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University of Florida Thesis Or Dissertation Formatting Template

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University of Florida Thesis Or Dissertation Formatting Template TOWARDS ORCHID IPM: TOOLS AND MOLECULAR TECHNIQUES FOR THE DIAGNOSIS AND MANAGEMENT OF SELECTED ORCHID ARTHROPOD PESTS AND DISEASES IN FLORIDA By ROBERT ALLEN CATING 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 2010 1 © 2010 Robert Allen Cating 2 To my Grandmother, who always believed in me 3 ACKNOWLEDGEMENTS There have been many people who have had an extraordinary influence on my research, the writing of this dissertation, and my development as a scientist, most notably my committee members Dr. Aaron Palmateer, Dr. Marjorie Hoy, Dr. Randy Ploetz, Dr. Robert McMillan, and Dr. Wagner Vendrame. I found that my committee was superbly balanced between those members who fostered my desire to get as much diagnostic, extension, and field experience as possible and those who guided me through daily laboratory methods, procedures, and lab management. In addition to my committee members, there were other faculty and staff who were crucial for the completion of this dissertation. Dr. Carol Stiles and Patti Rayside taught me laboratory procedures for working with fungi, Dr. Jeff Jones, Ellen Dickstein, and Jason Hong assisted me frequently with bacteriology, Dr. Jane Polston answered my questions about virology, and Dr. Janice Uchida, at the University of Hawaii, answered my questions directly related to orchid pathology. Dr. Ayyamperumal Jeyaprakash, who was always patient, assisted me in the lab on a daily basis teaching me molecular biology and phylogenetic analysis procedures. There were several commercial orchid growers who donated time and plants, and I am sincerely grateful for the time and interest each of them put into assisting me with my work: Kerry Herndon, Bill Peters, Dr. Martin Motes, Duane and Donna Goodwin, Suzanne Farnsworth, Robert Fuchs, Michael Coronado, Jose Esposito, and Jack Batchelor. I discovered early in my research that it was going to be difficult to obtain funding to perform orchid related research. I express my sincere gratitude to the Redland Orchid Festival, Inc. and the Redland Professional Orchid Growers for coming forward and providing the funding for my work. 4 Also, there were people who supported me in different ways at different times, and I am grateful for their assistance: Roger, Jesse, and Cielo Shannon, Heidi Bowman, Amit Gupta, Mauro Tudares, Rio St. John, Tara Tarnowski, Asha Brunings, Linley Dixon, and Dalia and Robert Stubblefield. 5 TABLE OF CONTENTS page LIST OF TABLES .........................................................................................................................10 LIST OF FIGURES .......................................................................................................................11 ABSTRACT ...................................................................................................................................13 CHAPTER 1 LITERATURE REVIEW .......................................................................................................15 Introduction .............................................................................................................................15 Integrated Pest Management ...................................................................................................18 What is Integrated Pest (Disease) Management? ............................................................18 Management Tactics of an Integrated Pest Management Program .................................20 Mechanical and physical control ..............................................................................21 Cultural control ........................................................................................................22 Chemical control ......................................................................................................22 Biological control .....................................................................................................23 Research Objective .................................................................................................................24 Chapter 2: Identification and Characterization of a Soft-Rot Pathogen Isolated from Vanda, Phalaenopsis, Oncidium, and Tolumnia Orchids in Florida...................................25 Objectives ...............................................................................................................................27 Chapter 3: Culture of Pseudocercospora Species that Affect Orchids ..................................28 Taxonomy and identification ...................................................................................28 Molecular systematics ..............................................................................................29 Growth and sporulation of Cercosporoid fungi in culture .......................................30 Objective .................................................................................................................................31 Chapter 4: A Comparison of the Standard and High-Fidelity Polymerase Chain Reactions (PCR) in the Detection of Pseudocercospora odontoglossi from Cattleya Orchids and its Use in the Diagnosis of Orchid Diseases ...................................................31 High-fidelity PCR ....................................................................................................32 Objectives ...............................................................................................................................33 Chapter 5: The use of Silwet L-77 as an Integrated Pest Management Tool .........................34 Objectives ...............................................................................................................................35 Appendices .............................................................................................................................35 2 IDENTIFICATION AND CHARACTERIZATION OF A SOFT-ROT PATHOGEN ISOLATED FROM Vanda, Phalaenopsis, Oncidium, AND Tolumnia ORCHIDS IN FLORIDA ...............................................................................................................................39 Introduction .............................................................................................................................39 Materials and Methods ...........................................................................................................41 Isolation and Initial Tests ................................................................................................41 Fatty Acid Analysis .........................................................................................................42 6 Carbohydrate Utilization Tests ........................................................................................42 Molecular and Phylogenetic Analysis .............................................................................42 Pathogenicity Tests ..........................................................................................................45 Results .....................................................................................................................................46 Isolation and Identification of Dickeya Strains ...............................................................46 Fatty Acid Analysis .........................................................................................................46 Carbohydrate Utilization Tests ........................................................................................46 Molecular and Phylogenetic Analysis .............................................................................47 Pathogenicity Tests ..........................................................................................................48 Discussion ...............................................................................................................................48 3 CULTURE OF Pseudocercospora SPECIES THAT AFFECT ORCHIDS ..........................80 Introduction .............................................................................................................................80 Materials and Methods ...........................................................................................................81 Growth and Sporulation of P. dendrobii on Different Media .........................................81 Effects of Temperature on Growth and Sporulation of Pseudocercospora spp. on V-8 Agar ......................................................................................................................83 Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid ..........................................................................................................83 Results .....................................................................................................................................84 Growth and Sporulation of P. dendrobii on Different Media .........................................84 Effects of Temperature on Growth and Sporulation of three Pseudocercospora spp. on V-8 Agar .................................................................................................................85 Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid ..........................................................................................................85
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