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University of Florida Thesis Or Dissertation Formatting IDENTIFICATION OF FUNGI CAUSING LEAF SPOT ON BERMUDAGRASS IN FLORIDA, AND SENSITIVITY OF BIPOLARIS CYNODONTIS TO AZOXYSTROBIN By PRASERT STAVORNVISIT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2015 © 2015 Prasert Stavornvisit To my family and friends ACKNOWLEDGMENTS I would like to thank Dr. Phil Harmon, my major advisor and Dr. Jeff Rollins, committee member for all their guidance and support throughout this process. I thank Dr. Brian Schwartz from University of Georgia for project support. I thank my colleagues, Brenda Rutherford, Anne Vitoreli, Dr. Sladana Bec, Jerry Dewberry and Lydia Munday for their help and instruction. Lastly I would like to thank my family for their patience and understanding. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT .....................................................................................................................................9 CHAPTER 1 LITERATURE REVIEW .......................................................................................................11 Brief History of Fungicide Use in Turfgrass ..........................................................................11 Fungicide Resistance ..............................................................................................................13 History of Fungicide Insensitivity in Turfgrass Pathogens .............................................13 Fungicide Resistance Concept .........................................................................................14 Pathogen Fitness ..............................................................................................................15 Mechanisms of Resistance ..............................................................................................15 Bipolaris Leaf Spot and Melting Out on Bermudagrass .........................................................19 Bipolaris Leaf Blotch and Melting Out ...........................................................................20 Pathogen, Host, Disease Cycle ........................................................................................20 2 SURVEY OF BIPOLARIS SPECIES ISOLATED FROM BERMUDAGRASS GOLF COURSES IN FLORIDA .......................................................................................................22 Introduction .............................................................................................................................22 Materials and Methods ...........................................................................................................23 Sample Collection, Isolation and Storage of Fungal Pathogens ......................................23 DNA Extraction and Amplification .................................................................................24 Sequence and Phylogenetic Analysis ..............................................................................25 Results.....................................................................................................................................25 Isolate Collection .............................................................................................................25 Molecular and Morphological Identification of Bipolaris spp. .......................................26 Discussion ...............................................................................................................................27 3 EVALUATION OF AZOXYSTROBIN RESISTANCE IN BIPOLARIS CYNODONTIS ......................................................................................................................34 Introduction .............................................................................................................................34 Materials and Methods ...........................................................................................................35 Whole Genome Sequencing ............................................................................................35 Primers Sets Used to Attempt Amplification of The Cytochrome b Gene .....................37 Amended Agar Assays ....................................................................................................38 Fungicide Sensitivity Assays ...........................................................................................38 5 In vivo Sensitivity of Bipolaris Isolates to Azoxystrobin Fungicides .............................38 Results.....................................................................................................................................40 Sequencing of Cytb Gene in Bipolaris cynodontis ..........................................................40 Azoxystrobin Sensitivity Assay ......................................................................................40 Pathogenicity Test ...........................................................................................................41 Discussion ...............................................................................................................................41 APPENDIX A: SURVEY FOR BIPOLARIS SPECIES ISOLATED FROM BERMUDAGRASS GOLF COURSES IN FLORIDA .......................................................................................................50 B: EVALUTION OF AZOXYSTROBIN RESISTANCE IN BIPOLARIS CYNODONTIS ....52 LIST OF REFERENCES ...............................................................................................................55 BIOGRAPHICAL SKETCH .........................................................................................................66 6 LIST OF TABLES Table page 2-1 Culture collection used in this study ..................................................................................29 2-2 References culture and accession number used in this study ............................................30 2-3 Number of samples with the indicated fungal species isolated by month .........................32 3-1 The primer sets from related fungi .....................................................................................43 3-2 The primer sets designed base on Cochliobolus heterostrophus, scaffold_47:34321- 34542..................................................................................................................................43 3-3 The primer sets designed using R_2014_07_11_13_39_47_user_LIL- 46_Rollins_contig_474 contig (Bipolaris cynodontis cytochrome b gene sequence) .......43 3-4 Results of azoxystrobin amended media assay ..................................................................44 3-5 Results of azoxystrobin amended media assay for species other than Bipolaris cynodontis ..........................................................................................................................45 7 LIST OF FIGURES Figure page 2-1 Distribution of the species identified in the samples submitted from golf course in 22 Florida counties ..................................................................................................................32 2-2 The phylogenetic tree constructed using studied isolates, reference sequences, and the maximum likelihood method .......................................................................................33 3-1 DNA sequence of the contig containing a partial cytochrome b gene sequence of Bipolaris cynodontis isolate 1318 (exon in red and intron in black). Primer Cytbfwd 4 is highlighted in yellow and Cytbrvs 4 reverse complement is highlighted in green. ....46 3-2 Alignment of DNA sequences coding for amino acid residues 127 to 143 of the cytochrome b gene of 38 Bipolaris cynodontis isolates .....................................................47 3-3 Partial amino acid sequences including residues 127 through 143 of the cytochrome b gene from 38 Bipolaris cynodontis isolates. Line 13 shows the F129L mutation found in isolate 1344 ..........................................................................................................48 3-4 Levels of disease severity from isolate 1326, 1344, and 1375. Control pots were inoculated with water only. The w treated pots were sprayed with water and the a treated pots were treated with Heritage fungicide (azoxystrobin). ....................................49 8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science IDENTIFICATION OF FUNGI CAUSING LEAF SPOT ON BERMUDAGRASS IN FLORIDA, AND SENSITIVITY OF BIPOLARIS CYNODONTIS TO AZOXYSTROBIN By Prasert Stavornvisit December 2015 Chair: Philip F. Harmon Major: Plant Pathology Bipolaris leaf blotch and melting out are destructive diseases of bermudagrass, paspalum, and zoysiagrass maintained as golf course putting greens and fairways. Bipolaris cynodontis is the most common fungal pathogen reported to cause the disease on bermudagrass (Cynodon spp.). In 2012, golf course superintendents who utilized the UF IFAS Plant Diagnostic Center (PDC) Rapid Turfgrass Diagnostic Service
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