University of Pretoria etd, Meyer J B (2006) i Banana streak badnavirus (BSV) in South Africa: incidence, transmission and the development of an antibody based detection system Jacolene Bee Meyer Submitted in partial fulfillment of the requirements of the degree Master of Science in the Faculty of Natural & Agricultural Science University of Pretoria Pretoria October 2005 i University of Pretoria etd, Meyer J B (2006) ii ACKNOWLEGEMENTS I would like to thank: My collegues and friends at Du Roi Invitrolab, especially Anne Davson, Mark Hassenkamp, Dr. John Robinson and the rest of the team for their advice, support, financial assistance as well as providing plant material and tissue culture equipment Prof. Gerhard Pietersen for his valuable inputs, proofreading of the thesis, advice and motivation The personnel at the University of Pretoria for their inputs with special thanks to Prof. Louis Nel and Wanda Markotter, as well as my co-students; Orienka Koch, Peter Coetzee and Liz Botha. The personnel at ARC-PPRI with special thanks to Anna Ndala for assistance in sample preparations and Elize Jooste for proofreading certain chapters of the manuscript Kassie Kasdorf for his inputs and for all Electron microscopy work done Connie Frazer for her assistance in the collection of samples from the Burgershall and Kiepersol areas as well as the banana growers who made their farms available for sampling; Rodney Hearne, Jan Prinsloo, Piet Knipe, Flip Basson,, Hannes van der Walt and Dave Hall. The personnel of OVI for their technical and practical assistance, especially Dr. Wouter van Wyngaardt and Cordelia Mashau The Banana Growers Association of South Africa for funding certain aspects of this work INIBAP for funding certain aspects of this work My husband George for his patience, love and support My sister and parents for their love and motivation My Creator for the opportunities He has given to me in life ii University of Pretoria etd, Meyer J B (2006) iii LIST OF ABBREVIATIONS A Absorbance µl Microlitre AP Aspartic protease ARC-PPRI Agricultural Research Council-Plant Protection Research Institute BanMMV Banana mild mosaic virus BBrMV Banana bract mosaic potyvirus BBTV Banana bunchy top babuvirus BC Buffer control BEL BSV express locus BEV Banana endogenous virus Bp Base pairs BSD Banana streak disease BSV Banana streak badnavirus BSV-Cav BSV strain isolated from Williams (Australia) BSV-GF BSV strain isolated from Goldfinger (FHIA 01), Australia BSV-IM / BSV-IRFA BSV strain isolated from Musa cultivar IRFA 914 BSV-Mys BSV strain isolated from Mysore (Australia) BSVMysV Banana streak Mysore virus BSV-Onnè / BSV-OL BSV strain isolated from Nigeria form a hybrid plantain TMP x 7002-1 BSV-RD BSV strain isolated from Red Dacca (Australia) C Antibody constant region CaMV Cauliflower mosaic caulimovirus CARBAP Centre African de Recherches sur Bananiers et Plantains CaVMV Cassava vein mosaic cavemovirus CaYMV Canna yellow mottle badnavirus cDNA Complimentary deoxyribonucleic acid CIRAD Centre de Coopèration Internationale en Recherche Agronomique pour le Dèveloppement CMV Cucumber mosaic cucumovirus CP Coat protein CSSV Cacao swollen shoot badnavirus CoYMV Commelina yellow mottle badnavirus CYMV Citrus yellow mosaic badnavirus DAS Double antibody sandwich DBV Dioscorea bacilliform badnavirus DEPC Diethyl pyrocarbonate DNA Deoxyribonucleic acid DTT Dithiothreitol ELISA Enzyme linked immunosorbent assay EM Electron microscopy EMBRAPA Empresa Brasiliera de Pesquisa Agropecuaria ERPV Endogenous pararetrovirus FHIA Fundacion Hondurena de Investigacion Agricola FHIA-01 Musa cultivar, synonym Goldfinger H Antibody heavy chain HBV Hepatitis B virus HC Healthy Control HeRV Human endogenous retrovirus IC Immunocapture Ig Immunoglobulin iii University of Pretoria etd, Meyer J B (2006) iv IITA International Institute for Tropical Agriculture INIPAB International Network for Banana and Plantain ISEM Immunosorbent electron microscopy J Joining segment on antibody KTSV Kalanchoe top-spotting badnavirus L Antibody light chain LTR Long terminal repeat M Molar Met Methionine mM Millimolar mRNA Messenger ribonucleic acid MWCO Molecular weight cut off point OD Optical density ORF Open reading frame OVI Onderstepoort Vetenary Institute PC Positive control PCR Polymerase chain reaction PVCV Petunia vein clearing petuvirus PYMV Piper yellow mottle badnavirus RH RNAse H RNA Ribonucleic acid RT Reverse transcriptase RTBV Rice tungro bacilliform tungrovirus Rubisco Ribulose-biphosphate carboxylase oxygenase SbCMV Soybean chlorotic mottle soymovirus SCBV Sugarcane bacilliform badnavirus SFV-3 Simian foamy virus SRV Schefflera ringspot badnavirus TAS Triple antibody sandwich TC Tissue culture tRNA Transcription ribonucleic acid TVCV Tobacco vein clearing cavemovirus UP University of Pretoria UV Ultraviolet V Antibody variable region V L1 One variable region on Chicken light chain v/v Ratio of volume added to volume VH Variable region on heavy chain of antibody VIC Virus Indexing Centre VL Variable region on light chain of antibody w/v Ratio of weight added to volume iv University of Pretoria etd, Meyer J B (2006) v INDEX LIST OF ABBREVIATIONS iii LIST OF FIGURES vii LIST OF TABLES x SUMMARY 1 CHAPTER 1 INTRODUCTION AND AIMS OF STUDY 3 CHAPTER 2 LITERATURE REVIEW 7 CHAPTER 3 DETERMINING BSV INCIDENCE ON TISSUE CULTURE DERIVED NON-CAVENDISH MUSA IN MPUMALANGA 27 CHAPTER 4 CHARACTERISATION OF BSV ISOLATES AND THE PRODUCTION OF AN ANTISERUM 43 CHAPTER 5 SCREENING OF BSV WITH A SYNTHETIC PHAGE- DISPLAYED LIBRARY OF ANTIBODIES DERIVED FROM CHICKEN IMMUNOGLOBULIN GENES 71 CHAPTER 6 TRANSMISSION OF BSV WITH FOUR MEALYBUG SPECIES 89 CHAPTER 7 INCORPORATION OF AN INTERNAL CONTROL FOR BSV IC-PCR 125 APPENDIX 1 LIST OF FINDINGS FROM WHICH SPECIFIC GUIDELINES FOR BSV CONTROL CAN BE FORMULATED 132 APPENDIX 2 SUMMARIZED DESCRIPTION OF LOCATIONS, INDICATING SITES SAMPLED AS WELL AS MUSA SELECTIONS SAMPLED AT EACH SITE 135 ANNEXURE A STANDARD BUFFERS, CHEMICALS AND MEDUIMS 140 ANNEXURE B NUCLEIC ACID EXRACTIONS AND PCR PROTOCOLS 146 ANNEXURE C TAS-ELISA PROTOCOL, DEVELOPED IN CHAPTER 4 OF THIS THESIS, FOR THE DETECTION OF BANANA STREAK BADNAVIRUS 152 ANNEXURE D PANNING WITH SYNTHETIC LIBRARIES 154 ANNEXURE E STANDARD METHODS 155 v University of Pretoria etd, Meyer J B (2006) vi REFERENCES 157 vi University of Pretoria etd, Meyer J B (2006) vii LIST OF FIGURES Figure 2.1 Classification of the Retroelements as published by Hansen and Heslop-Harrison (2004). 14 Figure 2.2 Organization of functional domains of ORF III of ScBV (Bouhida et. al., 1993). 16 Figure 3.1 Layout of Site K 33 Figure 3.2 Schematic representation of a cross section mode of sampling in large plantations. Positions at which samples were taken are indicated by an ‘x’. The figure is not to scale and data points may vary slightly between plantations 34 Figure 3.3 Photograph of a 1% agarose gel, stained with Ethiduim Bromide; a correct sized gel band of 476bp indicates the presence of BS-GF. 39 Figure 3.4 BSD symptoms observed on the leaf lamina of the cultivar High Noon 39 Figure 4.1 Graph of absorbance versus antiserum dilution illustrating the method used to determine specific and non-specific titres of R210 Bleed 1 against BSV infected and healthy plant material. 54 Figure 4.2 Graph of absorbance values obtained in TAS-ELISA with different dilutions for G1F Bleed 2 (Goat, diluted 1:2000 to 1:8000) and R210 Bleed 1 (Rabbit, diluted 1:2000 to 1:8000). 65 Figure 4.3 Photographs of banana streak symptoms on the leaf lamina of plants with accession numbers 00/3050 and 99/0169, illustrating yellow and chlorotic eye spots and streaks. 68 Figure 5.1 An ELISA protocol developed for the evaluation of biotinylated IgG on streptavidin beads. 81 Figure 5.2 Four selection rounds presented by different agar plates representing bacterial colonies carrying phages with antibodies, as signs of possible antigen specificity. 85 Figure 6.1 Photograph of insect cages in which mealybug colonies were reared. 97 vii University of Pretoria etd, Meyer J B (2006) viii Figure 6.2 Schematic representation of the schedule utilized on individual mealybug species, in order to demonstrate the transmissibility of expressed-episomal BSV. 100 Figure 6.3 Photograph illustrating the establishment of non-viruliferous mealybugs on the FH-4 donor plant. 100 Figure 6.4 Ethiduim bromide stained, 1 % agarose gel showing IC-PCR products for BSV-OL. 103 Figure 6.5 Absorbance values (OD-405nm) from TAS-ELISA on TC derived FHIA-21 plants measured four months after propagation. 105 Figure 6.6 Photograph of an ethidium bromide stained agarose gel (1%) showing IC-PCR amplicons of BSV-OL from TC derived FHIA-21 plants. 106 Figure 6.7 Photograph of an ethidium bromide stained agarose gel (1%) showing IC-PCR products of BSV-GF. 106 Figure 6.8 Photograph of an ethidium bromide stained agarose gel (1%) showing IC-PCR amplicons of BSV-RD in TC derived FHIA- 21 plants. 106 Figure 6.9 TAS-ELISA absorbance values (OD-405nm) of Williams plants to be used as recipient or control plants, to be used for the planned transmission studies. 107 Figure 6.10 Gel photograph of a 1% agarose gel stained with ethiduim bromide showing amplicons for BSV-RD after performing PCR on plant sap samples. 109 Figure 6.11 Gel photograph of purified pGEM plasmids digested with EcoR1 to reveal the sizes of DNA inserts. 109 Figure 6.12 Gel photograph of PCR products obtained by using primers T32 and BADNAT1. Lane 1: Molecular marker (Fermentas, SM 1113), Lane 2: FH-4 DNA, Lane 3: FHIA-21 TC1 plant DNA. 109 viii University of Pretoria etd, Meyer J B (2006) ix Figure 6.13 Diagram of the sequence alignment of Clone 1-3 (TC1-3RC), “Musa x paradise clone Musa 6 Banana streak virus” (AF106946) and BSV-OL (BSV-OL.txt). 110 Figure 6.14 Agarose gel showing a correct size amplicon (456bp), from PCR with P.longispinus-specific primers. 112 Figure 6.15 Agarose gel showing a correct size amplicon from PCR with P.