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Fitness, Molecular Characterization and Management of Bacterial Leaf Spot in Tomatoes

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Fitness, Molecular Characterization and Management of Bacterial Leaf Spot in Tomatoes FITNESS, MOLECULAR CHARACTERIZATION AND MANAGEMENT OF BACTERIAL LEAF SPOT IN TOMATOES By PETER ABRAHAMIAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Peter Abrahamian ACKNOWLEDGEMENTS I would like to thank my major advisor Dr. Gary Vallad for his extensive guidance, advising and help throughout my PhD studies. Dr. Vallad was always encouraging and supportive of pursuing new ideas. I also enjoyed all the times we discusses science. You are an awesome advisor! I also thank Dr. Jeffrey Jones my co-advisor for his extensive support, help and openness throughout my studies. I want to thank my committee members Drs. Erica Goss, Mathews Paret and Samuel Hutton for their constructive and insightful comments for improving the quality of my research. I would like to thank all the members of the Vallad Lab at GCREC. Thank you Ai-vy Riniker for being there for me when I first started working at Dr. Vallad’s lab. I also thank Rebecca Willis, Scott Hughes and Steve Kalb for their excellent technical help in securing, maintaining and harvesting tomato plants throughout the work conducted at GCREC. I also thank Dr. Aimen Wen, Heather Adkison and Late Wilson and my colleagues, Tyler Jacoby, Caroline Land, and Andy Shirley, for all the fun times and stimulating talks at the lab. Special thanks to Sujan Timilsina for all helping me with the bioinformatics work and to Sushmita KC for helping in collecting and processing samples in the field and greenhouse experiments. I want to thank also Dr. Neha Potnis and Jerry Minsavage at the Jones lab in Gainesville for training me in the techniques required for this study. Finally, I would like to thank my parents and family who accompanied me throughout this journey that started a long time ago. Thank you for your patience and continuous support! I also thank my brother, Roy, for his encouragement. 3 TABLE OF CONTENTS page ACKNOWLEDGEMENTS .............................................................................................................3 LIST OF TABLES ...........................................................................................................................6 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................................10 CHAPTER 1 INTRODUCTION ..................................................................................................................12 Hosts and Symptomology ......................................................................................................13 Distribution and Epidemiology ..............................................................................................14 Phenotypic and Genetic Diversity ..........................................................................................17 Pathogenicity and Effectors ....................................................................................................22 Disease Control ......................................................................................................................27 2 THE TYPE III EFFECTOR AVRBST ENHANCES XANTHOMONAS PERFORANS FITNESS IN FIELD-GROWN TOMATO ............................................................................32 Materials and Methods ...........................................................................................................36 Bacterial Strains, Plasmids and Plants ...............................................................................36 Mutant Construction ..........................................................................................................37 In planta Growth and Competition Assays ........................................................................38 Field Trials .........................................................................................................................39 Colony Testing ...................................................................................................................40 Statistical Analyses ............................................................................................................41 Results ....................................................................................................................................41 In planta Activity and Leaf Infiltration Assays .................................................................41 Disease Severity in Field Plants .........................................................................................42 Bacterial Populations in the Field ......................................................................................43 Effect of avrBsT on Movement..........................................................................................44 Effect of avrBsT on Strain Recovery .................................................................................45 Discussion...............................................................................................................................46 3 TRACING XANTHOMONAS PERFORANS POPULATIONS ON TOMATO FROM GREENHOUSE- TO FIELD BY WHOLE-GENOME SEQUENCING ..............................59 Materials and Methods ...........................................................................................................62 Bacterial Strains and Race Characterization ......................................................................62 Genome Sequencing and de novo Assembly .....................................................................63 Characterization with Multi-Locus Sequence Analyses ....................................................63 4 Core-Genome Multi-Locus Sequence Typing ...................................................................64 Single-Nucleotide Variations Calling ................................................................................64 Phylogenetic Analyses .......................................................................................................65 Effector Analyses ...............................................................................................................66 Results ....................................................................................................................................66 Genomes and Average Nucleotide Identities .....................................................................66 Multi-Locus Sequence Analyses........................................................................................66 Core Genome Comparison .................................................................................................67 Genome-Wide SNP Analyses ............................................................................................68 Race Characterization and Effector Profiles ......................................................................70 Discussion...............................................................................................................................71 4 EFFICACY OF COPPER ALTERNATIVES FOR MANAGEMENT OF BACTERIAL SPOT ON TOMATOES VARIES IN TRANSPLANT AND FIELD PRODUCTION ........90 Materials and Methods ...........................................................................................................93 Bacterial Strains, Inoculation and Plants ...........................................................................93 Greenhouse Trials ..............................................................................................................94 Field Trials .........................................................................................................................95 Statistical Analyses ............................................................................................................96 Results ....................................................................................................................................97 Efficacy of BST Reduction in Tomato Seedlings ..............................................................97 BST Disease Level in Field-Grown Tomato .....................................................................99 Discussion.............................................................................................................................101 5 SUMMARY AND DISCUSSION ........................................................................................114 APPENDIX A ENVIRONMENT AND STATISTICS TABLES ................................................................119 B ADDITIONAL STRAIN CHARACTERIZATION DETAILS ...........................................128 LIST OF REFERENCES .............................................................................................................144 BIOGRAPHICAL SKETCH .......................................................................................................160 5 LIST OF TABLES Table page 2-1 List of bacterial strains and plasmids .................................................................................51 3-1 List of Xanthomonas strains characterized and used in this study .....................................78 3-2 Average number of SNPs, coverage and effects on coding domain sequence for each phylogenetic group .............................................................................................................80
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