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Detection and Isolation of Homogeneous Genotypes of Citrus Tristeza Virus for Use in Virus Control Through Cross Protection

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Detection and Isolation of Homogeneous Genotypes of Citrus Tristeza Virus for Use in Virus Control Through Cross Protection Detection and isolation of homogeneous genotypes of Citrus tristeza virus for use in virus control through cross protection. By Kirsti Snyders (28121903) Submitted in partial fulfilment of the requirements for the degree Magister Scientiae Microbiology (MSc.) In the Faculty of Natural and Agricultural Science, Department of Microbiology and Plant Pathology University of Pretoria Pretoria, South Africa Supervisor: Prof. G. Pietersen Submitted: April 2015 Declaration of originality I, …………………………........... declare that the thesis/dissertation, which I hereby submit for the degree …………………............................ at the University of Pretoria, is my own work and has not previously been submitted by me for a degree at this or any other tertiary institution. SIGNATURE: ..………………………….. DATE: …………………………………. i Acknowledgements I would like to extend my most sincere gratitude to the following people, as this research project would not have been possible without them: First and foremost, to my heavenly father for giving me the talent and strength to pursue my dreams, for blessing me with amazing people in my life and carrying me through all the challenging times. My family for their unconditional love and support during this chapter in my life. Thank you for the everyday encouragement, especially through the testing parts. My supervisor, Prof. Gerhard Pietersen, for his continuous support and opportunities. Thank you for always making time to help in any possible way. My lab colleagues; David Read, Ronel Viljoen, Jennifer Wayland, Jackie Lubbe and Megan Harris for the support and friendship. I am extremely grateful to have such amazing friends in my life. Dr. Stephanus van Vuuren (Oom Fanie) for providing required information for the write up of my dissertation. Glynnis Cook for all your help and support throughout my entire MSc study. Jonathan Featherston at Onderstepoort, Agricultural Research Council (ARC) – Biotechnology platform for all your help and guidance with my Next-generation sequencing analysis. Citrus Research International (CRI) and University of Pretoria for their financial support throughout this project. The Department of Microbiology and Plant Pathology for the great memories, whether it was work related or a social event. Last, but not the least, all authors of the respective research articles I made use of within my dissertation. Thank you for making it possible to use your research to back my work. ii List of Abbreviations AC Average Coverage Amp Ampicilin AMV-RT Avian myeloblastosis virus - reverse transcriptase ARC Agricultural Research Council ARC-ITSC Agricultural Research Council – Institute for Tropical and Subtropical Crops BLAST Basic Local Alignment Search Tool bp Base pair BrCA Brown citrus aphids BSA Bovine serum albumin CA California CCPP Citrus Clonal Protection Program cDNA Complementary Deoxyribonucleic acid CL Consensus Length cm centimetre CP Major coat protein CPm Minor coat protein CREC Citrus Research Education Center CRI Citrus Research International CTV Citrus tristeza virus DNA Deoxyribonucleic Acid dNTP Deoxynucleotriphosphate iii dsRNA Double stranded Ribonucleic Acid DTT Dithiothreitol E. coli Escherichia coli ELISA Enzyme-linked Immunosorbent Assay FL Florida GFMS Grapefruit Mild Strains gRNA Genomic Ribonucleic Acid g/l grams/litre h hour HEL Helicase HSP Heat shock protein HSP70h Homologue of Heat shock protein 70 IDR Inter-domain region IPTG Isopropyl- ß-D-1 thiogalactopyranoside KCl Potassium Chloride kDa Kilodalton KH2PO4 Potassium Dihydrogen Phosphate km kilometres KPL Kirkegaard & Perry Laboratories, Inc. LB Luria-Bertani LMS Lime Mild Strains LNR-ITSG Landbounavorsingsraad - Instituut vir Tropiese en Subtropiese Gewasse iv M Molar MA Massachusetts Mab Monoclonal antibody MD Maryland MEGA Molecular Evolutionary Genetics Analysis mg milligram MgCl2 Magnesium Chloride min minutes ml millilitre mm millimetre mM millimolar MMM Multiple Molecular Markers MSCP Mild strain cross-protection MT Methyltransferase NaCl Sodium Chloride Na2CO3 Sodium Carbonate Na2HPO4 Disodium Hydrogen Phosphate NaHCO3 Sodium bicarbonate NCBI National Center of Biotechnology Information ng nanogram NGS Next-generation sequencing nm nanometres nt Nucleotide v NTR Non-translated region ORF Open reading frame PBST Phosphate-Buffered Saline/Tween PCR Polymerase Chain Reaction PDR Pathogen derived resistance pH Potential of Hydrogen pmol picomol PRO Papain-like proteases PVP-40 Polyvinylpyrolidone-40 QD Quick decline TRC Total Read Count RdRp RNA-dependent RNA polymerase RNA Ribonucleic acid rpm Revolutions per minute RT Reverse transcription RT-PCR Reverse transcription-Polymerase Chain Reaction SACIP South African Citrus Improvement Program SAT Single aphid transmissions sec seconds sgRNA Sub-genomic Ribonucleic acid siRNA small/short interfering RNA SP Stem pitting SSCP Single strand conformational polymorphisms vi ssRNA Single stranded Ribonucleic acid SY Seedling yellows Taq Taq polymerase TAS-ELISA Triple Antibody Sandwich Enzyme-linked Immunosorbent Assay tRNA Transfer Ribonucleic acid U Units UV Ultra-violet w/v Weight/volume wt Wild-type X-gal Bromo-chloro-indolyl-galactopyranoside µl microliters µM micro Molar µg microgram µg/µl micrograms per microliter µg/ml microgram per millilitre vii LIST OF FIGURES FIGURE NUMBER PAGE Review of literature (CHAPTER 1). 1. Schematic illustration CTV induced symptoms ........................................................ (6) 2. A schematic representation of the CTV genome organization ................................. (8) 3. [A] A colony of brown citrus aphids (Toxoptera citricida), herded by ants on Ladu tangerine (C. reticulata Ladu) foliage, and [B] a colony of melon aphids (Aphis gossypii) on Fairchild tangerine (C. reticulata Fairchild) foliage .......................................................................................................... (11) 4. The classic disease triangle followed by CTV ........................................................ (13) 5. A diagram explaining the technique used to neutralize severe CTV strains using Passiflora species . ................................................................................................ (17) 6. Step-by-step explanation of Illumina Next-generation high throughput sequencing procedure, using bridge amplification to produce millions of double stranded DNA copies ..................................................................................................................... (21) 7. Grouping of the three genotypes obtained from 5'-UTR sequence analysis (Sequence type I, II and III) .................................................................................... (28) 8. [A] Neighbor-net analysis of the complete genomes of the RB isolates from New Zealand and currently available complete CTV genomes. [B] Maximum-likelihood dendrogram of the complete genomes of the RB isolates from New Zealand and currently available complete CTV genomes .......................................................... (30) 9. Neighbor-Joining dendrogram of the currently available complete genomes of the CTV genotypes present, including the two CTV strains HA16-5 and HA18-9 ....... (32) 10. Neighbor-joining dendrogram based on the sequences of the A-region derived from full genome sequences of CTV, obtained from GenBank ...................................... (33) 11. Neighbor-joining dendrogram based on the sequences of the p23 gene derived from full genome sequences of CTV, obtained from GenBank ...................................... (34) CHAPTER 2. 12. Map of the experimental farm, University of Pretoria, showing the sites where random sampling of the different citrus trees was done ...................................................... (67) 13. TAS-ELISA fraction concentration results obtained after partial CTV particle purification, by using polyclonal antibodies CTV 1052 and G604 .......................... (86) viii 14. Bayesian dendrogram showing the phylogenetic relationship between the sequences of amplicons from the p33 RT-PCR of the 6 SAT positive Mexican Lime seedlings 14- 7102, 14-7103, 14-7104, 14-7105, 14-7107 and 14-7132 and the 45 CTV reference genomes (red rectangles) ...................................................................................... (92) 15. Distribution of reads of sub-isolate 14-7102 following the mapping against the p33 gene regions of 45 CTV reference genomes ......................................................... (94) 16. Distribution of reads of sub-isolate 14-7103 following the mapping against the p33 gene region of 45 CTV reference genomes ........................................................... (95) 17. Distribution of reads of sub-isolate 14-7104 following the mapping against the p33 gene regions of 45 CTV reference genomes ......................................................... (96) 18. Distribution of reads of sub-isolate 14-7105 following the mapping against the p33 gene regions of 45 CTV reference genomes ......................................................... (97) 19. Distribution of reads of sub-isolate 14-7107 [1] following the mapping against the p33 gene regions of 45 CTV reference genomes ......................................................... (98) 20. Distribution of reads of sub-isolate 14-7107 [2] following the mapping against the p33 gene regions of 45 CTV reference genomes ......................................................... (99) 21. Distribution of reads of sub-isolate 14-7107 (Combined results) following the mapping against
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