Laboratory Manual for the Diagnosis of Cassava Virus Diseases www.iita.org Laboratory Manual for the Diagnosis of Cassava Virus Diseases Compiled by P Lava Kumar and James Legg International Institute of Tropical Agriculture www.iita.org About IITA The International Institute of Tropical Agriculture (IITA) is an international non-profit R4D organization founded in 1967 as a research institute with a mandate to develop sustainable food production systems in tropical Africa. It became the first African link in the worldwide network of agricultural research centers supported by the Consultative Group on International Agricultural Research (CGIAR) formed in 1971. Mandate crops of IITA include banana & plantain, cassava, cowpea, maize, soybean and yam. IITA operates throughout sub-Saharan Africa and has headquarters in Ibadan, Nigeria. IITA’s mission is to enhance food security and improve livelihoods in Africa through research for development (R4D), a process where science is employed to identify problems and to create development solutions which result in local production, wealth creation, and the reduction of risk. IITA works with the partners within Africa and beyond. For further details about the organization visit: www.iita.org and www.cgiar.org. Headquarters: International mailing address: IITA, PMB 5320, Ibadan, Oyo State, Nigeria IITA, Carolyn House, Tel: +234 2 7517472, (0)8039784000 26 Dingwall Road, Croydon Fax: INMARSAT: 873761798636 CR9 3EE, England E-mail: [email protected] United Kingdom Copyright © 2009 IITA IITA holds the copyright to its publications but encourages duplication of these materials for noncommercial purposes. Proper citation is requested and prohibits modification of these materials. Permission to make digital or hard copies of part or all of this work for personal or classroom use is hereby granted without a formal request provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and full citation. Copyright for components not owned by IITA must be honored and permission pursued with the owner of the information. To copy otherwise, to republish, to post on servers, or to redistribute to lists, requires prior specific permission. Contact [email protected] for further details. ii Preface “Laboratory Manual for the Diagnosis of Cassava Virus Diseases” is prepared for the benefit of participants of the regional training for the Disease Objective of Great Lakes Cassava Initiative (GLCI) on ‘Cassava Viruses: Biology, Diagnostics and Management’ held from 28 October – 6 November, 2009 at IITA, Dar es Salaam, Tanzania. The objective of this course is to train the ‘trainers’ from the GLCI Project National Program Partners in Burundi, Democratic Republic of Congo, Kenya, Rwanda, Tanzania and Uganda, in cassava virus disease biology, diagnostics and management to facilitate the capacity building in cassava disease diagnostics and management within the six target countries. This manual, a modified version of our previous laboratory manual, provides basic principles and offers step-by-step protocols for virus diagnosis using molecular assays for the detection of major viruses infecting cassava in sub-Saharan Africa. The polymerase chain reaction (PCR)-based diagnostic methods described in this manual are based on our experience over the years and involve contributions from many of the past and present members of our research units. Some of the descriptions and protocols have been adapted from work done elsewhere and the source of this information has been duly credited. Literature pertinent to theoretical and practical aspects of plant virology and disease diagnosis has been provided. These methods can also be used with appropriate modifications for the diagnosis of plant virus infecting other crops. We sincerely thank Prof. Mike Thresh and Dr Maruthi MN Gowda (Natural Resource Institute, UK), Dr DJ Kim (IITA-Kenya), Dr Edward Kanju (IITA-Tanzania), Mr Innocent Ndyetabula (ARI-Tanzania) and Dr Julian Smith (FERA, UK), for providing expertise and support to the organization of the training course. We would also like to acknowledge support of IITA-Tanzania staff, Rudolph Shirima, Constantine Busungu, Simon Jeremiah, Simon Boniface, Neema Lazaro and Sophia Swai. We are grateful to Dr Paula Bramel, Deputy Director General (Research-for Development), IITA, and Dr Victor Manyong, Regional Director (Eastern and Central Africa), IITA, for their support and encouragement. This course is funded from the Disease Objective component of the CRS-led GLCI project. James Legg P Lava Kumar iii iv Index Content Pages Abbreviations 1 List of symbols 2 I Background on Virus Disease Diagnosis 3 - 22 Lava Kumar and P Sreenivasulu 1 Diagnosis of virus diseases 3 2 Plant virus isolation and purification 13 3 Serological and nucleic acid based methods for plant viruses 15 4 Screening germplasm for virus resistance 23 II Laboratory protocols 25 – 55 5 Nucleic acid based methods 27 Lava Kumar 5.1 Isolation of total RNA form leaf tissues 27 5.2. Procedure for the isolation of total DNA for virus detection by PCR 30 5.3. Direct sample preparation for PCR/RT-PCR Assays 30 6 Polymerase chain reaction (PCR) 31 Lava Kumar 6.1 PCR 32 6.2 Reverse transcription (RT)-PCR 33 6.3. Two step RT-PCR 34 6.4 One-step RT-PCR 35 7 Gel electrophoresis of PCR and RT-PCR products 37 Lava Kumar 8 Multiplex PCR/RT-PCR for the simultaneous detection of cassava mosaic 39 begomoviruses and Cassava brown streak virus Lava Kumar, Maruthi Gowda, DJ Kim and J Legg 9 Guidelines about taking GPS coordinates in the field 43 Kai Sonder 10 Protocols for whitefly identification, rearing, virus transmission and silver leaf 45 testing in laboratory conditions Maruthi Gowda 11 Protocol for cassava pest and disease monitoring and sampling procedure 49 James Legg III Appendices 59 – 81 Compiled by Lava Kumar and James Legg A1 List of commonly used methods for the detection of plant viruses 60 A2 Common conversions 61 A3 Basic requirements for establishing ELISA and PCR-based diagnostic facility 62 A4 Useful virology resources 64 A5 Glossary of common terms in virology and diagnostics 66 A6 Program of the training course 76 A7 Contact address of participants and resource persons 78 A8 Group photograph 80 A9 Cassava virus diseases in Africa 81 James Legg and Mike Thresh The epidemiology of African plant viruses: basic principles and practices Mike Thresh and Denis Fargette v vi Abbreviations cv Cultivar DNA Deoxyribonucleic acid dH2O Distilled water dNTPs Deoxynucleotide triphosphates ELISA Enzyme-linked immunosorbent assay EM Electron microscope IC-PCR Immuno Capture-Polymerase Chain Reaction IC-RT-PCR Immuno Capture-Reverse Transcription-Polymerase Chain Reaction Ig Immunoglobulin IgG Immuno-J-globulin mol. wt. Molecular weight kb Kilo base kbp Kilo base pair kDa Kilo Dalton PCR Polymerase chain reaction RNA Ribonucleic acid RT-PCR Reverse transcription-polymerase chain reaction SEM Scanning electron microscope TEM Transmission electron microscope VLP Virus-like particles ACMV African cassava mosaic virus CBSD Cassava brown streak disease CBSV Cassava brown streak virus CMD Cassava mosaic disease CMBV Cassava mosaic begomoviruses CMGs Cassava mosaic geminiviruses EACMV East African cassava mosaic virus EACMCV East African cassava mosaic Cameroon virus EACMKV East African cassava mosaic Kenya virus EACMMV East African cassava mosaic Malawi virus EACMV-UG East African cassava mosaic virus-Uganda EACMZV East African cassava mosaic Zanzibar virus ICMV Indian cassava mosaic virus SACMV South African cassava mosaic virus SLCMV Sri Lankan cassava mosaic virus 1 List of Symbols/Units A Absorbance cm Centimeter ºC Degree centigrade g grams h hours l Liter k Kilo lb/sq.in Pounds per square inch M Moles m Meter mM Millimoles mm millimeter min minutes ml Milliliter mg Milligram P Micro Pl Micro Pg Microgram ng Nanogram nm Nanometer OD Optical density pH Hydrogen ion concentration % Percent rpm Revolutions per minute sec Seconds v Volume w Weight 2 1. Diagnosis of Virus Diseases Plant viruses cause major losses to agricultural 4) Inoculate (using plant sap, by grafting crops around the world. Chemical agents or vector) to a range of test plants and similar to fungicides and bactericides are not back inoculate to a parallel range of effective to control virus diseases. Strategies test plants to check possible multiple for virus management are mostly aimed at infections and to determine host range eradicating the source of infection to prevent it and symptoms. Compare symptoms from reaching the crop and interfering with the observed on experimental host range movement of vectors to prevent the spread of in literature for clues to identify the the disease. However, the most effective probable virus. Select systemically means of controlling virus diseases is through infected host for virus propagation for cultivating the virus-resistant varieties. Precise purification purpose; local lesion host identification of the causal agent is the first step for virus assays; and diagnostic in management of virus diseases. Although species, which react uniquely to that accurate description of symptoms is necessary particular causal virus. to describe the disease, virus diagnosis should 5) Determine the persistence of infectivity not be based on symptoms alone, because in sap extracts (dilution end point, several unrelated viruses cause similar thermal inactivation point, stability and symptoms and same virus or its strains can retention