Exploring the drivers of Xanthomonas population dynamics on tomato and pepper By Rishi Ram Bhandari A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama December 14, 2019 Keywords: Xanthomonas, pathogen diversity, copper resistance, metagenomics, phage, integrative and conjugative elements Copyright 2019 by Rishi Ram Bhandari Approved by Neha Potnis, Assistant Professor of Plant Pathology Leonardo De La Fuente, Professor of Plant Pathology Mark R. Liles, Professor of Biology Abstract Bacterial leaf spot (BLS), caused by four species of Xanthomonas; X. euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri, is an endemic disease of tomato and pepper in Southeastern United States and can result in total crop loss and there are no clear management strategies for this disease. Host resistance is considered as most important and effective method of control of BLS disease. There are some resistant cultivars of peppers against BLS which but the occurrence of the pathogenic races overcoming the resistance genes in tomato resulted in no such resistant cultivars in tomato. Although chemical control based on copper bactericides has been used extensively since decades, occurrence of copper-resistant bacterial pathogens are reducing the efficacy of copper-based bactericides. As majority of antibiotics and heavy metal resistance genes are encoded in plasmids, frequent plasmid transfer among the strains plays an important role in spreading the resistance in field. Recently we found two copper resistant strains of X. perforans without these copper resistance plasmids and integration of copper resistance genes in the chromosome. The goal of this project was to identify the pathogen population diversity of BLS Xanthomonas in Alabama using culture dependent and culture independent (shotgun metagenomics) techniques. Moreover, we also studied the chromosomal copper resistance dynamics in X. perforans population and their transfer potential both in planta and in-vitro studies. Sequencing and phylogenetic classification of 8 representative strains from the collection of 150 strains of BLS Xanthomonas from different places of Alabama suggest the presence of two novel sequence clusters within X. perforans. Culture-independent study of ii diversity using shotgun metagenomics from tomato, pepper and weed samples showed X. perforans and X. euvesicatoria as a dominant pathogen in tomato and pepper respectively. Shotgun metagenomics also showed the co-infection by multiple species/genera in the field at a given time and provided strain-level resolution showing the presence of two or more lineages of X. perforans in tomato samples. The result further suggest that shotgun metagenomics can be used for higher resolution of pathogen population structure in diversity studies. We also found copper tolerant X. perforans strains Xp 2010 and GEV 904 contained chromosomal encoded copper resistance genes in the genomic island. We further studied the structure of this island and investigate its transfer potential under in-planta and in-vitro conditions. Failure to obtain transconjugants/transductants under both conditions indicates that either the conditions we used for the transfer were not conducive or the transfer frequency is very low. Overall, these study of the pathogen population dynamics helps in providing the insight of bacterial population situation in Alabama. Thus, protective measures can be taken before the outbreak of pathogen in a large scale. The long-term goal of this research is to improve our understanding about pathogen population in Alabama and pathogen dynamics in both host and non-host. This study will help the farmers and large-scale growers to select more resistant/tolerant cultivars and look forward for resistant breeding program based on pathogen population information. Keywords: Xanthomonas, pathogen diversity, copper resistance, metagenomics, phage, integrative and conjugative elements iii Acknowledgements I would like to express my sincere gratitude to my major advisor, Dr. Neha Potnis for giving me the great opportunity to work on this interesting project and for her guidance. I am thankful for her constant encouragement, constructive criticisms and suggestions throughout my research and scientific writing. I am also thankful to my committee members Dr. Leonardo De La Fuente and Dr. Mark R. Liles for their guidance, valuable suggestions and readiness to help me throughout my research and writing of thesis. Special thanks to Dr. Eric Newberry for teaching me the basics of lab and various bioinformatics tools. I would also like to thank my lab members Prabha Liyanapathiranage and Shreya Sadhukhan for suggestions, help and encouragement during my research work. I am grateful to Ambika and my whole Auburn Nepali family for their support and love. I owe all my success to my parents, brothers and sisters for their support and unconditional love. iv Table of Contents Abstract ...................................................................................................................................... ii Acknowledgements ................................................................................................................... iv List of Tables ........................................................................................................................... vii List of Figures ........................................................................................................................ viii 1. CHAPTER ONE ................................................................................................................. 2 Introduction and Literature Review ....................................................................................... 2 Introduction ............................................................................................................................ 2 Bacterial leaf spot of tomato and pepper: an endemic plant pathogen of Southeast United States ...................................................................................................................................... 3 Pathogen population diversity of BLS Xanthomonas in Florida ........................................... 4 BLS disease symptoms, epidemiology and disease cycle ...................................................... 5 Control strategies for BLS Xanthomonas .............................................................................. 7 Antibiotic and copper resistance in BLS Xanthomonas....................................................... 10 References ............................................................................................................................ 13 2. CHAPTER TWO .............................................................................................................. 28 Population structure of BLS Xanthomonas in Alabama ...................................................... 28 Abstract ................................................................................................................................ 28 Introduction .......................................................................................................................... 29 Materials and methods ......................................................................................................... 32 Bacterial strain collection, sequencing, and assembly for culture-based population diversity of Xanthomonas in Alabama ............................................................................. 32 Reconstruction of the X. perforans core genome ............................................................. 32 Sample collection and DNA extraction for culture independent method to understand the population diversity of Xanthomonas in Alabama ........................................................... 33 Databases preparation and metagenome read alignment for strain level profiling of metagenomic samples ....................................................................................................... 34 Results .................................................................................................................................. 35 Diversity of BLS reveals presence of novel sequence clusters in X. perforans population in Alabama ........................................................................................................................ 35 Microbial taxonomic abundance based on mOTU reveals mixed infection with different microbial genera ............................................................................................................... 36 v Strain level resolution of metagenomic samples reveals the presence of more than one SCs and co-infection with different species in the same field .......................................... 36 Discussion ............................................................................................................................ 39 References ............................................................................................................................ 44 3. CHAPTER THREE .......................................................................................................... 61 Characterization of chromosomally encoded copper resistance in Xanthomonas perforans and investigate its transfer potential under in-vitro and in planta conditions ...................... 61 Abstract ...............................................................................................................................