecules. silencing is involved in production of 20Ð25-nt- Transgenic plants expressing antisense coat long degraded RNA (siRNA), and both forms are protein gene of CMV (Cuozzo et al. 1988)and suppressible by the same viral proteins (Arif and PVX (Hemenway et al. 1988) have been found Hassan 2000; Dasgupta et al. 2003; Sreenivasulu to inhibit the replication of their respective virus and Subba Reddy 2006). at low inoculum levels. Rezaian et al. (1988) observed that inhibition could involve antisense 8.39.2.14 RNAi Mediated RNA binding with CMV RNA. There is a possi- RNA silencing is a novel gene regulatory bility that CMV antisense coat protein gene can mechanism which limits the transcript level compete for the viral or host factors involved in by two ways Ð the first mechanism is named the replication of CMV RNA from the minus- as transcriptional gene silencing (TGS) which strand templates. Further they also observed that suppresses the transcription and the second in transformed plants expressing antisense RNA, mechanism is named as post-transcriptional only one plant line which expressed relatively gene silencing (PTGS) or RNA interference low levels of one of the antisense RNAs showed (RNAi) which activates sequence-specific RNA resistance to CMV, but other plants expressing degradation process. RNAi-related events were the same or the other two antisense RNAs had understood in almost all eukaryotic organisms similar sensitivity to CMV infection as the non- (Agarwal et al. 2003). RNAi is mechanistically transformed plants. It is possible that the three similar forms but phenotypically showed three RNA regions used in this study together con- types Ð (a) co-suppression or PTGS in plants, stitute about 12% of the CMV genome, and it (b) quelling in fungi and (c) RNAi in the is possible that antisense RNA to other regions animals. Recently, micro-RNA formation and of CMV RNA may be active. Use of mic an- heterochromatisation have been revealed as other tisense RNA will provide basic information on facets of naturally occurring RNAi processes of the replication, expression and molecular basis of eukaryotic cells. the pathogenicity of viruses and viroids. Further Virus-induced Gene Silencing (VIGS) and progress of research in this direction is possibly homology-driven RNA degradation occur during 8.39 Molecular Approaches 265 the multiplication of viral genomes in infected 1983). These substances have been purified, and plants. Viruses can be either the source, the the IVR was characterised as proteinaceous in na- target or both the source and the target of the ture and was demonstrated to inhibit replication gene silencing. Virus-derived transgenes gave of TMV in protoplasts and tomato leaf discs, and protection against the challenge viruses even CMV and PVX replication in various host tissues. when no transgene protein was produced. Virus- The AVF also is a type of glycoprotein which resistant plant revealed that the transgenes are functions as an interferon-like molecule (Sela highly transcribed in the nucleus, whereas the 1981). Only minute amounts of AVF are pro- steady-state level of cytoplasmic mRNA was duced in plants, and when purified, they are still very low. Virus-induced gene silencing is the active at the picogram level. It was estimated that phenomenon of viral recovery. Virus-resistant AVF exerts its antiviral activity at a level of few plants revealed that the transgenes were highly molecules per cell. transcribed in the nucleus, whereas the steady- Other recent reports have indicated the state level of cytoplasmic mRNA was very involvement of active substances in N-gene- low. Some of the transgenic mRNA molecules related resistance. Wieringa-Brants (1983) assumed the confirmation of dsRNA, which trig- demonstrated that intercellular fluids from gered sequence-specific degradation of self and TMV-infected N-gene-carrying tobacco were other homologous or cRNA sequences in the cy- antivirally active when introduced to another toplasm. Based on the principles of virus-induced tobacco. Padgett et al. (1997) reported that gene silencing, vectors designed with the genome progeny seedlings of N-gene-carrying tobacco, sequence of RNA viruses Tobacco mosaic virus, obtained by selfing individual plants that had Potato virus XandTobacco rattle virus are been repeatedly inoculated with TMV, are being widely used to knock down the expression somewhat protected from TMV infection. This of host genes. Virus-induced gene silencing- indicated that the activation of the resistance based techniques are extremely useful for studies mechanism is seed transmissible. Since these related to functional genomics in plants. were selfed seedlings and the antiviral effect The product of RNA degradation as a small did not pass to the second generation, the RNA species (siRNA) of 25 nucleotides of involvement of a dilutable active substance was both sense and antisense polarity. siRNAs were implied. Antiviral factors in other localised detected first in plants undergoing either co- infections were also reported (Faccioli and suppression or virus-induced gene silencing and Capponi 1983; Capponi et al. 1984). were not detectable in control plants that were Orchanski et al. (1982) reported that animal not silenced. The generation of siRNA turned out interferon is able to inhibit plant virus replica- to be signature of any homology-dependent RNA tion. A number of human interferons, including silencing event (Agarwal et al. 2003). some recombinant species, were active in reduc- ing TMV infection. Interferon affects the level of 8.39.2.15 Antiviral Activities in Plants TMV RNA in infected cells. The antiviral effect Sela and Applebaum (1962) demonstrated that of interferon is transient, fading away within 48Ð plant-sap fractions from virus-infected plants ex- 96 h after application (Sela 1987). Interferon- hibit antiviral activity in excess of that of cor- treated plant tissues reacted similarly to AVF- responding control saps. It was assumed that treated tissues by stimulating ATP polymerisation infected plants develop an antiviral factor (AVF), to oligonucleotide with antiviral activity (Reich- which was found in a number of plantÐvirus man et al. 1983;Devashetal.1985). combinations (Sela 1981). This opened new perspective for control of The hypersensitivity reaction of tobacco plants plant viruses by substances associated with ac- carrying the N resistance gene to TMV has iden- tive resistance phenomena. Identification at the tified an antiviral factor (AVF) (Sela 1981)oran genome level of the responsible gene(s) could inhibitor of virus replication (IVR) (Gera et al. provide new possibilities of breeding for non- 266 8 Methods of Combating Seed-Transmitted Virus Diseases specific virus resistance by genetic engineering Some countries are in a pole position, while comprising cloning of these gene(s) and transfer the investment in such a research is still low in to plants devoid of such resistance gene(s). a major part of the world. An optimistic sight of Transformation strategies with either the coat the situation would hope that countries playing a protein or satellite RNA are quite useful in virus leading role will help the other part of the world disease control. The use of coat protein genes is to solve problems of malnutrition. However, a quite promising as it is effective against several number of years are necessary before the achieve- viruses without hazard. The satellite RNA strat- ment of a generalised biotechnological control egy still needs further development, for example, of diseases induced by the main seed-transmitted to remove the hazards associated with the use of viruses. certain strains of satellite RNA of CMV. It is now In the meantime, the classical quality certain that in the near future genetic engineering control which has proven very effective in of the plant and virus genome will provide ad- controlling some diseases like LMV/lettuce ditional possibilities for controlling plant viruses. and BSMV/barley is now simple. It does not Transfer and integration of virus information into necessitate any investment in research, only in the cell genome will provide specific control organisation. A very large field is open to this and very likely will never result in a complete methodology, both for the selection of healthy resistance. Therefore, these techniques will not seed lots in an agricultural context and for a replace the conventional methods of plant breed- guarantee of quality in seed trade. ing for resistance and the sanitation programmes. In view of satisfying both types of needs, the In these attempts, difficulties will probably come Working Group of Virology of the International from the vehicles, the transformation and the re- Seed Testing Association (ISTA) is considering generation of the plants. These will be eliminated the problem of standardisation of the quality by the removal of the responsible fragment of control for each virus/seed system by ensuring the T-DNA. In addition, use of plant transposable the same high quality reactive to each user, re- elements with site-specific insertions will con- active carefully selected by a few specialists for tribute in the future in overcoming these problems becoming ‘the ISTA reactive for this virus/seed and broadening the field of applications of these system’. A working sheet will then describe with approaches to introduce foreign genes into the the highest accuracy how to use this reactive plant genome. either for determining the seed transmission rate or for knowing the status of a seed lot with respect to a tolerance. This working sheet will 8.40 Conclusion be established with the complete agreement of laboratories having performed a successful com- For a number of important seed-transmitted parative testing on the same seed lots. ISTA and viruses, sources of resistance have been found. International Seed Trade Federation are jointly However, programmes of resistance have involved in the search of a financial support that only been achieved for a few crops (bean, will enable to establish a bank of reactive in view tomato, pepper, lettuce), and when we consider of promoting quality control of seed for viruses in the geography, these programmes have been industrialised as well as in developing countries. achieved in only a limited part of the world. A powerful approach by biotechnology is now favoured because it could confer more References rapidly a resistance and because this resistance could concern cultivars already agronomically Adams DB, Kuhn CW (1977) Seed transmission of peanut adapted in each country. However, the challenge mottle virus. Phytopathology 67:1126Ð1129 Agarwal N, Dasaradhi PVN, Mohmmed A, Malhotra A, for scientists is now to develop strategies that Bhatnagar RK, Mukherjee SK (2003) RNA interfer- broaden the effect and increase the degree of ence biology, mechanism and applications. Microbiol resistance. Mol Biol Rev 67:657Ð685 References 267

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The central seed certification Von Wechmar MBD, Kaufmann A, Desmarais F, Rybicki Board, Department of agriculture and cooperation, EP (1984) Detection of seed transmitted brome mosaic Ministry of Agriculture, Government of India, New virus by ELISA, radial immunodiffusion and immu- Delhi, p 388 noelectroblotting tests. Pytopathologische Zeitchrift Turner NE, O’Connell KMO, Nelson RS, Sanders PR, 109(4):341Ð352 Beachy RN, Fraley RT, Shah DM (1987) Expression Vovk AM (1961) Inactivation of tobacco mosaic virus in of alfalfa mosaic virus coat protein gene confers cross- tomato seed at different storage times. Trans Inst Genet protection in transgenic tobacco and tomato plants. Acad Sci USSR 28:269Ð276 EMBO J 6:1181Ð1188 Walkey DGA (1992) Zucchini yellow mosaic virus. Con- Udayashankar AC, Nayaka CS, Kumar BH, Mortensen trol by mild strain protection. Phytoparasitica 20:99Ð CN, Shetty HS, Prakash HS (2010) Establishing inocu- 103 lum threshold levels for Bean common mosaic virus Walkey DGA, Dance MC (1979) High temperature inac- strain Black eye cowpea mosaic infection in cowpea tivation of seed borne lettuce mosaic virus. Plant Dis seed. Afr J Biotechnol 9(53):8958Ð8969 Rep 63:125Ð129 Upstone ME (1974) Effects of inoculation with the Dutch Walkey DGA, Innes NL (1979) Resistance to bean com- mutant strain of tobacco mosaic virus on the crop- mon mosaic virus in dwarf beans (Phaseolus vulgaris ping of commercial glasshouse tomatoes. Report of L.). J Agric Sci 92:101Ð108 284 8 Methods of Combating Seed-Transmitted Virus Diseases

Walkey DGA, Pink DAC (1984) Resistance in vegetable Wisler GC, Duffus JE (2000) A century of plant virus marrow and other Cucurbita spp. To two British management in the Salinas valley of California, ‘East strains of cucumber mosaic virus. J Agric Sci 102: of Eden’. Virus Res 71:161Ð169 197Ð205 Wongkaew S, Dollet M (1990) Comparison of Peanut Walkey DGA, Brocklehurst PA, Parker JE (1983) Seed stripe virus strains using symptomatology on partic- transmission of viruses. In 33rd annual report for 1982. ular host and serology. Oleagineux 45:267Ð278 National Vegetable Research Section, Wellesbourne, Wood GA, Chamberlain EE, Atkinson JD, Hunter JA Warwick, pp 82Ð83 (1975) Field studies with apple mosaic virus. N Z J Walkey DGA, Ward CM, Phelps K (1985) Studies on Agric Res 18 lettuce mosaic virus resistance in commercial lettuce WTO (1995) Agreement on the application of sanitary and cultivars. Plant Phytopathol 34:545Ð551 phytosanitary measures. In: Results of the Uruguay Wang Y, Hill CB, Bernard RL, Pedersen WL (2003) round of multilateral trade negotiations: the legal texts. Occurrence of seed coat mottling in soybean plants World Trade Organization, Geneva inoculated with Bean pod mottle virus and Soybean Wylie S, Wilson CR, Jones RAC, Jones MGK (1993) A mosaic virus. Plant Dis 87:1333Ð1336 polymerase chain reaction assay for cucumber mosaic Wang QC, Panis B, Engelmann F, Lambardi M, Valkonen virus in lupin seeds. Aust J Agric Res 44:41Ð51 JPT (2009) Cryotherapy of shoot tips: a technique Xu Z, Chen K, Zhang Z, Chen J (1991) Seed transmission for pathogen eradication to produce healthy plant- of peanut stripe virus in peanut. Plant Dis 75:723Ð726 ing materials and prepare healthy plant genetic re- Yakovleva N (1965) Borba s zelenoi mazaikoi Ogurtsov. sources for cryopreservation. Ann Appl Biol 154(3): (Control of green mosaic of cucumber). Z Rast Vredit 351Ð363 Bolez 10:50Ð51 Wardlaw CW (1961) Banana diseases including plantain Yang WZ, Hsiao CH, Chang WN (1986) Screening cu- and abaca. Longmans, Green and Co. Ltd., London cumbers from resistance to viruses and inheritance Waterworth P, Kahn RP (1978) Thermotherapy and asep- to Zucchini yellow mosaic virus. J Agric Res China tic bud culture of sugarcane to facilitate the exchange 35(2):192Ð201 of germplasm and passage through quarantine. Plant Zaumeyer WJ, Harter LL (1943) Two new virus diseases Dis Rep 62:772Ð776 of beans. J Agric Res 67:305Ð327 Watson MA (1959) Cereal virus diseases in Britain. Zaunmeyer WJ, Meiners JP (1975) Diseases resistance in NAASQ Rev 43:93Ð102 beans. Annu Rev Phytopathol 13:313Ð334 Watterson JC (1993) Development and breeding of resis- Zettler FW, Eiliott MS, Purcifusn DE, Mink GI, Gorbet tance to pepper and tomato viruses. In: Kyle MM (ed) DW, Knauft DA (1993) Production of peanut seed free Resistance to viral diseases of vegetables: genetics and of peanut stripe and peanut mottle viruses in Florida. breeding. Timber Press, Portland, pp 80Ð101 Plant Dis 77:747Ð749 Webb RE, Bohn GW (1962) Resistance to cucurbit viruses Zimmerman GS, Pilowsky M (1975) Experiments for pro- in Cucumis melo. Phytopathology 52:1221 tecting tomatoes, from tobacco mosaic virus (TMV) Webster CG, Wylie SJ, Jones MGK (2004) Diagnosis of by prior infection with the virulent strain M II - 16. plant viral pathogens. Curr Sci 86(12):1604Ð1607 Phytopathology 3:75 (Abstract) Weintraub H, Izant JG, Harland RM (1985) Anti-sense Zink FW, Grogan RG, Welch JE (1956) The effect RNA as a molecular tool for genetic analysis. Trends of the percentage of seed transmission upon subse- Genet 1:22Ð25 quent spread of lettuce mosaic virus. Phytopathology Wells DG, Deba R (1961) Sources of resistance to cowpea 46:662Ð664 yellow mosaic virus. Plant Dis Rep 45:878Ð881 Zink FW, Grogan RG, Bardin R (1957) The comparative Welsh MF, James M (1965) Detection of virus infection effect of mosaic-free seed and roguing as a control in imported Cherry and Apricot clones. Can Plant Dis for common lettuce mosaic. Proc Am Soc Hortic Sci Surv 45:3 70:277Ð280 Whitham S, McCormick S, Baker B (1996) The N Zitter TA (1977) Epidemiology of aphid-borne viruses. gene of tobacco confers resistance to tobacco mosaic In: Harris KF, Maramorasch K (eds) Aphids as virus virus in transgenic tomato. Proc Natl Acad Sci USA vectors. Academic, London, pp 385Ð412 93:8776Ð8781 Zitter TA, Simons JN (1980) Management of viruses by Wieringa-Brants DH (1983) A model to stimulate ac- alteration of vector efficiency and cultural practices. quired resistance induced by localized virus infec- Annu Rev Phytopathol 18:289Ð310 tion in hypersensitive tobacco. Phytopathology Z Zschiegner HJ, Kramer W, Sabs O, Fritzsche R, Dubnik 106:369Ð372 H (1971) Successful restriction of the spread of non- Wilcoxson RD, Peterson AG (1960) Resistance of Dollard persistent viruses by methods of virus vector control. red clover to the pea aphid, Macrosiphum pisi.JEcon In: Proceedings of 6th British insecticide and fungicide Entomol 53:863Ð865 conference, Brighton, pp 319Ð323 Plant Virus Transmission Through Vegetative Propagules (Asexual 9 Reproduction)

Abstract Virus and virus-like diseases spread through vegetative propagules like tubers, rhizomes, stolons, corms, bulbs and buds of economically impor- tant crops like cassava, potato, sugarcane, banana, sweet potato, Dioscorea, beet root, onion and majority of fruit and ornamental plants. Throughout the world, one or two virus and virus-like diseases (viroid and phytoplasma) are threatening the economy of their country. Fruit tree propagation is usually carried out through asexual reproduction by grafting or budding of the desired variety, onto a suitable root stock as observed in citrus, apple, peach, plum, grape and others. Although the virus diseases spread through vegetative propagules to short distances and further through insect vectors. Man is responsible for the world wide movement of many virus and virus-like diseases through the vegetative propagules. Cassava is the major food crop in 39 African countries and some of the South Asian countries, and the cassava mosaic disease is the primary limiting factor. Similarly, sugarcane mosaic in sugarcane is wide spread where ever crop is grown. Even tristeza virus is devastating disease throughout the world. In South African countries, swollen shoot disease reduces the cocoa crop yields. To combat these virus diseases which are carried through vegetative propagules, the pathogen diagnosis is primary factor. Widely accepted techniques like ELISA, PCR, microarrays and also other molecular tests are well applicable in practice. For almost all crops, the virus-free plant certification schemes, clean seed certification schemes against each crop have been developed in every country. Based on the advanced techniques developed related to crop production, virus management measures are being applied in almost all countries.

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 9, 285 © Springer India 2013 286 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction)

The importance of virus infection of 9.1 Different Vegetative vegetatively propagated plants and methods Propagative Plant Materials that are being used to eradicate viruses and virus-like diseases from plant clones that are Plants have a number of mechanisms for totally infected is discussed in this chapter. Some asexual or vegetative reproduction. Vegetative of the highly economically important viruses reproduction uses plants parts such as roots, which are worldwide in distribution which are stems and leaves. It is a process by which new propagated through vegetative plant material individuals arise without production of seeds or such as banana, citrus, pineapple, cassava, spores. Plants are produced using material from a sugarcane and potato have been discussed in single parent, and as such there is no exchange of detail in different chapters. genetic material; therefore, vegetative propaga- tion methods almost always produce plants that are identical to the parent clonal propagation. Virtually all types of shoots and roots are capable 9.3 Different Virus, Phytoplasma of vegetative propagation, including stems, basal and Viroid Diseases shoots, tubers, rhizomes, stolons, corms, bulbs and buds. Some of the crops like potato, sweet The details of different virus, viroid and phyto- potato, cassava, Dioscorea, beet root, carrot, plasma diseases in some of the vegetatively prop- Colocasia, onion, garlic, ginger, sugarcane, agated food crops, ornamentals and commercial banana, pineapple, vanilla, strawberry and crops are furnished in the Table 9.1. ornamentals like canna, carnations and chrysan- themum are propagated vegetatively. Fruit tree propagation is usually carried out through asexual 9.4 Virus Transmission reproduction by grafting or budding the desired variety onto a suitable rootstock as observed in Some of the vectors like aphids, leaf-hoppers, citrus, apple, peach, plum, grape and others. thrips, whiteflies, beetles, mites, nematodes and fungi are transmitting the major economically important diseases of vegetatively propagated 9.2 Role of Vegetatively crops and are responsible for heavy yield Propagated Plant Materials losses. For example in potato, nearly 30 virus in Virus Spread and virus-like diseases affect the crop, and viruses like Alfalfa mosaic virus, Cucumber The widespread use of vegetative propagation for mosaic virus, Potato virus M, Potato virus A the multiplication of many horticultural crops re- and PVY Potato virus S are transmitted by sults in the spread of viruses through propagules different aphid species in nonpersistent manner, such as cuttings, tubers, runners and bulbs. Since whereas Potato leaf roll virus is transmitted infection by most viruses is completely systemic, by aphid in persistent manner. Other vectors any propagule is likely to be infected. Thus, like beetles spread Andean potato latent and vegetative propagation presents a very efficient Andean potato mottle viruses in nonpersistent method of vertical virus spread, without the virus manner. Leaf-hoppers transmit Beet curly top having the difficulty of entering and establishing virus in potato in persistent manner. Nematode infection in a new healthy plant. vectors transmit Tobacco black ring virus, Potato Although virus spread through vegetative black ring spot and other viruses to potato, propagation might be expected to occur over and fungal vectors transmit Tobacco necrosis short distances in nature by natural scattering of virus in persistent manner. Tomato spotted infected propagules such as tubers, man has been wilt virus that causes is highly destructive responsible for the worldwide movement of many disease in potato is transmitted by thrips viruses by carrying the vegetative propagules. vector. 9.4 Virus Transmission 287

Table 9.1 Certain virus and virus-like diseases transmitted through vegetative propagules Type of vegetative Crop Virus/phytoplasma/viroid disease propagules Amaryllis Cucumber mosaic, Necrotic spot bulbs Apple Apple chlorotic leaf spot, Apple mosaic, Grafting Apple scar skin, Apple stem pitting, Prunus necrotic ring spot Avocado Avocado sunblotch Grafting Banana Banana bunchy top, Banana bract Rhizome, suckers mosaic, Banana streak, Cucumber mosaic Cacao Cacao necrosis, Cacao swollen shoot, Cuttings, grafts, seeds Cacao yellow mosaic Carnation Carnation latent, Carnation mottle, Stem cuttings Carnation necrotic fleck, Carnation ring spot, Carnation etched ring Cassava African cassava mosaic, Cassava brown Planting segments of the streak, Cassava Indian mosaic, Cassava stem, tuberous root vein mottle, Cassava Common mosaic Chrysanthemum Chrysanthemum stem Suckers, cuttings necrosis,Chrysanthemum vein chlorosis, Chrysanthemum B Citrus Citrus tristeza, Citrus enation-woody gall, Grafting on root stocks, Citrus exocortis, Citrus mosaic, Citrus stem cuttings variegation Colocasia Colocasia bobone disease Corms Dahlia Dahlia mosaic, Tomato spotted wilt, Root tubers/bulbs Impatiens necrotic spot Elephant foot yam Colocasia bobone disease, Dasheen Corns mosaic virus Gladiolus Bean yellow mosaic, Cucumber mosaic, Corm and cormlets Tobacco ring spot, Ornithogalum mosaic Grapevine Grapevine fanleaf, Grapevine leafroll, Stem cuttings, grafting Grapevine stem pitting Lilies Lily symptomless, Lily mottle, Lily virus Bulb, bulblets X, Cucumber mosaic, Tulip breaking Onion/Garlic/Shallot Onion yellow dwarf, Iris yellow spot, Bulb, bulblets Onion mite-borne latent, Garlic common latent, Shallot latent, Shallot virus X, Shallot yellow stripe Peach Prune dwarf, Prunus necrotic ring spot Grafting on root stocks Plum Plum pox, Prunus necrotic ring spot Stem cuttings Potato Potato Y, Potato X, Peanut bud Stem tubers necrosis,Potato leafroll, Potato spindle tuber Rose Common rose mosaic, Rose leaf curl, Stem cuttings Rose ring pattern, Rose streak, Rose tobamo Passion fruit Passion fruit woodiness,Passiflora ring Cuttings, graftings spot, Passion fruit yellow mosaic Pine apple Pineapple yellow spot, Pineapple Suckers, slips chlorotic leaf streak, Pineapple wilt Strawberry Strawberry crinkle, Strawberry latent, Runners Strawberry mild yellow edge, Strawberry vein banding (continued) 288 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction)

Table 9.1 (continued) Type of vegetative Crop Virus/phytoplasma/viroid disease propagules Sugarcane Sugarcane mosaic, Sugarcane streak Stem cuttings mosaic, Sugarcane bacilliform, Sugarcane yellow leaf, Sugarcane grassy shoot Sweet potato Sweet potato feathery mottle, Sweet Tuberous roots potato chlorotic fleck, Sweet potato yellow dwarf, Sweet potato mild mottle, Sweet potato latent Tulip Tulip breaking, Tulip chlorotic Bulb and bulblets blotch,Tulip virus X, Tobacco necrosis, Tobacco rattle Yam Dioscorea green banding mosaic, Tuberous roots Dioscorea latent, Yam mosaic, Dioscorea bacilliform, Cucumber mosaic virus

In another commercial crop like sugarcane and productive life of crops like citrus, peach, infected by Sugarcane mosaic virus is worldwide plum, avocado and passion fruit is much reduced and spreads through aphid species in nonper- once viruses are affecting these crops. sistent manner. This crop is also infected with In almost all tropical countries, nearly 30 leaf-hopper-transmitted Sugarcane streak virus viruses and virus-like diseases affect potato, of and mealybug-transmitted Sugarcane bacilliform which 5Ð6 are very important (Khurana 1992; badnavirus. In almost all cassava-growing coun- Sastry and Saigopal 2010). The losses in potato tries, whitefly Bemisia tabaci transmits number yield due to one or more virus(es) infecting of begomoviruses in persistent manner. For infor- potatoes vary from low to very high (Garg 2005). mation on vector transmission for almost all veg- Infections of PVY and PLRV have potentiality etatively propagated plants, Brunt et al. (1996) to reduce the yield up to 60Ð80%, while mild compilation is one of the informative and reliable viruses like PVX, PVS and PVM also decreased sources. the yield from 10 to 30% in infected plants. Cassava is the major food crop in 39 African countries, and Cassava mosaic disease for which 9.5 Yield Losses Bemisia tabaci is the vector has caused catas- trophic yield losses. Padwick (1956) brought to- Even though intensive researches were carried gether the available information on yield losses out and novel technologies were developed, and estimated that the yearly loss in yield due to still one has to admit that virus and virus-like this disease was equivalent to about 11% of the diseases cause considerable economic yield crop in Africa. Yield losses on individual vari- losses throughout the world with different eties susceptible cassava range from 20 to 95% virusÐhost combinations. Many viruses are (Beck and Chant 1958). In Africa, African cas- latent in some of their host species yet have sava mosaic virus causes yield losses of 20Ð90% adverse effects. In the perennial crops which (Terry and Hahn 1980;Threshetal.1994). An- are vegetatively propagated like fruit crops, nual economic losses in East and Central Africa losses are enormous. Yield losses due to viruses are estimated to be 1.9Ð2.7 billion USD (Patil and have been reviewed by Duffus (1977), Agrious Fauquet 2009). (1990, 2005) and Waterworth and Hadidi (1998). Among fruit crops, tristeza is the most In general, viruses cause loss of plant vigour, devastating disease of citrus, and thousands of which automatically affects yield/production, citrus trees in several countries like the USA, 9.6 Virus Diagnosis 289

Brazil, Argentina, Israel, India and Uruguay were grapes which adversely affect fresh market sales affected by tristeza and also with greeningÐfungal and red wine colour (Goheen 1970). Even the complex. Bennet and Costa (1949) reported that commercial value of some of the ornamentals like in about 12 years, 60,00,000 or about 75% of tulip and gladiolus was reduced due to flower the orange trees were destroyed due to tristeza. break virus symptoms which have reduced flower In Argentina, also the losses were as high as 20 number and size. million bearing trees worth of approximately 500 million dollars (Nolla and Fernandez 1976). In two decades, following introduction of tristeza 9.6 Virus Diagnosis in South America, 20 million citrus trees were destroyed in Argentina alone (Klotz 1961). The Few decades ago, virus detection was based real magnitude of this dreadful disease is vividly mainly on biological techniques which are indicated by Wallace (1959) who estimated that too slow and not amenable to large-scale tristeza has threaten to destroy more than half of application. Advances in molecular biology the world’s citrus. and biotechnology over the last three decades In Ghana, cocoa which is one of the ma- were applied to develop rapid, specific and jor commercial crops was generally affected by sensitive techniques for the detection of plant Cocoa swollen shoot virus infection which has viruses. Serological or immunological assays reduced the yield of mature trees by 25% after have been developed and used successfully for 1 year, by 50% after second year and cent percent a number of years for the detection of plant after third year, by which time almost plants were viruses (Torrance and Jones 1981). These tests dead, and this disease has taken the livelihood are broadly subdivided into liquid and solid- of the people (Crowdy and Posnette 1947). This phase tests. In the former, both antigen and disease has killed half of the mature trees in antibody react in solution to form a visible a 250,000 acre area of cocoa (Wellman 1954). precipitate (precipitin or microprecipitin tests, Over 140 million trees of cocoa have been rouged gel diffusion assays) or agglutination of cells out in Ghana in eradication programme (Brunt (agglutination methods). In the latter, assays and Kenten 1971). In the eastern province of the are conducted on a solid surface such as on Gold Coast, 1,000,000 plants were destroyed by a microtitre plate or nitrocellulose membrane, this disease, and production was reduced from and the antigenÐantibody reaction is visualised 116,000 tons in 1936 to 64,000 tonnes of cocoa by means of a suitable detection system such in 1945. In one farm, the total production de- as an enzyme-labelled antibody. However, the creased from 30 tonnes per annum during 1926Ð use of advanced immune-diagnostic methods for 1929 to 20 tonnes in 1936Ð1939 and to only 6 the identification and detection of viruses made tonnes in 1943Ð1944, due to this disease (Pos- the detection easier, more sensitive and with nette 1945). reasonable cost (Lankow et al. 1987; Rao and Viruses also reduce yield in a number of un- Singh 2008). usual ways by affecting growth habit of their During the last three decades, enzyme- host crops. Viruses in strawberry reduced runner linked immunosorbent assay (ELISA) was the production (McGrew 1970); pox virus in plums widely used method for the detection of viruses caused 40Ð100% of fruit to drop before maturity that is highly sensitive, simple, fast and most among several cultivars (Nemeth 1986), as did importantly has the ability to quantify virus mosaic virus in apple trees (Posnette 1989). This content in plant tissue. The binding of the virus virus also was responsible for less branching in and specific antibody is made visible through young apple trees (Rebendel et al. 1979). an antibody tagged with an enzyme which can Viruses are also responsible for some unusual react with a substrate to produce a coloured, effects on crops such as greater nutrient require- water-soluble product. The first reported method ments of fruit trees and reduced coloration in was the double antibody sandwich ELISA 290 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction)

Table 9.2 Application of ELISA techniques in the detection of certain virus and phytoplasma diseases in vegetatively propagated plants Crop Virus Reference Almond Prune dwarf virus Fonseca et al. (2005) Apple Apple stem grooving virus Hassan et al. (2008a) Apple mosaic virus Bhardwaj et al. (1994) Apricot Plum pox virus Polak et al. (1995) Banana Banana bract mosaic virus Dhanya et al. (2007) Banana streak mosaic virus Thottappilly et al. (1998), Delanoy et al. (2003), Rajasulochana et al. (2008), Prakash et al. (2010) Banana bunchy top virus Geering and Thomas (1996) Cucumber mosaic virus Hu et al. (1995), Kiranmai et al. (1996), Rajasulochana et al. (2008) Black pepper Cucumber mosaic virus Sarma et al. (2001), de Silva et al. (2002), Bhat et al. (2004), Aglave et al. (2007) Piper yellow mottle virus Bhadramurthy et al. (2005) Cardamom Cardamom vein clearing virus Saigopal et al. (1992) Cassava African cassava mosaic virus Thomas et al. (1986), Malathi et al. (1988), Konate et al. (1995), Ogbe et al. (1997) Cassava X virus Martinez and Pinto (2001) Indian cassava mosaic virus Aiton and Harrison (1989), Harrison et al. (1991), Konate et al. (1995) Chickpea Chickpea chlorotic dwarf virus Kumari et al. (2004) Citrus Citrus tristeza virus Bar-Joseph et al. (1979), Cambra et al. (1991), Garnsey and Cambra (1991), Ochasan et al. (1996), Rustici et al. (2000), Avijit and Ramachandran (2002), Ahlawat and Pant (2003), Baranwal and Ahlawat (2008), Fisher et al. (2011) Citrus ring spot virus Hoa and Ahlawat (2004) Citrus tatter leaf virus Su and Tsai (1990) Grape Grape leaf roll closterovirus Hu et al. (1991) Iris Iris severe mosaic virus Kulshrestha et al. (2004), Zaidi et al. (2011) Onion Iris yellow spot virus Bulajic et al. (2009) Tobacco streak ilarvirus Sivaprasad et al. (2010) Peach Peach latent mosaic viroid Hassan et al. (2008b) Pepper Pepper yellow mottle virus de Silva et al. (2002) Potato Potato viruses de Bokx and Maat (1979), de Bokx et al. (1980), Singh and Somerville (1986), Jordan and Hammond (1991), Spiegel and Martin (1993), Barker et al. (1993), Salim Khan et al. (2003), Latvala-Kilby et al. (2009) Lilies Cucumber mosaic virus Chen et al. (2008) Sugarcane Sugarcane mosaic virus Chen et al. (1998), Rao et al. (2002a, b) Vanilla Cucumber mosaic virus Bhat et al. (2003)

(DAS-ELISA) where the antibody is bound to colour intensity, which is proportional to virus the solid phase (e.g. polystyrene microtiter plate), contents, can be measured spectrophotometri- then the test samples, enzyme-labelled antibody cally. Since the report of Clark and Adams in and the substrate are added sequentially, with 1977, many ELISA variants were reported, by unbound material removed by washing between using different enzymes or universal conjugates, steps (Clark and Adams 1977). In a positive and the successful attempts of ELISA in test, the substrate solution turns coloured, different virusÐhost combinations are presented whereas a negative test remains colourless. The in Table 9.2. 9.7 Vegetatively Transmitted Plant Virus and Virus-Like Disease Management. . . 291

The polymerase chain reaction (PCR) has a decline in yield production. Plant growers, been used as the new standard method for especially nursery men and horticulturists, detecting a wide variety of templates across have greatly contributed to the spread of these a range of scientific disciplines, including diseases inadvertently by using infected (scions virology (Mullis and Faloona 1987). The method or root stock) plant material. Because of the employs a pair of synthetic oligonucleotides catastrophic losses they cause, intensive attempts or primers, each hybridising to one strand were made to produce disease-free planting mate- of a double-stranded DNA target, with the rial. The production and distribution of virus-free pair spanning a region that will exponentially propagating materials has proved highly success- reproduced. The hybridised primer acts as ful in controlling virus diseases in many crops a substrate for a DNA polymerase, which and promises to be of wider application in others. creates a complementary strand via sequential The term ‘virus-free’ denotes material found to addition of deoxynucleotides. The process can be be free from known virus and virus-like diseases. summarised in three steps: (1) dsDNA separation The term is also misused today to label plants at temperatures above 90ıC, (2) primers that have been treated to free them from some annealing at 50Ð75ıC and (3) optimal extension of these viruses. Yet alternatives such as ‘virus at 72Ð78ıC. The rate of temperature change, the tested’ or ‘free from the following viruses :::’ length of the incubation at each temperature and are appropriate. Prof. Hollings defines virus-free the number of times each cycle are repeated and plants as ‘free from the known and specified controlled by a programmable thermal cycle. The viruses for which tests have been done’. Majority amplified DNA fragments will then be separated of the plants are raised through true seeds. More by agarose gel electrophoresis, and the bands are than two hundred and thirty-one (231) virus and visualised by staining the resulting bands with viroid diseases are seed transmitted, and no report ethidium bromide and irradiation with ultraviolet exists so far regarding the transmission of phyto- light. The specificity of PCR testing is dependent plasma diseases through true seeds. The true seed on the primer sets used. The diagnosis of certain and vegetative propagative plant materials which virus, virioid and phytoplasma diseases in some transmit viruses, their diagnosis and methods of the vegetative propagated crops by PCR and of making them disease-free are discussed in its variants is presented in Table 9.3. number of review and textbook chapters (Jones and Torrance 1985; Stace-Smith and Hamilton 1988; Roberts 1999; Jones 2000;Thresh2003; 9.7 Vegetatively Transmitted Albrechtsen 2006; Punja and De Boer 2007;Rao Plant Virus and Virus-Like and Singh 2008; Prakash and Singh 2010). Disease Management In recent years, the technique of cryotherapy by Certification Schemes of shoot tips has been used to eliminate virus and virus-like pathogens from the infected 9.7.1 Success Stories of Production germplasm of vegetatively propagated plants like of Virus-Free Plant Propagules banana and temperate fruits (Wang et al. 2009). Cryotherapy of shoot tips is a new method for Crop plants have a greater potential for improved pathogen eradication based on cryopreservation yield and quality when they are free from techniques. Cryopreservation refers to the storage harmful diseases. Stocks of the vegetatively of biological samples at ultra-low temperature, propagated crops like potato, sugarcane, cassava, usually that of liquid nitrogen (196ıC), and sweet potato, beet, onion, strawberry, blue berry, is considered as an ideal means for long-term mulberry, banana and certain ornamental plants storage of plant germplasm. In cryotherapy, have ample chances for infection with one or plant pathogens such as viruses, phytoplasmas more viruses/phytoplasmas/viroids which are and viroids are eradicated from shoot tips by multiplied continuously for many years and show exposing them briefly to liquid nitrogen. Uneven 292 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction)

Table 9.3 Application of PCR and its variants in the diagnosis of certain virus, viroid and phytoplasma diseases of vegetatively propagated crops (PCR) Crop Virus Reference Almond Prune dwarf virus Parakh et al. (1995), Fonseca et al. (2005) Apple Apple mosaic virus Choi and Ryu (2003) Apple scar skin viroid Sipathioglu et al. (2007) Apple stem pitting virus Ito et al. (2002), Kundu and Yoshikawa (2008), Hassan et al. (2008a) Apple chlorotic leaf spot virus Candresse et al. (1995) Apple proliferation Jarausch et al. (2004), Barbic and Dolla Via (Phytoplasma) (2008) Apple stem grooving virus Nemchinov et al. (1995), Kinard et al. (1996), James et al. (1997), Nickel et al. (2004), Hassan et al. (2008a) Apricot Plum pox virus Schneider et al. (2004) Prunus necrotic ring spot virus Sipathioglu et al. (2007) Alstroemeria Cucumber mosaic virus Verma et al. (2005) Banana Banana bunchy top virus Shamloul et al. (1995), Wanitchakorn et al. (1997), Selvarajan et al. (2007), Vishnoi et al. (2009), Prakash et al. (2010) Banana bract mosaic virus Thomas et al. (1997), Dassanayake (2001), Hassan et al. (2008b, 2008) Cucumber mosaic virus Singh et al. (1995), Kiranmai et al. (1998), Aglave et al. (2007) Banana streak virus Harper et al. (2002, 2004, 2010). James et al. (2011); Begonia Prunus necrotic ring spot virus Neeraj Verma et al. (2002) Black pepper Cucumber mosaic virus Bhat and Siju (2007) Piper yellow mottle virus Bhat and Siju (2007) Carrot Carrot motley dwarf virus Vereruysse et al. (2000) Cassava African cassava mosaic Alibi et al. (2008) Cassava brown streak virus Rwegasira et al. (2011) Cassava mosaic disease Berrie et al. (1997), Harrison et al. (1997), Rothenstein et al. (2006) East African cassava mosaic Alibi et al. (2008) cameron virus Indian cassava mosaic virus Makesh Kumar et al. (2005) Chrysanthemum Chrysanthemum stunt viroid Hooftman et al. (1996), Chung and Pak (2008), Zaidi et al. (2011) Tomato aspermy virus Verma et al. (2006) Tomato spotted wilt virus Fukuta et al. (2004) Citrus Citrus ring spot virus Hoa and Ahlawat (2004) Citrus yellow mosaic virus Baranwal et al. (2003, 2005), Borah et al. (2009), Ahlawat et al. (1996) Citrus exocortis viroid Duran-Vila et al. (1988), Ben-Shaul et al. (1995), Ramachandran et al. (2003), Bernard et al. (2006), Bagherian et al. (2009), Fisher et al. (2011) Citrus mosaic virus Baranwal et al. (2003) Citrus greening virus Baranwal et al. (2007),Gopaletal.(2007a, b) Citrus tristeza virus Fisher et al. (2011) Colocasia Konjac mosaic virus Padmavathi et al. (2011) Dahlia Dahlia mosaic virus Pahalawatta et al. (2007) Duranta Duranta leaf curl begomovirus Sharma et al. (2009) (continued) 9.7 Vegetatively Transmitted Plant Virus and Virus-Like Disease Management. . . 293

Table 9.3 (continued) Crop Virus Reference Fig Fig mosaic virus Walia et al. (2009) Garlic Leek yellow stripe virus Leisova-Svobodova and Karlova-Smekalova (2011) Onion yellow dwarf virus Leisova-Svobodova and Karlova-Smekalova (2011) Shallot latent virus Meenakshi et al. (2009), Leisova-svobodova and Karlova-Smekalova (2011) Gladiolus Bean yellow mosaic virus Katoch et al. (2002) Cucumber mosaic virus Raj et al. (1999, 2002) Grape vine Pierce’s disease Schaad et al. (2002) Arabis mosaic virus Ipach et al. (1992a, b) Bois noir Marzachi et al. (2003) Fan leaf virus Rowhanietal.(1993), Wetzel et al. (2002), Digiaro et al. (2007), Blahova and Pidra (2009) Flavescence doree Palermo et al. (2007), Margaria et al. (2007), Gori et al. (2007), Hren et al. (2007), Margaria et al. (2009) Grapevine fleck complex virus El-Beaino et al. (2001), Kopecky et al. (2004), Habili and Bogacz (2006) Grape vine leaf roll Routh et al. (1998), Acheche et al. (1999), Ling closterovirus et al. (2001), Niu et al. (2004), Faggioli and La Starza (2006), Osman et al. (2007), Ling et al. (2008), Margaria et al. (2009) Grapevine viroid Rezaian et al. (1988), Staub et al. (1995), Wah and Symons (1997) Grapevine virus A Pacifico et al. (2009) yellow speckle viroid Nakaune and Nakano (2006) Iris Iris mild mosaic virus Kulshrestha et al. (2005); Zaidi et al. (2011) Lily Phytoplasma Bertaccini et al. (2002) Onion Iris yellow spot virus Bulajic et al. (2009), Sivamani et al. (2009) Tobacco streak ilarvirus Sivaprasad et al. (2010) Orchids Cymbidium mosaic virus Wong and Seoh (2008) Peach, plum Plum pox virus Olmos et al. (1996), Candresse et al. (1998) Peach, plum, cherry Peach latent mosaic viroid Shamloul et al. (1995), Giunchedi et al. (1997), Hadidi et al. (1997) Pear Pear rusty skin Hadidi and Yang (1990), Zhu et al. (1995) Pear Pear decline (Phytoplasma)Lorenzetal.(1995), Davies et al. (1995) Pear and apple Apple scar skin Hadidi and Yang (1990), Hadidi et al. (1991) Pepper Pepper yellow mottle virus de Silva et al. (2002) Potato Potato viruses Barker et al. (1993), Hadidi et al. (1993), Spiegel and Martin (1993), Singh and Singh (1996), Singh et al. (1996), Schoen et al. (1996), Latvala-Kilby et al. (2009), Crosslin and Hamlin (2011) Potato spindle tuber viroid Singh et al. (2003), Boonham et al. (2004), Singh et al. (2006), Crosslin and Hamlin (2011) Prunus Prunus necrotic ring spot virus Spiegeletal.(1996), Rosner et al. (1997), Navarro et al. (1998), Spiegel et al. (2008) Raspberry Bushy dwarf virus Barbara et al. (1995) Sour cherry Plum pox virus Nemchinov et al. (1994) Stone fruits Plum pox virus Thomidis and Karajiannis (2003), Mavrodieva and Levy (2004) (continued) 294 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction)

Table 9.3 (continued) Crop Virus Reference Strawberry Strawberry mild yellow edge Kreuziger et al. (1995), Hadidi et al. (1991) potexvirus Sugar beet Beet mosaic virus Glasa et al. (2000, 2003) Beet necrotic yellow vein virus Kruse et al. (1994), Henry et al. (1995) Beet mild curly top virus Chen et al. (2008) Sugarcane Grassy shoot (phytoplasma) Rao et al. (2003), Srivastava et al. (2006), Marcone and Rao (2008) Sugarcane Fiji disease virus Smith and Van de Verde (1994), James et al. (2001) Sugarcane yellow leaf Gaur et al. (2008) phytoplasma Sugarcane mosaic virus Smith and Van de Verde (1994), Yang and Mirkov (1997), Gaur et al. (2003), Rao et al. (2006), Zhang et al. (2008), Subba Reddy et al. (2011) Sugarcane streak mosaic virus Smith and Van de Verde (1994), Hema et al. (1999, 2003), Rao et al. (2006), Hema et al. (2008), Subba Reddy et al. (2011) Sweet potato Sweet potato viruses Aritua et al. (2005) Sweet orange Citrus tristeza virus Cambra et al. (2000) Taro Taro bacilliform virus Rob Harding (2008) Tobacco Tomato spotted wilt virus Mumford et al. (1996), Pappu et al. (1998) Vanilla Cucumber mosaic virus Madhubala et al. (2005) Bean common mosaic virus Bhadramurthy and Bhat (2009) distribution of viruses and obligate vasculature- potato chlorotic stunt virus, Sweet potato little limited microbes in shoot tips allows elimination leaf phytoplasma and huanglongbing bacterium from the infected cells by injuring them with the causing ‘citrus greening’). Cryopreservation cryo-treatment and regeneration of healthy shoots protocols have been developed for a wide variety from the surviving pathogen-free meristematic of plant species, including agricultural and cells. Thermotherapy followed by cryotherapy horticultural crops and ornamental plants, and of shoot tips can be used to enhance virus can be used as such or adjusted for the purpose eradication. Cryotherapy of shoot tips is easy to of cryotherapy (Wang et al. 2009). implement. It allows treatment of large numbers The production of virus-free stock material of samples and results in a high frequency of depends on the following activities: (1) recog- pathogen-free regenerates. Difficulties related to nition of the virus and phytoplasma diseases by excision and regeneration of small meristems studying their characteristics, (2) development of are largely circumvented. To date, destructive reliable indexing methods to retrieve any exist- pathogens including viruses in banana (Musa ing healthy stock, (3) establishment of clones spp.), Citrus spp., grapevine (Vitis vinifera), of virus-free foundation stocks by employing Prunus spp., raspberry (Rubus idaeus), potato different techniques and (4) propagation of the (Solanum tuberosum) and sweet potato (Ipomoea disease-free plants under maximum hygiene and batatas) have been eradicated using cryotherapy. distribution of the planting material. If the nu- These pathogens include nine viruses (Banana cleus of the healthy stock obtained through the streak virus, Cucumber mosaic virus, Grapevine methods described earlier, stocks can be multi- virus A, Plum pox virus, Potato leaf roll virus, plied and kept healthy for an indefinite period. Potato virus Y, Raspberry bushy dwarf virus, Plant propagators by carefully selecting the best Sweet potato feathery mottle virus and Sweet looking and excellently performing plants for 9.7 Vegetatively Transmitted Plant Virus and Virus-Like Disease Management. . . 295 propagating new plants, may knowingly or un- guidelines for maintaining the health of mother- knowingly avoid many of these diseases. This stocks and propagated material, (6) guidelines visual selection process helps in diagnosis of dis- for labelling planting material for traceability, eased plants based on external visual symptoms. (7) guidelines for national and international dis- tribution of planting material (8) guidelines for infrastructure and technical competence and (9) guidelines for supervision and monitoring com- 9.7.2 Certification Schemes pliance by the producers. The systems of rules, regulations and check Certification schemes have been established to inspections designed for the production of comply with official standards/regulations set virus-free material constitute crop certification by the national and international authorities to schemes. Selection is a basic step in programme guarantee the quality of propagative material for clean stock production. It determines that to trueness-to-type (genetic purity) and absence a specific clone processes the desirable growth from specified pathogens in the micropropagated and yield characteristics of the cultivar and that plants (Golino and Savino 2005). Usually, it is a the cultivar is correctly identified. Selection is domestic programme consisting of multiplication the responsibility of horticulturists cooperating and distribution of plant material that involves a in the clean stock programme. It will be combination of activities; the process basically effective when all the virus and phytoplasma involves assessing the risks (pathogens and diseases are recognised. Indexing tests suggest pests), selection of clean-planting material, virus that many cultivars are wholly infected with testing, micropropagation and tests for genetic one or more viruses and a good success is fidelity. A certificate is only provided to the achieved in eliminating these diseases from the plants that are produced as per the directive of infected stock by the methods like thermotherapy, the scheme. chemotherapy and meristem tip culture. These Certification schemes depend on the objective methods have enabled plants to be freed from of the programme and vary considerably depend- more than 200 viruses. Disease recognition, ing on crops and countries. Initially, certification indexing and therapy are the responsibility of schemes originated as a method to control virus plant virologists. The recent reviews catalogue diseases in seed potatoes and tree crops (Anony- for more success than failures. These three mous 1992). In the absence of effective control methods are discussed in detail in the subsequent strategies and host resistance, cultivation of virus- chapters. Some of the outstanding examples free planting material has become the choice for of the crops where indexing and certification growers and vigorously promoted by the public schemes have helped the rehabilitation of the and private agencies associated with welfare of crop industry are citrus bud wood certification, agriculture sector. Certification scheme usually stone fruit virus diseases certification, strawberry involves several components to ensure success of certification, clear stock programme of grapes, the programme (Hollings 1965; Meijneke 1982; avocado certification schemes, etc. Even for the NCS-TCP 2008). Some of the key components tuber crops like potato, sweet potato and beet are (1) standards that clearly define the pur- root, the certification schemes are well worked pose of certification and protocols/guidelines for out and are being implemented wherever the producing certified planting material, (2) guide- crops are grown. The quality of these vegetative lines for selection of mother-stocks to be used in planting material depends on its trueness to the micropropagation programme, (3) availability cultivar type, its vigour and health. Each of these of reliable indexing methods for the detection characters are amenable to a certain extent to of regulated and unregulated quarantine pests control by certification, which may be regarded and pathogens, (4) protocols and procedures for as an administrative means of quality control production of virus-free planting material, (5) of planting materials. Some of the widely 296 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction) adopted certification schemes are (1) cassava the genetic diversity of crop gene pools and to seed certification scheme, (2) banana production the development of new concepts to overcome certification, (3) citrus budwood certification, these problems. In all three fields, viruses play an (4) strawberry certification, (5) certification important role (Diekmann 1998). of grapevine planting material, (6) EMLA scheme for virus-free fruit trees, (7) potato seed certification, (8) sugar beet stickling certification, (9) seed programmes for sugarcane, (10) EPPO’s 9.9 Conclusion certification schemes for ornamental plants and (11) bulb inspection service scheme. More details The seed certification schemes for vegetative on the production of virus-free certified crops can propagated plants like apple, peach, pear, plum, be obtained from some of these review articles grape and sugarcane are operating successfully (Calavan et al. 1970; Goheen 1989; Lazar et al. in various parts of the world. Presently sufficient 2002; Rowhani et al. 2005). disease-free plant material which was tested by molecular methods is being distributed. As new pathogens and new strains for the existing 9.8 IPGRI’S Role in Controlling viruses are continuously evolving, periodically Virus Diseases plant materials are to be evaluated, and there in Fruit Germplasm should be close cooperation between plant breeders, propagators and growers for successful The International Plant Genetic Resources Insti- implementation of the schemes. tute (IPGRI) is one of the 16 international agri- cultural research centers (IARCs) of the CGIAR (Consultative Group on International Agricul- References tural Research). It was established in 1974 as the Acheche H, Fattouch S, M’hirsi S, Marzouki N, Mar- International Board for Plant Genetic Resources rakchi M (1999) Use of optimized PCR methods for (IBPGR) and has its headquarters in Rome, Italy. the detection of GLRaV3: a closterovirus associated IPGRI’s mandate is to advance the conservation with grapevine leafroll in Tunisian grapevine plants. and use of genetic resources for the benefit of Plant Mol Biol Rep 17:31Ð42 Aglave BA, Krishnareddy M, Patil FS, Andhale MS present and future generations. (2007) Molecular identification of a virus causing IPGRI’s activities in the field of germplasm banana chlorosis disease from Marathwada region. Int health can be divided into three categories. J Biotechnol Biochem 3:13Ð23 The first activity relates to strategic research Agrious GN (1990) Economic considerations. In: Man- dahar CL (ed) Plant viruses, vol II, Pathology. CRC with the objective of developing techniques Press, Boca Raton, pp 1Ð22 that will improve the safety and efficiency of Agrious GN (2005) Plant pathology. Academic, New York germplasm movement and involves essentially Ahlawat YS, Pant RP (2003) Major virus and virus- biotechnology. The second, and perhaps most like diseases of citrus in India, their diagnosis and management. Ann Rev Plant Pathol 2:447Ð474 obvious, activity consists mainly of assembling Ahlawat YS, Varma A, Pant RP, Shukla A, Lockhart and disseminating information relating to quaran- BEL (1996) Partial characterization of a Badnavirus tine. In this area, IPGRI closely collaborates with associated with citrus yellow mosaic disease in In- the Plant Protection Service of FAO (Food and dia. Thirteenth IOCV conference, Riverside, USA, pp 208Ð217 Agriculture Organization of the United Nations). Aiton MM, Harrison BD (1989) Monoclonal antibodies to The third activity concerns the phytosanitary Indian Cassava mosaic geminivirus (ICMV). Report of aspects of gene bank management. In this area, the Scottish crop research institute for 1988, p 175 IPGRI contributes to improving the awareness Albrechtsen SE (2006) Testing methods for seed transmit- ted viruses: principles and protocols. CABI Publish- of curators and other scientists working with ing, Wallingford, pp 1Ð268 germplasm of the deleterious effect of seed Alibi OJ, Ogbe FO, Bandyopadhyay R, Lava Kumar P, transmitted pathogens in the conservation of Dixon AGO, Hughes Jd’A, Naidu RA (2008) Alternate References 297

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AIPUB, Trichus, pp 431Ð434 Routh G, Zhang YP, Saldarelli P, Rowhani A (1998) Shamloul AM, Minafra A, Hadidi A, Giunchedi L, Use of degenerate primers for partial sequencing and Waterworth HE, Allam EK (1995) Peach latent RT-PCR based assays of grapevine leafroll-associated mosaic viroid: nucleotide sequence of an Italian viruses 4 and 5. Phytopathology 88:1238Ð1243 isolate, sensitive detection using RT-PCR and Rowhani A, Chay C, Golino DA, Falk BW (1993) geographic distribution. Acta Hort 386:522Ð530 Development of polymerase chain reaction technique Sharma A, Shankarappa KS, Rangaswamy KT, Dubey for the detection of grapevine virus in grapevine RK, Maruthi MN (2009) First record of duranta leaf tissue. Phytopathology 83:749Ð753 curl, a begomovirus associated disease in India. Indian Rowhani A, Uyemoto JK, Golino DA, Martelli GP (2005) J Virol 20:88Ð89 Pathogen testing and certification of Vitis and Prunus Singh M, Singh RP (1996) Factors affecting detection species. Annu Rev Phytopathol 43:261Ð278 of PVY in dormant tubers by reverse transcription Rustici G, Accoto GP, Noris E, Masenga V, Luisoni polymerase chain reaction and nucleic acid spot E, Milne RG (2000) Indian citrus ringspot virus: hybridization. J Virol Methods 60:47Ð57 a proposed new species with some affinities to Singh BP, Somerville TH (1986) Factors effecting the potex-, Carla, foves- and allexi viruses. Arch Virol detection of potato virus Y in tubers by enzyme-linked 145:1895Ð1908 immunosorbent assay (ELISA) Ind. J Plant Pathol Rwegasira GM, Rey MEC, Nawabu H (2011) Approaches 4:75Ð81 to diagnosis and detection of cassava brown streak Singh Z, Jones RHC, Jones MGK (1995) Identification virus (Potyviridae: Ipomovirus) in field grown of cucumber mosaic virus subgroups Ð 1 isolates from cassava crop. AJFAND Scholarly, Peer Reviewed banana plants affected by infection chlorosis using 11(3):4739Ð4756 RT-PCR. Plant Dis 79:713Ð716 Saigopal DVR, Naidu R, Joseph T (1992) Early detection Singh RP, Kurz J, Boiteau G (1996) Detection of of ‘Katte’ disease of small cardamom through Enzyme stylet borne and circulative potato viruses in aphids Linked Immunosorbent Assay (ELISA). J Plant Crops by duplex reverse transcription polymerase chain 20(Suppl):73Ð75 reaction. J Virol Methods 59:189Ð196 Salim Khan M, Hoque MI, Sarker RH, Muehlbach H-P Singh RP, Ready KFM, Nie X (2003) Biology. In: (2003) Detection of important plant viruses in In vitro Hadidi A, Flores R, Randles JW, Semancik JS regenerated potato plants by double antibody sandwich (eds) Viroids. CSIRO Publishing/Science Publishers, method of ELISA. Plant Tissue Cult 13(1):21Ð29 Collingwood/Enfield, pp 30Ð48 304 9 Plant Virus Transmission Through Vegetative Propagules (Asexual Reproduction)

Singh RP, Dilworth AD, Singh M, Babcock KM reactivities with other whitefly transmitted gemini (2006) An alkaline solution simplifies nucleic acid viruses. J Gen Virol 67:2739Ð2748 preparation for RT-PCR and infectivity assay of Thomas JE, Geering ADW, Gambley CF, Kessling AF, viroids from crude sap and spotted membrane. J Virol White M (1997) Purification, properties and diagnosis Methods 132:204Ð211 of banana bract mosaic potyvirus and its distinction Sipathioglu HM, Ocak M, Usta M (2007) Comparison of from abaca mosaic potyvirus. Phytopathology three conventional methods for the detection of plant 87:698Ð705 virus/viroid RNAs from heat dried Ð high phenolic Thomidis T, Karajiannis I (2003) Using ELISA and PCR host leaves. Asian J Plant Sci 6(1):102Ð107 to test the potential for spread of plum pox virus by Sivamani S, Krishnaveni S, Usha ZB, Ravi KS (2009) seeds of different stone fruits cultivars. NZ J Crop Efficient mechanical transmission of Iris yellow spot Hort Sci 31:69Ð72 virus (IYSV) to onion and their detection by tissue Thottappilly G, Dahal G, Lockhart BEL (1998) Studies blot immunoassay (TBIA). J Insect Sci 10:166 on a Nigerian isolate of banana streak badnavirus: I. Sivaprasad Y, Bhaskara Reddy BV, Rekha Rani K, Raja Purification and enzyme-linked immunosorbent assay. Reddy K, Sai Gopal DVR (2010) First report of Ann Appl Biol 132:253Ð261 Tobacco streak ilar virus infecting onion (Allium Thresh JM (2003) Control of plant virus diseases in cepa). BSPP New Dis Rep 22:17 sub-Saharan Africa: the possibility and feasibility of Smith GR, Van de Verde R (1994) Detection of sugarcane an integrated approach. Afr Crop Sci J 11(3):199Ð223 mosaic virus and Fiji disease virus in diseased Thresh JM, Fargette D, Otim-Nape GW (1994) Effects sugarcane using polymerase chain reaction. Plant Dis of African cassava mosaic geminivirus on the growth 78:557Ð561 and yield of cassava. Trop Sci 34:43Ð54 Spiegel S, Martin RR (1993) Improved detection of Torrance L, Jones RAC (1981) Recent developments in potato leaf roll virus in dormant potato tubers and seedofical methods suited for use in routine testing for microtubers by the polymerase chain reaction and plant viruses. Plant Pathol 30:1Ð24 ELISA. Ann Appl Biol 122:493Ð500 Vereruysse P, Gibbs M, Tirty L, Hofte M (2000) RT-PCR Spiegel S, Scott SN, Bowman-Vance V, Tam Y, Gali- using redundant primers to detect the three viruses akparov MN, Rosmer A (1996) Improved detection associated with carrot motley dwarf disease. J Virol of Prunus necrotic ringspot virus by the polymerase Methods 88(2):153Ð161 chain reaction. Eur J Plant Pathol 102:681Ð685 Verma N, Hallan V, Ram R, Zaidi AA (2002) Detection of Spiegel S, Tam Y, Maslenin Y, Kolber M, Nemeth M, Prunus necrotic ringspot virus in begonia by RT-PCR. Rosner A (2008) Typing Prunus necrotic ringspot virus Plant Pathol 51(6):800 isolates by serology and restriction endonuclease anal- Verma N, Singh AK, Singh L, Raikhy G, Kulshrestha ysis of PCR products. Ann Appl Biol 135:395Ð400 S, Singh MK, Hallan V, Ram R, Zaidi AA (2005) Srivastava S, Singh V, Gupta PS, Baitha A (2006) Nested Cucumber mosaic virus (CMV) infecting Alstroemeria PCR assay for detection of sugarcane grassy shoot hybrids in India. Aust Plant Pathol 34:119Ð120 phytoplasma in the leafhopper vector Deltocephalus Verma N, Kumar K, Kulshrestha S, Raikhy G, Hallan vulgaris: a first report. Phytopathology 55:25Ð28 V, Ram R, Zaidi A, Garg ID (2006) Detection and Stace-Smith R, Hamilton RI (1988) Inoculum thresh holds molecular characterization of a Tomato aspermy virus of seedborne pathogens: viruses. Phytopathology isolate infecting chrysanthemums in India. Acta Hort 78:875Ð880 722:41Ð53 Staub U, Polivka H, Herrmann JV, Gross HJ (1995) Vishnoi R, Raj SK, Prasad V (2009) Molecular Transmission of grapevine viroids is not likely to occur characterization of an Indian isolate of Banana bunchy mechanically by normal pruning. Vitis 34:119Ð123 top virus based on six genomic DNA components. Su Hong-Ji, Tsai MC (1990) Distribution and detection of Virus Genes 38:334Ð344 citrus tattenleaf virus by ELISA test with monoclonal Wah Y, Symons RH (1997) A high sensitivity RT-PCR antibodies. In: Aubert B, Tontyaporn S, Buangsuuon assay for the diagnosis of grapevine viroids in D (eds) Proceedings of the Asian Pacific international field and tissue culture samples. J Virol Methods conference on citriculture, pp 171Ð174 63:57Ð69 Subba Reddy CV, Sreenivasulu P, Sekhar G (2011) Walia JT, Salem NM, Falk BW (2009) Partial sequence Duplex Ð immunocapture RT Ð PCR for detection and and survey analysis identify a multipartite Ð negative discrimination of two distinct potyviruses naturally severe RNA virus associated with fig mosaic. Plant infecting sugarcane (Saccharum spp. hybrid). Indian J Dis 93:4Ð10 Exp Biol 49:68Ð73 Wallace JM (1959) An unusual kind of seed transmission Terry ER, Hahn SK (1980) The effect of Cassava mosaic of the avocado sunblotch virus. Proc 9th Int Bot Congr disease on growth and yield of local and an improved Montreal 2:421 variety of Cassava. Trop Pest Manage 26:34Ð37 Wang QC, Panis B, Engelmann F, Lambardi M, Thomas JE, Massalski PR, Harrison BD (1986) Valkonen JPT (2009) Cryotherapy of shoot tips: Production of monoclonal antibodies to African a technique for pathogen eradication to produce cassava mosaic virus and differences in their healthy planting materials and prepare healthy plant References 305

genetic resources for cryopreservation. Ann Appl Biol of plant viruses. Studium Press LLC, Houston, pp 154(3):351Ð363 127Ð139 Wanitchakorn R, Harding RM, Dale JL (1997) Banana Yang ZN, Mirkov TE (1997) Sequence and relationships bunchy top virus DNA-3 encodes the viral coat of sugarcane mosaic and sorghum mosaic virus strains protein. Arch Virol 142:1673Ð1680 and development of RT-PCR-based RFLPs for strain Waterworth HE, Hadidi A (1998) Economic losses due discrimination. Phytopathology 87:932Ð939 to plant viruses. In: Hadidi A, Khetarpal RK, Ko- Zaidi AA, Hallan V, Raikhy G, Singh AK, Ram R (2011) ganezawa H (eds) Plant virus diseases control. Ameri- Viruses of ornamental plants in India Current status can Phytopathological Society Press, St. Paul, pp 1Ð13 and future prospects. Acta Hort 901:67Ð76 Wellman FL (1954) Some important diseases of Cacao. Zhang M-Q, Rao GP, Gaur RK, Ruan M-H, Singh FAO Plant Protect Bull 2:129Ð133 M, Sharma SR, Singh A, Singh P (2008) Sugarcane Wetzel T, Jardak R, Meunier L, Ghorbel A, Reustle mosaic virus. In: Rao GP, Paul Khurana SM, Lenardon GM (2002) Simultaneous RT/PCR detection and SL (eds) Characterization diagnosis and management differentiation of arabis mosaic and grapevine fanleaf of plant viruses, vol I, Industrial crops. Studium Press nepoviruses in grapevine with a single pair of primers. LLC, Houston, pp 111Ð144 J Virol Methods 101:63Ð69 Zhu SF, Hadidi A, Hammond RW, Yang X, Hansen AJ Wong SM, Seoh ML (2008) Detection of Cymbidium (1995) Nucleotide sequence and secondary structure mosaic virus and Odontoglossum ringspot virus using of pome fruit viroids from dapple apple diseased a single pair of PCR primers. Chapter 7. In: Rao GP, apples, pear rusty skin diseased pears, and apple scar Valverde RA, Dovas CL (eds) Techniques in diagnosis skin symptomless pears. Acta Hort 386:554Ð559 Future Strategies and Conclusions 10

Abstract The virus and virus-like diseases are transmitted through true seed (sexual) in certain crops and through vegetative propagules like tuber, rhizome, bulb, suckers and bud sticks in some other crops. For framing suitable virus management measures, the disease diagnosis is prerequisite. Early detection of virus and virus-like diseases is critical to prevent or minimise the spread of the virus diseases. If virus and virus-like symptoms are suspected, then it is critical to confirm the presence of it by following a proper identification process through a recognised diagnostic laboratory by specialised techniques like ELISA and PCR. When the identification of the virus is confirmed, specific management strategies must be implemented immediately. For almost all crops against major virus diseases, definite management measures including production of virus-free planting materials are well worked out and are implemented in day-to-day agricultural operations. As single control measure will not give maximum disease management, IDM methods which are sound and environmentally acceptable are widely applied. The word integrated in IDM initially referred to the simultaneous use or integration of many number of tactics in combination that are focused on maintaining the disease below its economic threshold level. Chemical control is generally compatible with host resistance. Thus, a management strategy integrates one or several compatible tactics in to a single package. When diverse virus management measures that act in different ways are combined and used together, their effects are complimentary resulting in far more effective overall control. Such experiences have lead to the development of integrated management concepts for seed-borne virus diseases that combine available host resistance, cultural, chemical and biological control measures. Selecting the ideal mix of measures for each pathosystem and production situation requires detailed knowledge of the epidemiology of the causal virus and mode of action of each individual management measure so that diverse responses can be devised to meet the unique features of each of the different scenarios considered.

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6 10, 307 © Springer India 2013 308 10 Future Strategies and Conclusions

and specific assays that have opened the doors 10.1 Introduction to a greater use of these tests for detecting seed-transmitted viruses. These assays will help Although viruses have been recognised over the tree growers, crop consultants and plant several decades, they were considered not to be protection personnel not to rely excessively seed transmitted before 1910 and subsequent on symptomatology and/or time-consuming studies revealed that percentages of seed diagnostic procedures and permit early detection transmission varied depending upon virusÐhost of viruses. The advantages of having different combinations. Furthermore, new viruses are methods are obvious as they not only result in continuously being discovered and a portion increased sensitivity but also expand the range of of them are seed transmitted. To date, more than applications. Each detection procedure is directed 231 viruses are found transmitted through seed in towards a special characteristic of the virus different plant species. It would not be surprising transmissibility, antigenic properties, nucleic acid that with further research this number may characteristic and sequences. These detection increase more than one-fourth of the recognised techniques will increase the reliability of virus- plant viruses to be seed transmitted in at least one free seed production and also in screening of of their hosts. germplasm. At present, there is no universal strategy The latest developments in molecular biol- for the detection of plant viruses. The main ogy/biotechnology have an impact on seed health methods currently in use for the detection and testing, especially for seed-transmitted viruses. diagnosis of seed-transmitted viruses can be These new methods are generally much more grouped as biological, physical, serological rapid and sensitive than the older techniques. and molecular. Bioassays based on knowledge But the main disadvantages with these techniques of symptoms produced and the diversity of are more complicated and often require the use experimentally infectable hosts provide an of sophisticated and expensive laboratory facil- understanding for detection or diagnosis of ities. Nevertheless, seed testing stations having virus/viroid diseases. The biological methods responsibility for indexing a large number of seed are labour intensive and require a lot of space. lots for economically important viruses may find Physical methods like electron microscopy of it advantageous to adopt these new procedures. seed extracts are more rapid and frequently allow The increasing knowledge along with additional tentative identification which can be confirmed adaptation and modification makes these methods by other criteria. However, these methods require more suitable for routine seed health testing. costly equipments. The serological methods However, they should be viewed as management overcome the disadvantages of biological tools in conjunction with other diagnostic proce- and physical methods since they use only a dures, crop acquaintance, biology of the virus and small proportion of the information encoded the ecology of the vector as well as the disease. by the virus. In serology, a diverse array of tests exploiting antigenÐantibody recognition There is an important distinction between are available to detect viruses even at lower viruses that are retained on the seed surface or in concentrations and in few cases, it also helps in the endosperm or perisperm from those that infect detection of localised infections. Enzyme-linked embryo. Embryonic infection virtually assures immunosorbent assays using chromogenic, transmission in seedlings, whereas viruses fluorogenic, chemiluminescent or radioactive occurring in other parts of the seed do not result substrates are some of the choices. Molecular into cent percent seedling infection. When the methods involving nucleic acid hybridisation, virus survives as a contaminant in or on the seed, which can use whole or selected parts of the viral it may be eliminated or significantly reduced by genome, have been developed. heat therapy or chemical treatments. On the other Recent developments in molecular detection hand, embryonic infection of viruses cannot be technology resulted in more convenient, effective prevented without loss in viability of seed. 10.1 Introduction 309

Seed transmission provides an ideal starting breeders have introduced a considerable number point for the establishment of a disease in field of resistance genes into crops, but unfortunately crops. Firstly, it enables infection to occur at the pathogen often has a remarkable ability to the earliest possible time in the development adapt through gene-for-gene type of resistance. of the young seedling, a factor that frequently A prerequisite to the breeding for durable governs the severity of virus infection in an in- resistance against seed-transmitted viruses is dividual plant. Secondly, seed infection results through understanding of the hostÐparasite in individual infected seedlings being scattered relationships. With this the breeders can put widely throughout a crop field, with each infected their efforts to develop methods of controlling seedling providing a virus reservoir for subse- virus disease epidemics by means of non-race- quent secondary spread by vectors. specific resistance. Indeed, durable resistance Certain changes have occurred in recent years, seems more readily achievable to viruses than to due to advancements in computer applications many fungal pathogens. This is possibly because and their fruitful collaboration between virus epi- although viruses have considerable opportunities demiologists and bioinformaticians. Now, only for mutations, the nature of the genome limits the a few simulation models have been constructed recombination and hence the ability to combine for predicting seed transmission and the rate a resistance-breaking gene array with the overall of spread in fields. However, further research fitness for survival. is necessary to connect links so that simulation Several breeding programmes for resistance will then be closer to the real spread. Validation to virus or to its vector transmission have been of models under field conditions may serve to developed, but so far very limited success has improve existing models and suggest the factors been achieved. Availability of new technologies that are still incomplete. The prospects of suc- in molecular biology and genetic engineering cess in forecasting largely depend on the level now makes it possible to explore new approaches of understanding of insectÐvector relationships, in plant virus management. Now that transforma- influence of environmental stimuli for activation tion of a number of plants has become routine, and role of atmospheric system in their transport. it is possible to manipulate some of the defence Since definite viricides are not yet available, and attenuation mechanisms to improve the virus an effective control of seed-transmitted virus dis- resistance properties in various crops. So far, two eases is possible by development and implemen- strategies Ð one based on coat protein genes and tation of seed certification schemes along with the other as satellite RNA Ð have been proved destroying the sources of infections like weed successful in experimental situations. The use and volunteer plants, roguing the infected plants of coat protein gene is quite promising as it wherever disease incidence is lower and control- is effective for several viruses in different crop ling the virus spread through oils and/or insec- plants. ticides in reducing vector population. In case of The satellite RNA strategy still needs further infection among fruit crops, sometimes the virus development in removing the hazards associated may be latent and propagation from the infected with the use of certain strains of satellite RNA of plant is done inadvertently. Since some of the CMV. The uses of coat protein and satellite RNA fruit tree viruses are seed transmitted, seedlings sequence have created novel type of resistance used as root stocks serve as sources of inoculum. genes, since they involve non-plant sequences Hence, in the production of nursery stocks, it expressed from the plant genome. Additional is essential that both scion and root stocks be type of novel mechanisms involves the use of derived from trees that are indexed and known to antisense RNA. Integration of a copy of short be virus-free. sequence of the viral genome in the opposite The spread of the disease if not arrested by orientation would lead to transcription in a mic- any of the measures cited above, crop resistance RNA (antisense RNA) which will be able to becomes the only permanent solution. Plant interfere with the infecting virus RNA. 310 10 Future Strategies and Conclusions

Natural resistance genes have not yet been In recent years ISEM, ELISA, SPRIA, the targets of thinking by molecular biologists, cDNA and RTÐPCR techniques are being because of their inaccessibility. The gene that widely used for seed-transmitted virus detection. codes resistance against Cowpea mosaic virus The combined use of simple biological tests (CPMV) in cowpea (cv. Arlington) may be one and newly developed serological detection of the first resistance genes to be isolated. With techniques would be of immense value for other types of resistance gene, the specificity is reliable virus detection in the imported seed not necessarily in the action of the resistance lots. Developing countries need to cooperate gene but in recognition between the resistance with developed countries at a regional and gene product and the virus. It may be possible, global level. An institute which can provide therefore, to exploit these nonspecific resistance the necessary materials, expertise, advice and mechanisms through genetic engineering. training on the application of these tests for At present, new methods for the analysis of quarantine inspections needs to be set up. plant viral genomes are exemplified by the anal- ysis of avirulence genes in TMV. The informa- tion gained from the application of these new 10.2 Plant Virus Management methods is likely to generate new types of resis- by Integrated Approach tance mechanism that may be effective against a wide range of viruses. More and more novel Excellent information has been generated in developments are likely to emerge through the the USA against flare up of Peanut stripe use of biotechnological techniques. This new area virus (PStV) in peanuts. This disease is seed of research is already well developed enough transmitted to a tune of 37% and spreads for single genes to be transferred at least in through efficient vectors like A. craccivora and dicotyledons but poses some difficulties in han- M. persicae under field conditions. This virus dling monocotyledonous plants. However, it is disease is currently regarded as predominantly likely that suitable methods will soon be devised affecting the peanut production in Southeast to overcome these problems, which opens more Asia. In India, only during 1987, PStV was options for virus management. noticed among some peanut entries of AICORPO Intensive researches have been carried out trials (Prasada Rao et al. 1988). Its establishment involving chemicals, sometimes with the combi- will become a threat to peanut production nation of irradiation or heat, and elimination of and additionally pose serious problems in the viruses from the infected seed without loss of exchange of peanut germplasm. In order to viability, but in many cases, the control obtained combat the spread of this disease, a collaborative have not been suitable. project was jointly made by ICRISAT, NBPGR There is a consistent exchange of germplasm and DOR (Hyderabad). Regular monitoring was throughout the world. In order to ensure made to destroy the entries showing symptoms. healthy germplasm stocks, genetic resources In this connection, the suggestions made by should adopt strict quarantine procedures while Demski and Lovell (1985) for managing PStV introducing new seed accessions that may carry in peanut hold good for new virus diseases seed-transmitted viruses. Rigorous application inadvertently introduced into a country and of quarantine measures as discussed in Chap. 8 consist of: would also help to restrict the virus introductions 1. Only certified seed should be used for planting through seed into countries where they are not in research plots. prevalent. Therefore, methods used at quarantine 2. The seed in the experimental trials at the stations should be rapid, reliable and sensitive first time of disease appearance should be but at the same time simple too to undertake and processed or sold for consumption and not to provide desirable test results. be used for sowing, except in breeding tests. 10.2 Plant Virus Management by Integrated Approach 311

3. All the residual seed and debris should be Successful virus management of TSWV removed from the harvesting place and trans- in tomato, watermelon and potato; PStv in porting equipments. groundnut; sunflower necrosis disease (TSV) in 4. All the research plots containing plants sunflower and CMV in muskmelon was achieved inoculated with viruses should be grown by integrated approach. It is clear that in all the under screen cages, with further precautions above-cited virusÐhost combinations, the disease to prevent spread and should be planted only management was possible through integrating at experimental stations. two to three methods of virus management. 5. Breeders should release the tested seed which At present, integrated management of seed- was found free from all the viruses. transmitted viruses like Lettuce mosaic in lettuce, 6. The breeding plots of the crop in question Zucchini yellow mosaic in vegetable cucurbit and should be isolated from other research plots to CMV and BYMV in lupins was well worked out, the extent practically possible. and similar approaches have to be extended to the 7. Infected seed, except breeder’s seed, should be other seed-transmitted virus diseases which are destroyed either by burning or autoclaving. causing heavy economic losses. Generally no single procedure offers maxi- From the experimental data for the man- mum virus control. A rational combination of agement of virus and virus-like diseases in procedures weighing the efficacy of one pro- tropics, different crops have given partial virus cedure against others and utilising their sup- management by the application of one of plementary effects is the strategy for an inte- the measures like use of cultural practices or grated management programme, and encourag- utilisation of disease-free planting material or ing results have been obtained to combat virus application of insecticides/oils in vector control. diseases. Coutts et al. (2011) have minimised Since the pathologists/virologists/entomologists losses caused by Zucchini yellow mosaic virus felt that no single strategy will help in in vegetable cucurbit crops in tropical, subtrop- achieving maximum virus and virus-like disease ical and Mediterranean environment through the management, research was also carried out by combination of cultural methods and host re- embracing multidisciplinary strategy indexing sistance. Earlier, Lecoq and Pitrat (1983)have virus and vector identification, ecology and also successfully reduced the spread of CMV epidemiology of virus and vector, chemical in muskmelon by using an integrated approach methods, genetic methods and implementation of through use of resistant cultivars and weed con- exclusion and eradication techniques along with trol and by plastic mulching. Since virus-free phytosanitary measures, which is nothing but seed production has paramount importance, one multidisciplinary approach and is being named should apply the general virus control princi- as integrated disease management (IDM). ples discussed earlier for preventing reinfection In entomology, the term ‘integrated pest as far as possible. In the German Democratic management’ (IPM) concept was being used Republic, LMV was successfully managed by which combines and integrates biological and using an integrated approach comprising some chemical control measures for most effective of the techniques discussed earlier (Vetten 1984; management of different pests and diseases; Carroll et al. 1990). Integrated management of similarly, the pathologists have coined the Soybean mosaic virus in soybean was success- technology as ‘integrated disease management’ fully achieved by following host plant resistance (IDM). Even for plant virus disease management, and vector control (Pedersen et al. 2007). various workers have used IPM techniques for From Australia, Jones (2001) has developed virus disease management in different virus integrated disease management strategies against and host combinations. Some of the available nonpersistent aphid-borne viruses like CMV and information on plant virus and virus-like diseases BYMV in lupin (Lupinus angustifolius)which is presented here comprising the aspects like use are also seed transmitted and the details are fur- of resistant/tolerant varieties, biological controls, nished herein. crop rotations, selected planting and harvesting 312 10 Future Strategies and Conclusions

Table 10.1 Integrated disease management (IDM) approaches to minimise infection with Tomato spotted wilt virus in vegetable crops Control measures Nurseries Protected crops Field crops Phytosanitary Avoid spread from finished crops No Yes Yes Avoid spread from ornamental plants Yes Yes Yes Minimise spread from weeds or ‘volunteer’ crop plants Yes Yes Yes Use rouging within crop No Yes Yes Introduce healthy transplants No Yes Yes Avoid spread within seedlings trays Yes No No Certification of seedlings nurseries Yes No No Cultural Isolate from other susceptible crops No (Yes) Yes Promote early canopy cover and high plant density No Yes Yes Manipulate planting date No No Yes Use mulches or minimum tillage No No Yes Employ windbreaks, and barrier or cover crops No No Yes Diminish vector population growth (Yes) Yes No Install fine nets (Yes) Yes No Obtain advance warning of outbreaks Yes Yes Yes Institute susceptible crop and seed-free period’ (ultimate No Yes Yes measure when all else fails) Resistance Deploy virus-resistant cultivars No Yes Yes Chemical Apply insecticides Yes Yes Yes Apply oils or film-forming products (Yes) (Yes) No Biological Introduce thrips predators Yes Yes Yes Source: Jones (2004), Pappu et al. (2009) dates and cultural or environmental controls. losses in majority of the crops, and this has The government of every country should been discussed in the first chapter. The type provide grants to the agricultural departments member of Tospoviruses, Tomato spotted wilt and research organisations for developing virus (TSWV), has a very wide host range and innovative IDM techniques and subsidies to infects vegetables, fruit crops and ornamentals. the farming community to meet the exorbitant Looking into the seriousness of TSWV in production expenditure and training on recent Australia and also in different countries, Jones IDM technology to the farming community (2004) has proposed various effective integrated through extension education programmes. virus disease management strategies based on Most definitions of IDM further stress that epidemiological data and is presented in the table IDM should first rely as far as possible on cul- (Table 10.1). tural and biological methods. The real challenge Perusal of the table gives that all the aspects of IDM is how the various methods discussed of plant virus management techniques were sug- above can be best combined to give the farmers gested and some suggestions are quite effective an acceptable and socially and environmentally at nurseries, some against protected crops and desirable form of prevention of crop losses (Van some against field crops. With vast experience, Emden 1982; Maelzer 1986; Jones 2004). Prof. R.A.C. Jones of Australia has suggested ap- Throughout the globe, Tospoviruses and proaches that can be planned for other virus vec- Ilarviruses transmitted by thrips vectors infect tor and host combinations in different countries. a large number of host plants and cause heavy No single control measure is likely to be effective 10.3 Challenges for the Future 313 on its own in field situations, and so to ensure direction, virologists of all the countries have that an IDM approach is effective, there is need to combine two or three successful treatments to adopt a diverse array of control measures to for effective plant virus disease management. To be applied before, during and after planting and quote a few, in India, the incidence of Papaya to provide effective education and information ring spot virus in papaya was delayed by a com- transfer to growers. bination of reflective row cover, mineral oil and Another member of Tospovirus, Watermelon imidacloprid and deltamethrin sprays (Kallesh- bud necrosis virus (WBNV), is responsible for waraswamy et al. 2009). Another success is of the severe outbreaks wherever the watermelon crop management of sunflower necrosis disease trans- is grown, and Rajasekharan et al. (2010)have mitted by thrips that was effectively minimised recommended that the integrated approach com- by bordering the sunflower crop with sorghum prising of seed treatment with imidacloprid plus 3 and sunflower seeds treated with imidacloprid rows of maize and use of silver colour UV reflec- (Gaucho 70 W.S., 5 g/kg) along with sunflower tive mulch plus alternate sprays of imidacloprid spraying with imidacloprid (Confidor 200 S.L., and thiamethoxam plus spraying of imidacloprid 0.05%), three sprays at 15, 30 and 45 days af- on bunds plus keeping weed free has proved to be ter sowing. The IDM also involves sowing of effective. In these treatments, the initial PDI was sorghum of 6 rows, sown 15 days prior to the sun- 1.25 at 30 DAS and has postponed the WBNV flower crop. Both the thrips and TSV incidence incidence beyond flowering initiation stage; only were reduced with increased yields (Shirshikar 10% at 60 DAS and 34.3% disease incidence was 2008). recorded at 90 DAS, while the WBNV incidence in control treatment was 95%. In central India, even stem necrosis disease in 10.3 Challenges for the Future potato caused by PBNV was successfully reduced by integrated approach comprising of (1) delay- In considering future avenues of research, it ing the planting of potato crop from the 4th of may be prudent to distinguish between seed- October to the 3rd of November, (2) two foliar transmitted viruses in general and viruses sprays of imidacloprid (0.07%) at 21 and 35 days that are dependent upon seed transmission for after planting and (3) use of polyethylene sheet or perpetuation. Such a distinction may be useful in paddy straw. considering some of the variations evident among At Malawi, Subrahmanyam et al. (2002)have interactions between seed-transmitted viruses successfully reduced the groundnut rosette dis- and their hosts. Co-evaluation of relatively ease by following cultural practices and use of re- few virusÐhost combinations has proceeded to sistant genotypes. The readers may also get more the extent that seed transmission is crucial for information about integrated approach for virus virus perpetuation. Future research should focus management from the review articles of Varma on identification of the sequences and genes (1993), Thresh (2003), Jones (2004), Pedersen involved in determining seed transmissibility et al. (2007), Ambang et al. (2009), Pappu et al. and, subsequently, how viral genes interact with (2009), and Fereres and Moreno (2011). each other as well as with the host. After go throwing the earlier chapters, the Even regarding the mode of transmission, cer- readers would have got the clarity that generally tain elementary points raised by Johansen et al. no single procedure offers maximum control of (1994)havetobeclarified.Theyareasfollows: virus and virus-like diseases. A rational combi- (a) During the translocation of assimilates, how nation of procedures weighing the efficacy of one does any virus move from infected maternal tis- procedure against others and utilising their sup- sues to an ovule (single cell) or to host zygotic plementary effects is the strategy for an integrated cells/tissues of the developing embryo? (b) When control programme, and encouraging results have unassisted pollen transmission of viruses occurs, been obtained to combat virus diseases. In this by what specific route does virus move from 314 10 Future Strategies and Conclusions internally infected pollen grains to intracellular of some instances where resistance to aphids is sites in the ovule? (c) During the development of characterised by tolerance to infection in which the ovule/embryo, by what mechanisms are most aphids have difficulty in recovering viruses from viruses excluded from the embryo or otherwise infected plants not because of the lack of pro- prevented from being transmitted through seeds? duction of viruses “per se” but because of helper Research on the ecology of directly or po- material. tentially important seed-transmitted viruses is es- The potentiality of limiting the virus spread sential for finally developing methods of virus through searching plants low in the production disease control. A limited number of viruses of helper materials needs to be investigated. Al- like Soybean mosaic virus, Pea seed-borne virus most all our research efforts to date have been and Peanut stripe virus have been systematically devoted to study the diseased plants and the studied for their ecology. Virus ecology does virus particles. The tritrophic interactions among not merely consist of the viruses, the crops that the weed crop hosts, viruses and aphid vectors are subject to infection and the environment as remain largely unexplored. This might be due in a simple disease triangle. It is tremendously to complexity of the subject, which requires an complex and consists of (1) the viruses that are interdisciplinary knowledge in distinct fields of continuously changing, and of which several re- virology, entomology, meteorology and mathe- main unknown; (2) the sources of infection, be matics. No one discipline alone will be able to they other crops or wild species; (3) the vectors, achieve the major progress in seed-transmitted each having complicated life cycles and ecologies virus research. Even the virological risks due of their own; (4) the crops that genetically differ to human interference with nature in handling in susceptibility and sensitivity and continuously plant material from new places are needed to change with age, season and cropping system; constantly be on alert. The more dynamic the and (5) the often capricious environment that cropping systems are, the more rapidly new prob- particularly affects vectors and crops. Ecological lems are found to emerge. Any interference with studies include quantification in order to express, natural or agroecosystems entails unpredictable and hopefully to predict, how epidemics will risks, including the spread and upsurge of viruses. develop (epidemiology) and whether and when Without accepting risk in dealing with the nature measures should be taken. and in developing agriculture, there would be no Even seed-transmitted viruses transmitted progress. However, the risks posed by the seed- by aphids in a nonpersistent manner remain transmitted viruses must be restricted to an ac- the weakest part in the disease cycle; gaining ceptable minimum by proper knowledge of crop control through use of effective oils, insecticides, and pathogen ecology. pheromones or other biological agents needs Since viruses having no physiology of their further exploration. Synthetic organic chemicals own, cannot be controlled directly by chemical that interfere with the probing and feeding applications, though a number of attempts were activity of aphids too exist. These chemicals used made. Therefore, until an effective viricide is de- in conjunction with oils increase their efficacy veloped against the virus diseases, indirect meth- than oil formulations alone. ods involving cultural practices like crop rotation, Basic research on the chemistry and mode plant population and date of planting have shown of action of naturally occurring antifeeding sub- to be promising in minimising the virus spread stances from plant species needs to be carried out. under field conditions. If these cultural practices Aphids depend upon the presence of helper mate- are well planned, it would be low cost manage- rials produced by plants themselves for the acqui- ment tactics in minimising the vector population sition of many nonpersistent viruses. Plants ought and in turn virus spread. These practices should to exist that are deficient in these helper materials be factored into management decisions that are and that would therefore be poor sources of made prior to the crop production season. Success virus inoculum for aphids. Indeed, we are aware in virus disease management so far gained with a References 315 few crops augurs well, but it cannot be assumed Lecoq H, Pitrat M (1983) Field experiments on the inte- that methods successful at one place will work at grated control of aphid-borne viruses in muskmelon. another, necessitating to study the epidemiology In: Plumb RJ, Thresh JM (eds) Plant virus epidemiol- ogy Ð the spread and control of insect Ð borne viruses. of virus disease wherever they are prevalent at Blackwell Scientific Publication, London, pp 169Ð176 higher incidence. Maelzer DA (1986) Integrated control of vectors of plant virus diseases. In: McLean GD, Garrett RG, Ruesink WG (eds) Plant virus epidemics: monitoring, modelling and predicting out breaks. Academic, New References York, p 550 Pappu HR, Jones RAC, Jain RK (2009) Global status Ambang Z, Ndongo B, Amayana D, Djile B, Ngoh JP, of tospovirus epidemics in diverse cropping systems: Chewachong GM (2009) Combined effect of host success achieved and challenges ahead. Virus Res plant resistance and insecticide application on the 141:219Ð236 development of cowpea viral diseases. Aust J Crop Sci Pedersen P, Grau C, Cullen E, Koval N, Hill JH (2007) 3(3):167Ð172 Potential for integrated management of soybean virus Carroll TW, Hockett EA, Zaske SK (1990) Elimination of disease. Plant Dis 91:1255Ð1259 seed borne barley stripe mosaic virus (BSMV) from Prasada Rao RDVJ, Reddy AS, Chakravarthy SK (1988) barley. Seed Sci Technol 18:405Ð414 Survey for peanut stripe virus in India. Indian J Plant Coutts BA, Kehoe MA, Jones RAC (2011) Minimising Protect 16:99Ð102 losses caused by Zucchini yellow mosaic virus in Rajasekharam T (2010) Biological and molecular charac- vegetable cucurbit crops in tropical, subtropical and terization and management of watermelon bud necro- Mediterranean environment through cultural methods sis virus. PhD thesis, University of Agricultural Sci- and host resistance. Virus Res 159:141 ences, Dharwad, pp 142 Demski JW, Lovell GR (1985) Peanut stripe virus and the Shirshikar SP (2008) Integrated management of sunflower distribution of peanut seed. Plant Dis 69:734Ð738 necrosis disease. Helia 31(49):27Ð34 Fereres A, Moreno A (2011) Integrated control mea- Subrahmanyam P, van der Merwe PJA, Chiyembekeza AJ, sures against viruses and their vectors. In: Caranta C, Chandra S (2002) Integrated management of ground- Aranda MA, Tepfer M, Lopez-Moya JJ (eds) Recent nut rosette disease. Afr Crop Sci J 10:99Ð110 advances in plant virology. Caister Academic Press, Thresh JM (2003) Control of plant virus diseases in sub- Norwich, p 412 Saharan Africa: the possibility and feasibility of an Johansen IE, Edwards MC, Hampton RO (1994) Seed integrated approach. Afr Crop Sci J 11(3):199Ð223 transmission of viruses: current perspectives. Ann Rev Van Emden HF (1982) Principles of implementation of Phytopathol 32:363Ð386 IPM. In: Cameron P, Wearing CH, Kain WM (eds) Jones RAC (2001) Developing integrated disease manage- Proceeding of the Australian workshop on develop- ment strategies against non-persistently aphid Ð borne ment and implementation of IPM. Government Printer, viruses Ð a model programme. Integr Pest Manag Rev Auckland, p 9 6:15Ð46 Varma A (1993) Integrated management of plant virus Jones RAC (2004) Occurrence of virus infection in seed diseases. In: Crop protection and sustainable agricul- stocks and 3 year Ð old pastures of Lucerne (Medicago ture. Wiley, Chichester, pp 140Ð157 (Ciba Foundation sativa). Aust J Agric Res 55:757Ð764 symposium-177) Kalleshwaraswamy CM, Krishna Kumar NK, Dinesh MR, Vetten HJ (1984) Lettuce mosaic virus information Chandrashekar KN, Manjunatha M (2009) Evaluation on integrated control. Das salat mosaik virus in- of insecticides and oils on aphid vectors for the man- formation on integrated, control. Pflanzen senschutz agement of papaya ringspot virus (PRSV). Karnataka Nachrichtenblatt des Deutschen Pflanzen schutz dien- J Agric Sci 22(3-Spl. Issue):552Ð553 sters 36(9):141Ð142 Index

A Array technologies, 137, 256 Abutilon mosaic virus (AbMV), 8, 78 Artichoke Italian latent, 11, 59 ACLSV. See Apple chlorotic leaf spot virus (ACLSV) Artichoke latent, 11, 60 Aegilops, spp., 11 Artichoke yellow ring spot, 11, 59 Agarose gel electrophoresis, 134Ð135, 293 Aseptic plantlet culture, 252Ð253 Agropyron elongatum,11 Asparagus bean mosaic virus,11 Alfalfa cryptic virus (ACV), 5, 91 Asparagus latent, 11, 59 Alfalfa mosaic virus (AMV), 68, 69, 76, 90, 91, 107, 109, Asparagus officinalis, 11, 252 140, 141, 169, 173, 175, 203, 244, 261Ð264, 288 Asparagus stunt, 28, 59 Alfalfa temperate, 10, 57 Asparagus virus I (AV1), 11, 60 Alfamo virus,63 Asparagus virus II (AV2), 11, 59 Alliaria petiolata,29 Assessment of crop losses, 67Ð69 Allium cepa, 19, 21, 174 Atriplex pacifica,25 Alphacryptovirus,57 Aureusvirus,57 Alphaflexiviridae,60 Australian Lucerne latent, 11, 59 Aluminum mulches for vector control, 196, 197 Avena fatua,11 Amaranthus albus,10 Avena sativa, 21, 192 Amaranthus caudatus,15 Avocado sun-blotch, 6, 11, 57, 104, 132, 286 Amaranthus hybridus, 15, 27, 168, 169 Avocado viruses 1Ð3, 11, 57 Amaranthus viridis,26 Avoidance of virus inoculum from infected seeds, AMV. See Alfalfa mosaic virus (AMV) 186Ð189 Andean potato latent virus (APLV), 18, 61, 169, 288 Avoiding of continuous cropping, 189Ð190 Antisense RNA, 259, 264Ð265, 311 Avoid spread from finished crops, 314 Antiviral activities in plants, 259, 265Ð266 Avoid spread from ornamental plants, 314 Anulavirus,57 Avoid spread within seedlings trays, 314 Aphid vectors, 8, 70, 75, 77, 92, 170, 171, 190Ð192, 195, Avsunviroid, 5, 57, 60 218, 316 Avsunviroidae, 5, 57, 60 Aphis gossypii, 171 Azuki bean mosaic, 12, 60 Apium graveolens, 15, 26 Apple chlorotic leaf spot virus (ACLSV), 7, 8, 10, 61, 174, 289, 294 B Apple dapple viroid, 6, 10, 57 Badnavirus, 57, 141, 290 Apple mosaic virus (ApMV), 10, 59, 123, 209, 289, 292, Banana streak virus (BSV), 294, 296 294 Banana viruses, 11, 58 Apple scar skin viroid, 6, 10, 57, 128, 289, 294, 295 Barley false stripe, 11, 58, 105 Application of DIBA, 127 Barley mottle mosaic, 7, 8, 11, 62 Application of ELISA, 122Ð126, 292 Barley yellow dwarf virus (BYDV), 8, 63 Approved seed certification standards, 209Ð210 Barrier and cover crops, 192 Apricot, 6, 23, 247, 248, 292, 294 Bean common mosaic (BCMV), 4, 6, 12, 13, 60, 65, 68, Apricot gummosis,10 69, 87Ð92, 94, 103, 104, 107Ð109, 111, 112, 122, Apscaviroid,57 123, 127, 130, 139, 140, 142, 166Ð168, 173Ð175, Arabidopsis thaliana, 15, 26, 29, 87, 90, 240 177, 189, 191, 197Ð198, 202Ð205, 207, 212, 213, Arabis mosaic virus (ArMV), 141, 167, 295 216, 217, 219, 223, 242, 244, 260, 296 Arachis hypogaea, 14, 16, 17, 106 Bean common mosaic necrosis, 13, 60, 123, 139, 203 Arracacha virus A, 11, 59 Bean pod mottle virus (BPMV), 68, 77, 90, 105, 261 Arracacha virus B, 11, 59, 174 Bean red node, 28, 59

K.S. Sastry, Seed-borne Plant Virus Diseases, DOI 10.1007/978-81-322-0813-6, 317 © Springer India 2013 318 Index

Bean southern mosaic, 13, 61, 89, 174, 207 Bunyaviridae,61 Bean western mosaic, 12, 60, 89 Bymovirus,57 Bean yellow dwarf,8 BYMV. See Bean yellow mosaic virus (BYMV) Bean yellow mosaic virus (BYMV), 13, 21, 60, 61, 69, 90, 104, 106, 111, 123, 140, 141, 174, 202, 205, 207, 216, 242, 244, 264, 289, 295, 313 C Beet 1 alpha crypto, 13, 57 CABMV. See Cowpea aphid-borne mosaic (CABMV) Beet 2 alpha crypto, 13, 57 Cacao necrosis virus (CNV), 14, 59, 289 Beet 3 alpha crypto, 13, 57 Cacao swollen shoot virus (CSSV), 14, 57, 130, 289 Beet cryptic virus (BCV), 5, 91 Camelina sativa,29 Beetle vectors, 77, 170 CaMV. See Cauliflower mosaic (CaMV) Beet mild yellowing, 13, 60 Cannabis sativa,19 Beet temperate, 13, 57 Capillovirus, 57, 62, 63 Beet western yellows virus (BWYV), 8 Capsella bursapastoris,91 Beet yellows, 63, 174 Capsicum annuum, 10, 17, 23, 26 Beet 41 yellows,13 Capsicum frutescens, 17, 26, 27 Begomovirus, 57, 78, 260, 290, 294 Capsid protein mediated resistance, 257Ð258 Benincasa hispida,17 Carica papaya,21 Berseem mosaic, 10, 57 Carlavirus, 14, 25, 58, 62, 63, 76, 78, 110, 111, 177 Betacryptovirus,57 Carmovirus, 17, 58, 62, 63, 77, 177 Betaflexiviridae, 58, 61, 62 Carrot motley dwarf,8,294 Beta vulgaris (B. vulgaris,), 9, 10, 13, 20, 24, 27, 28, 91, Carrot motley leaf, 14, 62 174 Carrot red leaf (CaRLV), 8, 14, 60 Betula pendula,15 Carrot temperate virus 1 (CTeV1), 5, 14, 57 BICMV. See Blackeye cowpea mosaic (BICMV) Carrot temperate virus 2 (CTeV2), 5, 14, 57 Biological methods, 105Ð109 Carrot temperate virus 3 (CTeV3), 5, 14, 57 Biological properties, 109, 263 Carrot temperate virus 4 (CTeV4), 5, 14, 57 Biosafety regulations, 227, 228 Carthamus tinctorius, 25, 27 Blackeye cowpea mosaic (BICMV), 13, 60, 65, 69, 106, Cassia occidentalis,14 108, 111, 112, 115, 141, 191, 203, 205, 207, 212, Cassia yellow spot (poty), 14, 60 216 Cauliflower mosaic (CaMV), 7, 8, 15, 58, 63, 167 Black gram leaf crinkle,62 Caulimoviridae, 57, 58 Blackgram mild mottle, 13, 123 Caulimovirus, 58, 62, 63, 76, 141 Blackgram mottle virus (BMoV), 13, 58, 107, 117, 123, cDNA probes, 133, 136Ð137 130, 202 Celery latent, 15, 60 Black raspberry latent virus (BRLV), 14, 24, 59, 172, 175 Celosia argentea,20 Blue international seed sample certificate, 214, 215 Celosia cristata,25 Brassica chinensis var. parrachinensis,29 Cerastium holosteoides,17 Brassica napus var. silvestris,29 Cerastium viscosum,20 Brassica rapa var. amplexicaulis sub var. dentata,25 Cerastium vulgatum, 28, 91 Brassica rapa var. laciniifolia,20 Certification schemes, 114, 131, 213, 217Ð220, 293Ð298, Brassica vulgaris,24 311 Brinjal mosaic, 24, 61, 189 CF-PCR, 139, 142 Brinjal ring mosaic, 14, 62 CGMMV. See Cucumber green mottle mosaic virus Brinjal severe mosaic, 14, 62 (CGMMV) Broad bean mild mosaic, 14, 60 Challenges for the future, 315Ð317 Broad bean mottle, 14, 57 Challenges in diagnosis of pests in quarantine, 241 Broad bean stain, 14, 58, 69, 77, 89Ð91, 106, 123, 130, Chemical seed disinfection, 186Ð187 141, 166, 170, 174, 198, 240 Chenopodium album, 10, 25, 28, 169 Broad bean true mosaic, 14, 18, 58, 77, 115, 123, 127, Chenopodium amaranticolor, 15, 18, 19, 29, 109 130, 166, 170 Chenopodium murale, 21, 88, 89 Broad bean wilt, 14, 58 Chenopodium quinoa, 14, 15, 19, 20, 22, 24Ð26, 28, 174 Brome mosaic virus (BMV), 14, 57, 63, 91, 106, 115, Chenopodium serotinum (C. serotinum),18 123, 127, 130, 174 Cherry leaf roll virus (CLRV), 15, 59, 104, 111, 120, 123, Bromoviridae, 57Ð59 142, 144, 175Ð177, 187, 198, 240 Bromovirus, 57, 62, 63, 77, 110, 111, 141, 172, Cherry necrotic rusty mottle, 8, 15, 62 177 Cherry rasp leaf virus (CRLV), 15, 59, 174 Bromus inermis,11 Cherry ring mottle, 24, 59 Bromus, spp., 11 Cherry ring spot, 15, 62 Bumblebees, 79 Chicory yellow mottle (ChYMV), 15, 59 Index 319

Choice of enzyme labels and substrates, 120Ð121 Cowpea mosaic virus (CPMV), 16, 63, 68, 77, 105, Chrysanthemum coronarium,19 112, 115, 117, 141, 166, 186, 188, 202, 203, 212, Chrysanthemum morifolium,15 262Ð263, 312 Chrysanthemum stunt viroid (CSV), 6, 15, 60, 132, 140, Cowpea mottle virus (CPMoV), 16, 58, 77, 140, 198, 226, 294 202, 219, 240, 242 Cicer arietinum, 10, 17, 22, 106 Cowpea ring spot virus (CPRSV), 17, 58 Cichorium intybus,15 Cowpea severe mosaic virus (CPSMV), 17, 58, 124, 167 Cineraria mosaic, 15, 62 Cowpea severe mottle,17 Citrullus vulgaris, 17, 20 CPMoV. See Cowpea mottle virus (CPMoV) Citrus aurantifolia,15 CPMV. See Cowpea mosaic virus (CPMV) Citrus exocortis viroid, 6, 15, 60, 142, 226, 289, 294 Crambe hispanica,29 Citrus huanglongbing, 7 Crimson clover latent, 17, 59 Citrus leaf blotch, 15, 58 Crinivirus,58 Citrus mosaic, 15, 57, 289, 294 Crop rotation, 168, 191, 313, 316 Citrus psorosis, 8, 15, 60 Crops hygiene, 193Ð195 Citrus sinensis,15 Cross-banded masses in phloem, 110 Citrus sinensis x Poncirus trifoliata,15 Crotalaria juncea,26 Citrus tatter leaf, 15, 40, 292 Cryptovirus, 5, 57, 62, 63, 65, 76, 133, 177 Citrus veinal chlorosis,15 Crystalline aggregates, hexagonal, 110 Citrus xyloporosis,15 Crystalline arrays of virus particles, 110 Citrus yellow mosaic virus (CIYMV), 10, 58, 294 Crystalline, monolayer, 110 Classification of viruses, 56Ð62 Cucumber cryptic,5,18 Closed quarantines, 197, 250, 254 Cucumber green mottle mosaic virus (CGMMV), 17, 61, Closteroviridae,58 69, 80, 87, 89, 111, 127, 130, 140, 186, 188, 189, Closterovirus, 62, 63, 76, 110, 111, 295 194, 200, 202 Clover (red) mosaic, 16, 60 Cucumber leaf spot carmovirus (CLSV), 17, 57 Clover (white) mosaic, 16, 60 Cucumber mosaic virus (CMV), 4, 6, 17, 58, 63, 68, 69, Clover (red) vein mosaic, 16, 58 77, 80, 87, 89Ð91, 103, 106, 108, 115, 124, 127, Clover yellow mosaic virus (CLYMV), 16, 60, 108 128, 134, 140, 141, 166Ð169, 171, 174, 177, 178, CLRV. See Cherry leaf roll virus (CLRV) 191Ð193, 196, 198, 202, 203, 205Ð207, 211, 212, Clumping of virus particles, 129Ð130 216, 219, 244, 258, 259, 261Ð264, 266, 288Ð290, CMV. See Cucumber mosaic virus (CMV) 292, 294Ð296, 311, 313 Coat protein genes, 201, 259, 261Ð262, 264, 266, Cucumber pale fruit, 6, 19, 59, 226 311 Cucumis melo, 17, 20, 21, 27, 65, 79, 89, 106, 206 Cocadviroid,58 Cucumis melo var. flexaosus,20 Cocksfoot alpha cryptovirus,16 Cucumis pepo,30 Coconut cadang cadang viroid, 6, 16, 58, 132 Cucumis sativus, 17, 18, 27, 80, 89, 95 Cocos nucifera,16 Cucumis sativus cryptic,18 Coffea excelsa,16 Cucumovirus, 58, 62, 63, 76, 110, 111, 141, 167, 172, Coffee ring spot, 16, 60 177 Coleus blumei viroid 1, 6, 16, 58 Cucurbita flexuous,21 Coleus blumei viroid 2, 6, 16, 58 Cucurbita maxima, 24, 175 Coleus blumei viroid 3, 6, 16, 58 Cucurbita moschata,17 Coleus scutellarioides, 16, 141 Cucurbita pepo, 26, 30, 65, 88, 89, 106, 141, 174, 188, Coleviroid,58 202 Commelina communis,11 Cultivars with low seed transmission, 205Ð206 Comovirus, 58, 62, 63, 76, 77, 110, 111, 127, 177 Curtovirus,58 Conditions for ISTA certificates, 214Ð215 Cuscuta californica,18 Control measures for virus diseases, 185, 186 Cuscuta campestris,89 Control of the vectors, 195Ð197 Cyamopsis tetragonoloba, 12, 19 Conventional breeding of resistance, 203Ð205 Cycas necrotic stunt, 18, 59 Cowpea aphid-borne mosaic (CABMV), 16, 60, 68, 89, Cylindrical, pinwheels, 110 120, 130, 140, 141, 174, 203, 205, 207, 212, 242, Cynara scolymus,11 244 Cowpea banding mosaic, 17, 58, 91, 92, 174, 187 Cowpea chlorotic spot virus, 16, 57, 91, 92 D Cowpea green vein-banding (CGVBV), 16, 61 Dahlia mosaic virus (DMV), 140, 141, 294 Cowpea mild mottle virus (CPMMV), 16, 58, 78, 123 Dahlia pinnata, 18, 141 Cowpea moroccan aphid-borne mosaic, 17, 61 Datura quercina, 28, 59, 174 320 Index

Datura stramonium, 23, 28, 78, 173, 174 Exchange of legume germplasm, 234Ð235 Daucus carota,14 Exclusion of exotic plant viruses through quarantine, Decoration or antibody coating, 130 238Ð241 Desmodium canum, 18, 168 Extent of seed transmission, 6Ð7, 169, 205 Desmodium mosaic, 18, 61 Desmodium trifolium,18 Desmodium trifolium mottle,18 F Detection of latent infection, 122, 126 Fabavirus Detection of plant viroids in seeds, 143 , 58, 62, 63, 77, 111 Detection of plant viruses in seeds, 101Ð145, 310 Factors affecting yield losses, 69Ð70 Determination of seed transmission rate, 220Ð221 Factors influencing rate of seed transmission, 88Ð93 Determining the tolerant limits, 126 Factors influencing vector movement, 170Ð172 Development of seed structures, 85Ð87 FAO-IBPGR, 233Ð236 Fescue cryptic Dianthovirus, 62, 63 , 5, 19, 57 Festuca pratensis Different vegetative propagative plant materials, 288 ,19 Ficus carica Direct binding PCR, 139, 143 ,19 Direct ELISA, 119 Fig latent virus 1, 19, 130 Direct immunostaining assay (DISA), 116 Fijivirus, 64 Disperse dye immunoassay (DIA), 128 Flask-shaped double membrane, 110 Distribution of virus in the seed, 87Ð88, 216 Flexiviridae, 57 DNA viruses, 132, 141 Flocculation tests in liquid media, 114 Dodder latent mosaic, 18, 89 Fluorescent antibody techniques, 116Ð117 Dot-blot hybridization assay, 132Ð133, 136, 143 Fluorescent staining of nucleic acids, 117 Foxtail mosaic potexvirus Dot immuno binding assay (DIBA or DIA), 116, 126Ð128 , 19, 60 Fragaria chiloensis Double stranded DNA (dsDNA), 63, 144, 293 ,19 Fragaria chiloensis ilarvirus Double stranded RNA (dsRNA), 3, 5, 63, 64, 129, , 19, 59 Fragaria vesca 132Ð135, 264, 265 , 26, 28, 29 Fragaria x ananassa Dulcamara mottle, 18, 61 , 10, 24, 28, 29 Duplex RT-PCR, 141 French bean mosaic, 19 Functions of plant quarantine, 231Ð232 Furovirus, 58, 62, 63, 79, 177 E Echinocystis lobata, 4, 17, 30, 168 Echtes Ackerbohnen mosaic, 18, 58, 115 G Ecology and epidemiology of seed-transmitted viruses, Garland chrysanthemum temperate, 19, 57 165Ð179 Gel double immuno diffusion (Ouchterlony), 115Ð116 Effective methods of plant importations, 249Ð253 Gel electrophoresis, 133Ð135, 293 Eggplant mosaic, 18, 61, 89 Geminiviridae, 57, 58, 78 Electron microscopy, 7, 56, 87, 104, 110, 111, 116, 129, Geminivirus, 8, 62, 63, 141 131, 233, 249, 255, 310 General principles for the overall effectiveness of Eleusine coracana,22 quarantines, 253Ð254 Elimination of weed, volunteer and wild hosts, 190 Genetics of seed transmission, 88 ELISA. See Enzyme linked immunosorbent assay Geranium dissectum,28 Elm mosaic, 18, 174 Gladiolus spp., 27, 29 Elm mottle virus (EMoV), 18, 59, 174 Glycine max, 10, 13Ð16, 19, 20, 23Ð25, 27Ð29, 78, 89, Embryo-borne viruses, 86, 87, 189 106, 174 Embryo culture, 108, 199, 253 Glycine soja, 24, 25 Enamo virus, 58, 62, 63 Gomphrena globosa, 27, 29 Environmental factors, 92Ð93, 105, 166, 192 Granular and crystalline in cytoplasm and nuclei, 110 Enzyme linked immunosorbent assay (ELISA), 116, Grape decline,19 118Ð126, 216, 291, 310 Grapevine Bulgarian latent virus (GBLV), 19, 59 Epidemiological role of pollen-transmitted viruses, Grapevine fanleaf virus (GFLV), 19, 59, 289 173Ð177 Grapevine viroid, 6, 19, 59, 295 Epidemiology, 4, 6, 145, 165Ð179, 185, 217, 224, 225, Grapevine yellow mosaic,19 313, 316, 317 Grapevine yellow speckle, 6, 19, 57 Erroneously listed viruses, 1, 7Ð31 Grapevine yellow vein, 29, 60 Eucharis candida,19 Group A mastro,63 Eucharis mottle nepovirus,19 Group B begomo,63 Euonymus europaeus,19 Grow-out test, 78, 87, 94, 107Ð108, 111, 122, 125 Euonymus mosaic,19 Guar symptomless, 19, 61 Index 321

H International scenario, 238Ð329 Helianthus annuus,26 International seed health initiative (ISHI), 102Ð103 Hemp streak,19 International seed testing association (ISTA), 105, 210, Hibiscus cannabinus,19 213Ð215, 249, 267 Hibiscus latent ring spot, 19, 59, 124 IPGRI role, 298 High degree of seed transmission, 6, 91 Ipomoea batatas, 26, 296 High plains virus, 19, 62, 104, 124 Irradiation effect, 189 Hippeastrum hybridum,19 Isolate from other susceptible crops, 314 Hippeastrum mosaic, 19, 61 ISTA certificates, 214Ð215 History of biosafety, 227Ð228 History of seed-transmitted plant virus, 4Ð5 Hop chlorosis,19 J Hop stunt viroid, 6, 19, 59, 143 Juglans regia, 15, 175 Hop trefoil cryptic virus 1 (HTCV-1), 5, 19, 57 Hop trefoil cryptic virus 2 (HTCV-2), 5, 19, 57 Hop trefoil cryptic virus 3 (HTCV-3), 5, 19, 57 K Hordeivirus, 58, 62, 63, 88, 110, 258 Kalanchoe blossfeldiana,19 Hordeum depressum,11 Kalanchoe top-spotting, 19, 57 Hordeum vulgare, 11, 89, 106, 166, 174 Horizontal transmission, 172, 177 Hosta longipes,19 L Hosta virus x, 19, 60, 140 Labelled antibody techniques, 113, 114, 116Ð126, 129, Host plant, 56, 65, 91, 108, 109, 113, 116, 167Ð170, 173, 130 199, 201, 207, 227, 234, 245, 313, 314 Lactuca sativa, 10, 19, 27, 28, 106, 174 Host resistance, 201, 206, 297, 313 Lagenaria siceraria,17 Host species, 6, 90Ð91, 168, 290 Lamium amplexicaule, 10, 26Ð28 Hostuviroid,59 Lamium purpureum,17 Humulus Japonicas, 19, 59, 591 Lathyrus,21 Humulus lupulus, 19, 89 Lathyrus cicera,10 Hydrangea mosaic virus (HdMV), 19, 59 Lathyrus clymenum,22 Lathyrus ochrus,21 Lathyrus sativus,10 Leafhopper vectors, 63, 64, 248, 249, 288 I Leek yellow stripe virus (LYSV), 295 IBPGR in germplasm exchange, 234Ð236 Lens culinaris, 10, 13, 14, 17, 22, 106 IC-PCR, 139, 142, 143, 216 Lens esculenta, 13, 14 Idaeovirus,59 Lettuce mosaic virus (LMV), 4, 19, 61, 68, 69, 80, 87, Ilarvirus, 9, 59, 62, 63, 77, 111, 172, 173, 177, 314 90Ð93, 103, 106Ð109, 111, 113, 118, 122, 124, Immunity, 205 126, 128Ð130, 140, 141, 166Ð170, 174, 188, 190, Immunization, 112, 207Ð209 191, 202, 204, 211, 212, 216, 217, 221, 259, 267, Immuno diffusion tests, 114Ð116 313 Immunosorbent electron microscopy (ISEM), 129Ð131, Lettuce yellow mosaic,20 312 Ligustrum vulgare,28 Impatiens walleriana,15 Lilac ring mottle virus (LRMV), 20, 59 Important cases of introduction, 246Ð248 Lima bean mosaic,4,20 Important diseases restricted to some countries, 248Ð249 Liquid hybridisation assay, 132, 133 Improvements in ELISA, 121Ð122 Loganberry degeneration, 24, 59 Inability to infect embryos, 93Ð94 Lolium multiflorum,25 Inability to survive in embryos, 94Ð95 Loop mediated isothermal amplification (LAMP), 139, Inclusion bodies, 87, 109Ð111 142 Indicator hosts, 56, 104, 108Ð109, 245, 246, 249, 256 Low seed transmission / symptomless carriers, 103Ð104 Indirect ELISA, 113, 119Ð120, 122, 127 Lucerne Astralian latent, 11, 20, 59, 60 Infection status of a bulk seed, 222 Lucerne (Australian) symptomless, 201 Inoculum threshold, 166, 210Ð213 Lucerne transient streak, 20, 61 Insecticides for vector control, 195Ð196, 313 Lupinus albus, 13, 25 Integrated cultural practices, 192Ð193 Lupinus albus (white lupin),23 Integrated disease management (IDM), 313Ð315 Lupinus angustifolius, 10, 17, 87, 192, 203, 212, 313 Integrated pest management, 313 Lupinus luteus, 12, 17, 106 The intermediate quarantine, 251Ð252 Luteoviridae, 58, 60, 62, 81 322 Index

Luteovirus, 8, 62, 63, 76, 134, 140 Myosotis arvensis, 11, 27, 28, 91, 169 Lychnis divaricata, 20, 89, 174, 189 Myzus persicae, 169, 193 Lychnis ringspot, 20, 58, 89, 174, 189 Lycopersicon esculentum, 10, 15, 17, 19, 23, 27Ð29, 89, 106 N Nanovirus, 141 National scenario, 239Ð241 M Necrovirus, 55, 59, 62, 63, 75, 79, 104 Machlomovirus, 59 Need for networking, 254Ð255 Macroarrays, 137 Nematode vectors, 169, 195, 288 Macroptilium lathyroides,12 Nepovirus, 6, 19, 25, 55, 59, 60, 62, 64, 75, 76, 78, 79, Maize chlorotic mottle machlomovirus (MCMV), 20, 59 104, 107, 109, 111, 122, 167Ð169, 177, 258 Maize dwarf mosaic virus (MDMV), 20, 61, 68, 104, 240 Nested PCR, 139, 142 Maize leaf spot, 20 Nicandra physaloides, 10, 23, 173, 174 Maize mosaic virus (MMV), 20, 60 Nicotiana clevelandii, 11, 15, 18, 25, 28 Malus platycarpa,27 Nicotiana debneyi,24 Malus pumila, 27, 29 Nicotiana glutinosa, 21, 23, 27, 109, 174, 259 Manipulate planting date, 314 Nicotiana megalosiphon, 15, 26 Marafivirus, 63 Nicotiana rustica, 26, 28, 111 MCMV. See Maize chlorotic mottle machlomovirus Nicotiana tabacum, 15, 24, 26Ð29, 106, 109, 116, 187 (MCMV) Nicotiana velutina mosaic, 21, 58 MDMV. See Maize dwarf mosaic virus (MDMV) Non-persistent viruses, 76, 107, 195, 316 Mealybug, 76, 79, 290 Non-radiolabelled probes, 136 Mechanical spread, 80 Non-vector transmission, 80Ð81 Mechanism of seed transmission, 85Ð95 Nucleic acid specific hybridisation, 135Ð137 Medicago lupulina, 10, 19 Nucleorhabdovirus, 60 Medicago polymorpha,10 Number of infection sources, 90 Medicago sativa, 10, 11, 17, 20, 68, 89, 174 Medicago scutellata,10 Melilotus albus, 20, 28 O Melon necrotic spot virus (MNSV), 79 Oat mosaic virus (OMV), 57 Melon rugose mosaic, 20, 62, 124 Objectives of ISTA, 214 Mentha arvensis, 26, 169 Objectives of NBPGR, 243Ð245 Methods of testing at quarantine stations, 131, 233, Obligate symbiosis, 80Ð81 245Ð246 Obtain advance warning of outbreaks, 314 Mibuna temperate, 20, 57 Oils for vector control, 196, 313 Microarrays, 114, 137Ð138, 287 Olive latent virusˆu1 (OLV-1), 21, 59, 104 Mineral oils, 185, 196, 315 Olpidium brassicae,79 Mite transmission, 76 OLV-1. See Olive latent virusˆu1 (OLV-1) Mixed infections, 69, 90, 108, 134, 142, 178, 260 OMV. See Oat mosaic virus (OMV) MMV. See Maize mosaic virus (MMV) Onion mosaic, 21, 174 MNSV. See Melon necrotic spot virus (MNSV) Onion yellow dwarf virus (OYDV), 21, 61, 124, 174, 289, Molecular approaches, 258Ð266 295 Molecular beacons, 143Ð144 Open quarantine, 251 Molecular hybridization, 132Ð133, 136 Ophioviridae, 60 Molecular interactions, 258 Ophiovirus, 60 Molecular markers, 144Ð145, 204 Orange international seed lot certificate, 214, 215 Monoclonal antibodies, 112Ð114, 122, 131, 132, 246, 255 Morphological abnormalities, 105, 186 Movement of germplasm, 234Ð237, 243, 256, 298 P Movement protein genes (MP genes), 262 Pachyrrhizus erosus,25 MRSV. See Mulberry ring spot virus (MRSV) PAGE. See Polyacrylamide gel electrophoresis (PAGE) Mulberry ring spot virus (MRSV), 20, 60 Panicum mosaic virus (PMV), 21, 61, 68Ð70, 87, 90, 104, Multiplex PCR, 101, 139, 142 105, 122, 133, 166Ð168, 186, 190, 191, 196, 202, Multi-vesicular-body, 110 205, 212, 219 Mung bean isometric yellow mosaic, 20, 62 Papaver rhoeas,27 Mung bean mosaic potyvirus, 20, 60, 106 Papaya ring spot virus (PRSV), 21, 61, 315 Musa acuminate, 11 Paprika mild mottle tobamovirus, 21, 61 Muskmelon mosaic, 20, 58, 89 Parsley latent, 21, 62 Muskmelon necrotic spot, 21, 58 Parsnip yellow fleck virus (PYFV), 55, 62, 64 Index 323

Parthenium hysterophorus,9,28 Physical methods, 109Ð111, 310 Partitiviridae,4,5,57 Phytosanitory certificates, 199, 219, 230, 231, 233, 235, Pastinaca sativa,26 236, 239, 240, 249Ð250, 254 Pathways of spread of pests and pathogens, 232 Phytosanitory measures, 221, 224, 238, 239, 256, 313 PDV. See Prune dwarf virus (PDV) Pinwheels, 110, 111 Peach latent, 9, 22, 60, 292, 295 Piper nigrum,23 Peach necrotic leafspot, 9, 24, 59 Piper yellow mottle, 23, 57, 79, 140, 292, 294 Peach ringspot, 24, 59 Pisum arvense,22 Peach rosette mosaic virus (PRMV), 9, 24, 59 Pisum sativum, 13, 14, 17, 21, 22, 88, 90, 106, 145 Pea early browning virus (PEBV), 6, 87, 109, 128, 186, Plantago major, 11, 27 206, 257, 258, 262 Plant density, 191, 314 Pea enation mosaic virus (PEMV), 21, 58, 63, 69, 81, Planting dates, 191, 314 142, 261 Plant quarantine, 110, 198, 225Ð227, 229Ð232, 234, Pea false leaf roll, 21 238Ð241, 243, 244, 248Ð250, 252, 255Ð257 Pea fizzle top, 61, 107 Plant quarantine measures, 230, 256 Pea leaf rolling virus, 22, 61 PLRV. See Potato leafroll virus (PLRV) Pea mild mosaic virus (PMiMV), 21, 58 Plum, 23, 141, 245, 247, 248, 287Ð290, 295, 298 Pea mosaic virus, 196 Plum pox virus (PPV), 23, 61, 125, 140, 141, 224, 247, Peanut clump (African) (PCV), 22 248, 265, 289, 292, 294Ð296 Peanut clump (Indian) (PCV), 22, 58, 68, 91, 124, 130 PMiMV. See Pea mild mosaic virus (PMiMV) Peanut marginal chlorosis, 23, 62 PMV. See Panicum mosaic virus (PMV) Peanut mild mottle, 23, 61, 68, 124 PNRSV. See Prunus necrotic ringspot (PNRSV) Peanut mottle virus (PeMoV), 68, 70, 140Ð142, 203, 205, Polerovirus,60 216, 217, 220, 223, 260, 261 Pollen transmission, 172Ð177, 315 Peanut stripe virus (PStV), 65, 68, 80, 92, 109, 122, 133, Polyacrylamide gel electrophoresis (PAGE), 118, 134, 135, 136, 140Ð142, 166, 190, 198, 202, 203, 205, 135 207, 217, 219, 223, 242, 244, 260, 312, 313, 316 Polyclonal antibodies, 112Ð115, 122 Peanut stunt virus, 68, 140, 186 Polygonum aviculare,28 Pea stem necrosis, 22, 58, 79 Polygonum persicaria, 11, 91 Pea streak, 22, 58, 94 Polymerase chain reaction (PCR) based detection, 6, 7, PEBV. See Pea early browning virus (PEBV) 114, 131, 138Ð144, 199, 216, 246, 258, 287, 293, Pelamoviroid, 60 294 Pelargonium hortorum, 27, 29, 174 Pospiviroidae, 5, 57Ð60 Pelargonium zonate spot, 23, 57, 174 Potato Andean latent tymovirus, 23 PeMoV. See Peanut mottle virus (PeMoV) Potato leafroll virus (PLRV), 136, 262, 288, 290 PEMV. See Pea enation mosaic virus (PEMV) Potato spindle tuber, 5, 6, 23, 60, 88, 141, 169, 172, 187, Pennisetum glaucum,22 194, 226, 233, 247, 289, 295 Pepino mosaic virus (PepMV), 79, 80, 144, 186 Potato virus T (PVT), 23, 62, 169, 173, 174 PepMV. See Pepino mosaic virus (PepMV) Potato virus U (PVU), 24 Pepper chat fruit viroid, 23, 60, 140 Potato virus X (PVX), 24, 60, 64, 79, 80, 87, 113, 136, Persea americana,11 174, 194, 262, 264, 266, 290 Perspectives, 255Ð256, 266 Potato virus Y (PVY), 24, 61, 64, 113, 262, 263, 288, Pest and pathogen risk analysis, 225, 226, 254 290, 296 Pest risk analysis (PRA), 221, 224Ð227, 229, 238Ð241 Potato yellowing, 24, 57 Petroselinum crispum, 21, 26 Potexvirus, 19, 60, 62, 64, 110, 111, 177, 262, 296 Petroselinum hortense,26 Potyviridae, 57, 60, 61, 111 Petunia hybrida, 11, 18, 174 Potyvirus, 20, 26, 60Ð62, 64, 65, 76, 88, 104, 110, 111, Petunia violacea, 10, 24, 27, 28, 89, 107 134, 139, 141, 177, 203Ð205, 216, 257, 258 Phaseolus aborigineus,12 PPV. See Plum pox virus (PPV) Phaseolus acutifolius var. latifolius,12 PRA. See Pest risk analysis (PRA) Phaseolus angularis,16 Primary inoculum source, 166Ð167 Phaseolus aureus, 27, 92, 106 PRMV. See Peach rosette mosaic virus (PRMV) Phaseolus coccineus,25 Provisional certificate, 214 Phaseolus limensis,20 PRSV. See Papaya ring spot virus (PRSV) Phaseolus lunatus,14 Prune dwarf virus (PDV), 120, 188, 189, 292, 294 Phaseolus radiata,13 Prunus americana, 10, 24 Phaseolus vulgaris, 10, 12Ð17, 19, 23, 25, 28, 78, 89, Prunus amygdalus, 10, 24 109, 174, 191 Prunus avium, 6, 15, 24, 29, 120 Physalis peruviana,23 Prunus cerasus, 24, 174, 175 324 Index

Prunus necrotic ringspot (PNRSV), 4, 6, 9, 24, 59, 69, 88, Resistance, 4, 91, 94, 114, 126, 144, 145, 178, 187, 196, 89, 125, 134, 172, 173, 175Ð177, 189, 209, 213, 199Ð207, 212, 219, 226, 228, 242, 251, 257Ð266, 289, 294, 295 297, 311Ð314, 316 Prunus serotina,15 in cultivated species, 201Ð202 Psophocarpus tetragonolobus,30 in fruit crops, 202 PStV. See Peanut stripe virus (PStV) Resistant genes, 200, 208 PVT. See Potato virus T (PVT) Return-polyacrylamide gel electrophoresis (R-PAGE), PVU. See Potato virus U (PVU) 135 PVX. See Potato virus X (PVX) Reverse transcription PCR (RT-PCR), 139Ð144, 199, 216, PVY. See Potato virus Y (PVY) 255 PYFV. See Parsnip yellow fleck virus (PYFV) Reverse transcription-PCR-DBH, 143 Pyrus communis,27 Rhabdoviridae,60 Rhabdovirus, 62, 64 Rhubarb temperate, 25, 57 Q RIA. See Radio immunoassay (RIA) The quality control by ELISA, 216Ð217 RIPA. See Rapid immune filter paper assay (RIPA) Quality control of bulk seed lots, 220Ð222 Risks associated with GMO, 228Ð229 Quantity of plant materials, 250 RNAi mediated, 265 Quarantine, 101, 103, 104, 108, 110, 111, 115, 131, RNA Viruses, 132, 134, 136, 137, 139, 140, 216, 258, 145, 169, 172, 185, 197Ð199, 220, 221, 223Ð227, 264, 265, 311 229Ð246, 248Ð258, 297, 298, 312 Roguing, 190, 217Ð219, 235, 311 Quarantine facilities, 234, 244, 250, 254 Role of FAO, 234Ð236 Quarantine for germplasm, 241Ð244 Role of IPGRI and NBPGR, 243Ð246 Quarantine status of plant importations, 233 Role of vegetatively propagated plants, 288 Rosa multiflora,26 Rosa rugosa, 11, 26 R R-PAGE. See Return-polyacrylamide gel electrophoresis Radial immuno diffusion test, 115 (R-PAGE) Radio immunoassay (RIA), 113, 116Ð118 RPCV-1. See Red pepper cryptic-1 (RPCV-1) Radiolabelled probes, 136 RPCV-2. See Red pepper cryptic-2 (RPCV-2) Radish yellow edge virus (RYEV), 24, 57, 91 RP genes. See Replicase protein genes (RP genes) Raphanus raphanistrum, 15, 29 RRSV. See Raspberry ring spot virus (RRSV) Raphanus sativus, 24, 91 RT-PCR. See Reverse transcription PCR (RT-PCR) Rapid immuno filter paper assay (RIPA), 128Ð129 Rubus Chinese seed-borne nepovirus, 25, 60 Raspberry bushy dwarf virus (RBDV), 24, 59, 69, 175, Rubus idaeus, 24, 26, 28, 29, 91, 175, 176, 202, 296 176, 202, 295, 296 Rubus longanobaccus,24 Raspberry latent(Black raspberry latent), 14, 24, 59, 172, Rubus occidentalis, 14, 175, 176 175 Rubus rigidus,14 Raspberry ring spot virus (RRSV), 24, 60, 89, 90, 93, Runner bean mosaic, 25, 108 169, 173, 174, 178, 198, 240, 248 Rye grass cryptic virus (RGCV), 5, 25, 57, 91 RBDV. See Raspberry bushy dwarf virus (RBDV) RYEV. See Radish yellow edge virus (RYEV) RCCV. See Red clover cryptic virus (RCCV) RCMV. See Red clover mottle como virus (RCMV) RCVMV. See Red clover vein mosaic carlavirus S (RCVMV) Safflower mosaic,25 Real-time PCR, 139, 143Ð144 Sambucus nigra,28 Reasons for failure of seed transmission, 93Ð95 Sambucus racemosa, 15, 174 Red clover cryptic virus (RCCV), 5, 24, 57 Sambucus spp.,29 Red clover mottle como virus (RCMV), 25, 58 Santosai temperate, 25, 57 Red clover vein mosaic carlavirus (RCVMV), 6, 16, 25, Satellite RNA, 81, 132, 134, 259, 263Ð264, 266, 311 58 Satsuma dwarf, 25, 60 Red pepper cryptic-1 (RPCV-1), 25, 57 SbMV. See Soybean mosaic virus (SbMV) Red pepper cryptic-2 (RPCV-2), 25, 57 SCMV. See Sugarcane mosaic virus (SCMV) Reducing the rate of virus spread through vector Scopolia sinensis, 23, 172 management, 189Ð192 Scorpion probes, 143 Removal of infected seeds, 186 Secale cereale,30 Reovirus, 62, 64 Sechium edule,21 Repelling surfaces, 196Ð197 Secoviridae, 58Ð61 Replicase protein genes (RP genes), 262Ð263 Seed (sexual propagule), 2 Index 325

Seed certification against plant virus diseases, 215Ð216 Strawberry pallidosis, 26, 58 Seed disinfection by heat, 187Ð188 Subterranean clover mottle, 26, 61, 90, 140, 141, 216 “Seed germplasm” exchange, 236 Success stories of production of virus-free plant Seed health testing, 102, 199, 310 propagules, 293Ð297 Seed transmission of partitiviridae, 5 Sugarcane mosaic virus (SCMV), 6, 8, 69, 104, 140, 178, Seed transmission of viroids, 5Ð7 202, 252, 290, 292, 296 Seed transmission of viruses, 2Ð4, 6, 77, 86, 90, 91, 104 Sunflower mosaic potyvirus, 26, 61 Seed-transmitted plant viruses, 4, 5, 8, 62, 76, 140, 144, Sunflower ringspot ilarvirus,26 172, 206 Sunflower rugose mosaic, 26, 62 Senecio cruentus, 9, 15, 29 Sunn-hemp mosaic virus (SHMV), 111, 262 Senecio vulgaris, 11, 19, 26Ð28, 80, 168 Sunn-hemp rosette, 26, 61 Serological diagnosis, 162, 304 Sweet potato ring spot, 26, 60 Serological techniques, 111Ð132, 198 SYBR green dyes, 144 Setaria italica, 19, 22 Symptomless carriers, 103Ð104, 245, 246 SHMV. See Sunn-hemp mosaic virus (SHMV) Synergism, 69 Silene gallica,20 Sincomas mosaic,24 Single diffusion in tubes, 115 T Single stranded DNA (ssDNA), 137 TaqMan assay, 143 Single stranded RNA (ssRNA), 136, 261 TaqMan real time PCR, 143 SMV. See Squash mosaic (SMV) Taraxacum officinale, 15, 22, 27, 29, 168 Sobemovirus, 9, 61, 62, 64, 76, 77, 110, 177 TBIA. See Tissue blot immuno binding assay (TBIA) Solanum acaule,15 TBSV. See Tomato bushy stunt virus (TBSV) Solanum brevidens,24 Technical guidelines for exchange, 235 Solanum demissum-A,23 Telfairia mosaic, 26, 61 Solanum dulcamara,18 Telfairia occientatis,26 Solanum incanum,23 Tenuivirus, 62, 64 Solanum melongena, 17, 23, 27, 89 Theobroma cocao,14 Solanum nigrum, 24, 26 Thrips vectors, 17, 288, 314 Solanum tuberosum, 11, 18, 23, 24, 27, 174, 296 Tissue blot immuno binding assay (TBIA), 127Ð128 Solanum tuberosum cv. Cara,23 TMV. See Tobacco mosaic virus (TMV) Solanum tuberosum cv.D 42/8,23 TNV. See Tobacco necrosis virus (TNV) SoMV. See Sowbane mosaic virus (SoMV) Tobacco mosaic virus (TMV), 64, 80, 187, 265 Sources of resistance, 201, 203Ð205, 242, 251, 266 Tobacco necrosis virus (TNV), 63, 288 Southern bean mosaic, 13, 61, 64, 68, 77, 89, 90, 93, 94, Tobacco rattle virus (TRV), 64, 109, 144, 167, 171, 264, 123, 174, 207 265 Sowbane mosaic virus (SoMV), 88 Tobacco ring spot virus (TRSV), 64, 69 Soybean mild mosaic, 25, 61 Tobacco streak (Black raspberry latent strain), 175 Soybean mosaic virus (SbMV), 4, 68, 69, 105, 139, 145, Tobacco streak virus (TSV), 9, 63, 69, 90, 95, 172, 175, 186, 198, 202, 207, 223, 313, 316 202, 203, 219 Soybean streak,25 Tobamovirus, , 55, 62, 64, 75, 76, 109, 110, 116, 177, Soybean stunt, 6, 17, 58, 106, 186, 219 187, 204, 205, 257, 259, 262 Spergula arvensis, 17, 28, 69 Tobravirus, 6, 55, 61, 62, 64, 78, 79, 88, 131, 167, 168, Spinach latent virus (SpLV), 25, 59, 187 258 Spinach temperate crypto virus (SpTV), 26, 57 Tomato apical stunt viroid, 6, 28, 60, 79 Spinacia oleracea, 18, 26 Tomato aspermy, 28, 58, 247, 294 SpLV. See Spinach latent virus (SpLV) Tomato black ring, 28, 60, 69, 80, 89, 91, 125, 167, 169, SpTV. See Spinach temperate crypto virus (SpTV) 174, 248 Squash mosaic (SMV), 20, 58, 65, 77, 88, 89, 106, 125, Tomato bushy stunt virus (TBSV), 64, 79 130, 174, 189, 247 Tomato chlorotic dwarf viroid, 60, 64, 79 Stage of infection, 70, 91, 111, 261 Tomato mosaic (ToMV), 29, 61, 80, 87, 109, 111, 125, Stages of seed multiplication, 210 141, 187, 191, 208, 211 Stellaria media, 11, 17, 24, 26, 28, 89, 93, 168, 169, 174 Tomato planta macho viroid, 29, 60 Stone fruit ringspot, 9, 26 Tomato ringspot virus (ToRSV), 168, 173, 176, 198, 240, Storage effect, 189 247Ð249, 262 Stranded and fibrous masses of LIC, 110 Tomato spotted wilt virus (TSWV), 3, 64, 77, 78, 245, Strawberry latent ringspot, 26, 62, 80, 93, 125, 169, 260, 288, 294, 296, 314 248 Tomato streak virus,29 326 Index

Tombusviridae, 57Ð59, 61 Vicia palastina,14 Tombusvirus, 55, 61, 62, 64, 79, 110, 111, 132 VIDE database, 5, 65 ToMV. See Tomato mosaic (ToMV) Vigna angularis, 12, 68 ToRSV. See Tomato ringspot virus (ToRSV) Vigna catjang,16 Tospovirus, 9, 55, 61, 62, 64, 314, 315 Vigna mungo, 12, 13, 29, 77, 92 Transgenic approach, 258Ð259 Vigna radiata, 12, 13, 18, 20, 106 Trapping in electron microscopy, 130 Vigna sesquipedalis, 11, 12, 16, 17 Trichovirus,61 Vigna sinensis, 13, 14, 17, 27, 28, 109 Trifoliate orange,15 Vigna unguiculata, 10, 12, 14, 16Ð18, 23, 26Ð29, 89, 106, Trifolium alexandrinum,10 174, 205 Trifolium hybridum,21 Viola arvensis,27 Trifolium incarnatum,17 Viola tricolor,15 Trifolium michelianum,10 Virgaviridae, 58, 61 Trifolium pratense, 13, 16, 24, 25, 29, 108 Viroids, 5Ð7, 56, 80, 88, 131, 132, 134Ð137, 139, 140, Trifolium repens,30 227, 264, 293Ð295 Trifolium subterraneum, 18, 26 Virus avoidance, 197Ð199 Trigonella balansae,10 Virus-derived resistance, 202, 257Ð258 Trigonella foenumgraecum,15 Virus diagnosis, 113, 131Ð144, 291Ð293 Triticum aestivium, 12, 14, 22, 30, 89, 106 Viruses and seed viability, 69, 189 Tritimovirus,61 Viruses erroneously listed as seed-transmitted, 7Ð31 TRSV. See Tobacco ring spot virus (TRSV) Virus free seed, as control measures, 227 TRV. See Tobacco rattle virus (TRV) Virus-free vegetative planting material, 171, 297 TSV. See Tobacco streak virus (TSV) Virus longevity in seeds, 88 TSWV. See Tomato spotted wilt virus (TSWV) Virus management, 192, 199, 311Ð315 TuMV. See Turnip mosaic virus (TuMV) Virus resistance, 145, 200, 202, 258, 259, 261Ð263, 266, Turnip mosaic virus (TuMV), 111, 192 311 Turnip yellow mosaic (TYMV), 29, 62, 64, 87, 90, 91, Virus resistant cultivars, 314 125 Virus strain / isolate, 90 Tymoviridae, 61, 62 Virus transmission, 75Ð81, 87, 93, 95, 105, 107, 126, 167, Tymovirus, 55, 61, 62, 64, 77, 110, 177 171Ð173, 177, 189, 205, 216, 237, 258, TYMV. See Turnip yellow mosaic (TYMV) 287Ð298 Types of materials received, 233Ð234 Visual examination, 105Ð107 Vitis labrasca,14 Vitis vinifera, 19, 22, 27, 245, 296 U Voandzea subterranea,16 Ulmus americana, 15, 18, 174 Ulmus glabra,18 Umbravirus,62 Urdbeanleafcrinkle, 29, 62, 69, 77, 125 W Use mulches or minimum tillage, 314 Waikavirus, 62, 64 Use of shoot tip grafting or micrografting, 253 Watermelon mosaic virus (WMV), 111, 192, 261 WCCV-1. See White clover cryptic 1 (WCCV-1) V WCCV-2. See White clover cryptic 2 (WCCV-2) Vaccinium corymbosum,14 WCCV-3. See White clover cryptic 3 (WCCV-3) Variability in certain seed-transmitted viruses, 62Ð65 WCLMV. See White clover mosaic virus (WCLMV) Variants of PCR and their applications, 139 Wheat mosaic, 30, 58, 117 Variation in viruses, 178, 221 Wheat soil-borne mosaic, 30, 58, 63, 79, 111 Vector, 2, 62, 67, 75, 92, 109, 166, 186, 288, 310 Wheat streak mosaic (WSMV), 30, 61, 76, 78, 125, 140, resistant cultivars, 206Ð207 141, 240, 262 transmission, 75Ð76, 109, 171, 262, 290, 311 Wheat striate mosaic, 30, 60 Vegetative propagation, 113, 118, 131, 145, 225, 233, White clover cryptic 1 (WCCV-1), 5, 30, 57, 63 237, 244, 250, 251, 254, 288, 290, 292Ð294 White clover cryptic 2 (WCCV-2), 5, 30, 57, 63 Vegetative transmission, 293Ð297 White clover cryptic 3 (WCCV-3), 5, 30, 57 Verbena x hybrida,15 White clover mosaic virus (WCLMV), 108, 140, 262 Vertical transmission, 75, 172, 177 White clover temperate,30 Vicia cryptic, 5, 30, 57, 91, 130 Whitefly vector transmission, 78 Vicia faba, 11, 13, 14, 16, 18, 21, 22, 25, 30, 89, 91, 106, Winged bean ringspot, 30, 58 170, 173, 174 WMV. See Watermelon mosaic virus (WMV) Index 327

World Trade Organization (WTO), 199, 221Ð224, 238, Z 239, 256 Zea mays, 19, 20, 26, 27, 30 WSMV. See Wheat streak mosaic (WSMV) Zinnia elegans, 11, 27 Zucchini yellow mosaic (ZYMV), 30, 61, 140, 141, 207, 259, 261, 313 Y Yield losses, 9, 67Ð70, 126, 185, 196, 198, 212, 217, 223, 259, 288, 290Ð291