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Xanthomonas in Florida MULTI-LOCUS AND WHOLE-GENOME SEQUENCE ANALYSIS OF PSEUDOMONADS AND XANTHOMONADS IMPACTING TOMATO PRODUCTION IN FLORIDA By SUJAN TIMILSINA 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 2016 © 2016 Sujan Timilsina To my Family ACKNOWLEDGMENTS I would like to take this opportunity to express my gratitude towards Dr. Gary E. Vallad, committee chair and Dr. Jeffrey B. Jones, co-chair, for their constant support, encouragement and guidance throughout my graduate studies. I couldn’t have done this without their scientific inputs and personal mentorship. I would also like to thank Dr. Erica M. Goss for all her advice, recommendations and support. I would also like to extend my gratitude to my committee members, Dr. Bryan Kolaczkowski and Dr. Sam Hutton for their valuable suggestions and guidance. Very special thanks to Gerald V. Minsavage, for all his expertise, creativity, recommendations and constructive criticism. We collaborated with Dr. Frank White, Dr. Brian Staskawicz and Dr. Jim Preston for some aspects of my research and to write articles and reviews. I would like to thank them all for providing me the opportunity. During my PhD, I had the privilege to work with colleagues from the 2560 Jones lab in Gainesville and Vegetable Pathology lab in Balm and I thank the lab family. I appreciate the time and technical support from Dr. Neha Potnis. I thank my labmates Amanda Strayer, Juliana Pereira, Serhat Kara, Eric A. Newberry, Alberto Gochez, Yang Hu, and Deepak Shantaraj and numerous lab mates over the years for their co- operation and assistance. I would also like to thank all my Nepalese friends in Gainesville for their lively company during my stay here. I also want to thank my parents and brother, who helped me to shape my career. I specially thank my wife, Naweena, for her undying support, encouragement and patience. Thank you all for making this possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 12 Pseudomonas in Florida ......................................................................................... 12 Pseudomonas Characterization .............................................................................. 13 Taxonomy of Pseudomonas ................................................................................... 13 Xanthomonas in Florida .......................................................................................... 14 Population Studies of Bacterial Spot Causing Xanthomonas Species .................... 16 Type III Secreted Effectors ..................................................................................... 17 Project Goals and Objectives .................................................................................. 19 2 PHYLOGENETIC STUDY REVEALS A DISTINCT PHYLOGROUP OF PSEUDOMONAS CICHORII IN FLORIDA ............................................................. 21 Introduction ............................................................................................................. 21 Materials and Methods............................................................................................ 23 Bacterial Strains and Isolation .......................................................................... 23 Pathogenicity Tests .......................................................................................... 23 PCR Amplification and Gene Sequencing ........................................................ 24 Phylogenetic Analysis ...................................................................................... 25 GenBank Accession Numbers .......................................................................... 26 Results .................................................................................................................... 26 Isolation and Phenotypic characterization ........................................................ 26 Pathogenicity Test ............................................................................................ 26 Genetic Characterization .................................................................................. 27 Discussion .............................................................................................................. 29 3 PSEUDOMONAS FLORIDAE SP. NOV., A NOVEL BACTERIAL PATHOGEN ISOLATED FROM TOMATO .................................................................................. 39 Introduction ............................................................................................................. 39 Materials and Methods............................................................................................ 41 Bacterial Strains and Preliminary Characterization .......................................... 41 Biochemical and Physiological Tests. .............................................................. 41 Pathogenicity Assay ......................................................................................... 42 5 Gene Sequencing and Sequence Analysis ...................................................... 42 Biolog Assay and Fatty Acid Profiles ................................................................ 43 Whole Genome Sequence Comparison ........................................................... 44 Results .................................................................................................................... 44 Bacterial Characterization ................................................................................ 44 Pathogenicity Assay ......................................................................................... 45 Sequence Analysis ........................................................................................... 45 Biolog Assay and FAME-ID .............................................................................. 46 Whole Genome Sequence Analysis ................................................................. 47 Discussion .............................................................................................................. 48 4 ANALYSIS OF SEQUENCED GENOMES OF XANTHOMONAS PERFORANS IDENTIFIES CANDIDATE TARGETS FOR RESISTANCE BREEDING IN TOMATO ................................................................................................................ 60 Introduction ............................................................................................................. 60 Materials and Methods............................................................................................ 65 Bacterial Strains Collection and Pathogenicity Testing .................................... 65 Gene Sequencing ............................................................................................. 65 Sequence Analysis ........................................................................................... 66 Sequence Submission ...................................................................................... 67 Results .................................................................................................................... 67 Pathogenicity and Race Determination ............................................................ 67 Grouping of T3 Strains by gapA Sequence ...................................................... 67 Effector Gene Sequences ................................................................................ 68 Presence of AvrBsT in T3 X. perforans ............................................................ 69 Discussion .............................................................................................................. 70 5 CORE GENOME MULTILOCUS SEQUENCE TYPING OF XANTHOMONAS PERFORANS ......................................................................................................... 87 Introduction ............................................................................................................. 87 Materials and Methods............................................................................................ 90 Bacterial Strains and Genome Sequencing ...................................................... 90 Multilocus Sequence Analysis .......................................................................... 91 Results .................................................................................................................... 93 Bacterial Strains and Multilocus Sequence Analysis ........................................ 93 Core Gene Comparisons .................................................................................. 93 Phylogenetic Analysis ...................................................................................... 95 Discussion .............................................................................................................. 96 6 SUMMARY AND CONCLUSION .........................................................................
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