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Fusarium Oxysporum FORMAE SPECIALES ASSOCIATED with ORNAMENTAL PALMS

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Fusarium Oxysporum FORMAE SPECIALES ASSOCIATED with ORNAMENTAL PALMS DIAGNOSTICS, GENOMICS AND POPULATION STUDIES ON Fusarium oxysporum FORMAE SPECIALES ASSOCIATED WITH ORNAMENTAL PALMS By SUSHMA V. PONUKUMATI 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 Sushma V. Ponukumati To my family for their unconditional love and support ACKNOWLEDGMENTS I would like to express my gratitude to Dr. Monica Elliott, my advisor, for supporting me in my research in all possible ways. She always encouraged me to be a passionate, independent researcher and gave me freedom to explore new research fields. She helped me to develop the habits of logical thinking and proper planning. Dr.Elliott selfless time and care were sometimes all that kept me going during the writing of this dissertation. I would like to thank Dr. Jeffrey Rollins for inducing passion towards bioinfomatics and helping me understand the importance of critical thinking. I am also appreciative to Dr. Rollins for his constant guidance and dedication in his role as co-advisor. Profound gratitiude goes to my other Ph.D committee members, Dr. Mathew Smith and Dr. Robin Giblin Davis, for serving on my committee and for providing their time and expertise. Their advice and recommendations were valuable resources for my dissertation. I will always cherish Dr. Smith for his inspirational chats. Dr.Smith has been a good role model in teaching, who encouraged and expected students to think independently with a purpose regarding any experiment or project. I am also thankful to Dr. Robin Giblin Davis for his constant support and motivation. My profound gratitude goes to Dr. Jose C. Huguet Tapia who took extreme care and time to teach me bioinformatics. He was always available to trouble shoot and advise me regarding any bioinformatics related questions during the dissertation. A special mention goes to Ms. Beth Des Jardin for providing me a good support system in Dr. Elliott’s lab. She encouraged me and gave fantastic plant pathology skills training. 4 I would like to extend thanks to many people (friends, Dr. Elliott’s interns and co- graduate students) both at Fort Lauderdale Research and Extension Center and Plant Pathology Department in Gainesville who have supported me by their friendship and their advice. I would like to thank my husband Rajasekhar Ponukumati for his unconditional love and support; without it, this dissertation would not be possible. Finally, I express my gratitude to my parents and in-laws, my brother and sister for their belief in me and for their unending love and encouragement. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .......................................................................................................................... 4 LIST OF TABLES ...................................................................................................................................... 9 LIST OF FIGURES .................................................................................................................................. 11 ABSTRACT ............................................................................................................................................. 13 CHAPTER 1 LITERATURE REVIEW .................................................................................................................. 15 Introduction on Palms ....................................................................................................................... 15 Fusarium oxysporum ......................................................................................................................... 16 Fusarium Wilt Pathogens in Palms ............................................................................................ 18 Fusarium oxysporum Pathogenesis ........................................................................................... 23 Cell wall degrading enzymes ............................................................................................. 24 Signaling proteins .............................................................................................................. 24 Transcriptional factors ....................................................................................................... 25 Blocking host defenses ...................................................................................................... 25 Fusarium oxysporum Genomic Studies ..................................................................................... 26 Secreted in Xylem Gene Family ................................................................................................. 28 Objectives ......................................................................................................................................... 30 2 DIAGNOSTIC PRIMER PAIR TO IDENTIFY FUSARIUM WILT PATHOGENS ON ORNAMENTAL PALMS IN FLORIDA ......................................................................................... 32 Introduction ....................................................................................................................................... 32 Material and Methods ....................................................................................................................... 35 Isolates ...................................................................................................................................... 35 Genomic DNA and EF-1α Sequencing ..................................................................................... 36 Primer Design ............................................................................................................................ 36 Genomic DNA Amplification with PFW Primer Pair ............................................................... 37 6 DNA Amplification with Agar Plug and PFW Primer Pair ....................................................... 38 DNA Amplification with the FOA Primer Pair ......................................................................... 39 Phylogenetic Analysis ............................................................................................................... 39 Results............................................................................................................................................... 40 Genomic DNA and EF-1α Sequencing ..................................................................................... 40 Primer Design ............................................................................................................................ 40 Genomic DNA Amplification with PFW Primer Pair ............................................................... 40 DNA Amplification with Agar Plug and PFW Primer Pair ....................................................... 41 DNA Amplification with FOA Primer Pair ............................................................................... 41 Phylogenetic Analysis ............................................................................................................... 41 Discussion ......................................................................................................................................... 43 3 GENOME CHARACTERIZATION OF Fusarium oxysporum f. sp. palmarum .............................. 61 Introduction ....................................................................................................................................... 61 Material and Methods ....................................................................................................................... 66 Isolates ...................................................................................................................................... 66 DNA Extraction and Whole Genome Sequencing ..................................................................... 66 Genome Sequencing .................................................................................................................. 67 Benchmarking Universal Single Copy Orthologs ...................................................................... 68 Comparative Genomics to Identify Orthologous Gene Families ............................................... 68 Identification of Secretory Proteins ........................................................................................... 69 Detection of Carbohydrate Active Enzymes .............................................................................. 69 Detection of Secondary Metabolite Clusters ............................................................................. 70 Secreted in Xylem (SIX) Genes in FOC and FOP ...................................................................... 70 Detection of Virulence Genes in FOP and FOC ........................................................................ 71 Results............................................................................................................................................... 71 Genome Sequencing .................................................................................................................. 71 Benchmarking Universal Single Copy Orthologs ...................................................................... 72 Proteome Analysis of FOC and FOP ......................................................................................... 72 Identifying Orthologous Families among Fusarium Genomes .................................................
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