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University of Florida Thesis Or Dissertation Formatting ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF CUCURBIT POWDERY MILDEW IN NORTH CENTRAL FLORIDA By GABRIELLA SILVEIRA MAIA 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 2012 1 © 2012 Gabriella Silveira Maia 2 To my husband Alberto Azeredo and my daughter Anna Azeredo To my mother Arlete Silveira 3 ACKNOWLEDGMENTS I thank my past advisor, Dr. Amanda J. Gevens for her patience, encouragement financial support and dedication throughout my research program. I thank my current committee chair, Dr. Jeffrey A. Rollins for his guidance and valuable suggestions throughout my degree program. I am sincerely grateful to my committee member Dr. Phillip Harmon and past committee member Dr. Eileen Kabelka for their thoughtful commentaries, their guidance and mentorship throughout the course of this research. I also thank Dr. Stephen A. Jordan for taking time to show and teach me about PCR and molecular biology. It has been a privilege to learn from all of them. I am especially grateful to Dr. Raghavan Charudattan (Dr. ‘Charu’), Mr. Jim DeValerio, Mr. Mark Elliott and Dr. Ernest Hiebert for giving me the opportunity to work with them when I first moved to Gainesville. They always cheered me on and inspired me to pursue graduate school. I would also like to thank my undergraduate advisor, Dr. Robinson A. Pitelli, for his guidance in my early schooling and for encouraging me to come to Gainesville. Much appreciation is given to Natalia Nequi, Kris Beckham, Harold Beckham, Eldon Philman and Hermon Brown for their assistance in laboratory, field, and greenhouse work. I thank Donna Perry, Lauretta Rahmes, Jan Sapp, Gail Harris and Jessica Ulloa for making my time as a graduate student enjoyable and well-ordered. I would also like to thank Dr. James D. McCreight (USDA) for his helpful commentaries, suggestions and for kindly providing muskmelon seeds for this study. I am appreciative of my class mates for the study groups, laughter, parties and for sharing these wonderful years in Gainesville with me. I am thankful to the University of Florida, the UF International Center and to the Department of Plant Pathology and its 4 faculty members who in their unique ways, taught me more than plant pathogens and plant diseases. Without the unconditional love and persistent encouragement of those closest to me, I simply would not have accomplished this goal. The love and gratitude I have for all of them simply cannot be put into words. I am grateful to my parents Jaime Maia dos Santos and Arlete Silveira, for introducing me to the field of Plant Pathology, for being passionate about their work and for teaching me the value of hard work. I am especially thankful to my mother who sacrificed so much to provide me with an education. She has been my best friend and never stopped believing in me. I thank my wonderful husband, Alberto Azeredo for his love, friendship, and unending support and for helping me find strength and motivation when I had none. I thank my lovely little daughter, Anna Azeredo, who has given me more love and joy than she will ever know. I thank my sister, Fabiola Silveira Maia, for her constant thoughtfulness and uplifting words. I am grateful to my wonderful in-laws Raquel Azeredo and Gino Ceotto Filho and for their love and constant encouragement. I am eternally grateful to our American family: Shirley and Carl Romey, The Walters (Valerie, Chris, Ethan, Isaac and Caleb) and The Wises (Harriet and Buddy) for the fellowship, support and for ‘adopting’ us in Christian love. Above all, I am most thankful to God who blessed me with a beautiful family and gave me the ability to complete this degree. Without Him, none of this would have been possible. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 LITERATURE REVIEW .......................................................................................... 22 The Cucurbitaceae.................................................................................................. 22 Uses and Economic Importance ....................................................................... 22 Taxonomic Classification .................................................................................. 26 Cucurbit Breeding and Resistance to Powdery Mildew .................................... 27 Cucurbit Powdery Mildew ....................................................................................... 29 Importance ....................................................................................................... 29 Causal Organisms ............................................................................................ 32 Morphology ................................................................................................ 34 Taxonomic classification ............................................................................ 36 Signs and symptoms .................................................................................. 37 Powdery mildew on watermelon ................................................................ 38 Epidemiology and disease cycle ................................................................ 39 Ecology ...................................................................................................... 41 Host range ................................................................................................. 43 Population biology and genetic diversity .................................................... 45 Powdery Mildew Diagnosis and Research ....................................................... 48 Disease Management....................................................................................... 49 Host resistance .......................................................................................... 50 Temporal avoidance (avoiding high risk seasons) ..................................... 50 Scouting and early prevention in high risk areas and crops ....................... 51 Application of fungicides ............................................................................ 51 Fungicide resistance .................................................................................. 57 3 CUCURBIT POWDERY MILDEW ISOLATE COLLECTION, MAINTENANCE AND CHARACTERIZATION ................................................................................... 64 Materials and Methods............................................................................................ 65 Field Site Trials ................................................................................................. 65 Plant Material for Field Experiments ................................................................. 66 Origin and Collection of Cucurbit Powdery Mildew Samples ............................ 67 6 Plant Material for Laboratory Experiments ....................................................... 69 Optimization of Living Culture Technique for Cucurbit Powdery Mildew .......... 70 Cucurbit Powdery Mildew Isolation, Multiplication and Maintenance ................ 71 Cucurbit powdery mildew isolation from field samples ............................... 72 Multiplication and maintenance of cucurbit powdery mildew isolates ......... 73 Cucurbit Powdery Mildew Isolate Characterization .......................................... 74 Morphological analysis ............................................................................... 74 Molecular analysis ..................................................................................... 74 Statistical Analysis ............................................................................................ 77 Results and Discussion........................................................................................... 77 Cucurbit Powdery Mildew Isolation, Multiplication and Maintenance ................ 77 Morphological Analysis ..................................................................................... 78 Molecular Analysis ........................................................................................... 80 4 DETERMINATION OF POWDERY MILDEW PHYSIOLOGICAL RACES ............ 108 Materials and Methods.......................................................................................... 109 Plant Material and Growth Conditions ............................................................ 109 Sample Collection and Pathogen Identification .............................................. 110 Host Reaction ................................................................................................. 110 Disease Evaluation ......................................................................................... 113 Disease severity ....................................................................................... 114 Pathogen status ......................................................................................
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