Isolation, Identification and Characterization of Cucurbit Powdery Mildew in North Central Florida

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Isolation, Identification and Characterization of Cucurbit Powdery Mildew in North Central Florida
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english
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Maia, Gabriella S
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Degree:
Master's ( M.S.)
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University of Florida
Degree Disciplines:
Plant Pathology
Committee Chair:
Rollins, Jeffrey A
Committee Members:
Harmon, Phillip
Gevens, Amanda

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Subjects / Keywords:
cucurbit -- florida -- mildew -- podosphaera -- powdery -- xanthii
Plant Pathology -- Dissertations, Academic -- UF
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Plant Pathology thesis, M.S.
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Abstract:
Powdery mildew is a common and economically important foliar disease in vegetable production throughout the world. On cucurbitaceous crops, the disease can reduce yield by decreasing fruit size, number of fruits, and length of time fruits can be harvested. Fruit quality and marketability can also be affected due to premature leaf senescence causing fruits to become exposed and more susceptible to sunburn. Additionally, powdery mildew infection can predispose cucurbit plants to other diseases. Cucurbit powdery mildew is most frequently caused by two obligate fungal pathogens, Podosphaera xanthii (Castagne) U. Braun & Shishkoff 2000 and Golovinomyces cichoracearum (DC.) V.P. Heluta 1988. The most commonly identified pathogen; particularly in warmer production regions has been P. xanthii. Recently, there has been an increase in occurrence and severity of the disease in Florida, resulting in heightened concern with fungicide resistance and potentially a shift or displacement of the pathogen population. In this study, we identified and characterized single colony isolates of cucurbit powdery mildew from multiple sites, dates, and cucurbit hosts. A method for living culture maintenance of cucurbit powdery mildew isolates was determined. Two butternut winter squash ('Butterbush') fields at Live Oak and Citra, FL, were sampled during spring and fall 2009. For comparison, additional cucurbit isolates were collected from south west and north east FL. Microscopic observations of all 297 isolates sampled from butternut winter squash 'Butterbush' at Live Oak and Citra revealed hyaline conidia, ellipsoid to ovoid in shape, with conidial dimensions of 31-44 x 15-24 micrometer (n = 100) and footcells of 45-67 x 10-13 micrometer (n = 25). Conidial length to width ratios varied from 1.4-2.6. All isolates exhibited fibrosin bodies and conidia edge lines were crenate. Commercial winter squash resistance lines were evaluated for disease response and pathogen characterization. Isolates from butternut winter squash ('Butterbush') and additional cucurbit hosts from varied dates and locations around Florida (FL) were subjected to multiplex polymerase chain reactions (PCR) with species-specific primers S1/S2 (for P. xanthii) and G1/G2 (for G. cichoracearum). With S1/S2, a specific PCR product of 454 bp (base pairs) was amplified from genomic DNA of most isolates. In total, based on morphological and genetic analysis, all cucurbit powdery mildew isolates were identified as Podosphaera xanthii.
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Includes vita.
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by Gabriella S Maia.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
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Adviser: Rollins, Jeffrey A.
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1 ISOLATION, IDEN TI FICATION AND CHARACTERIZATION O F CUCURBIT POWDERY MILDEW IN NO R T H 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

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2 2012 Gabriella Silveira Maia

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3 To my husband Alberto Azeredo and my daughter Anna Azeredo To my mother Arlete Silveira

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4 ACKNOWLEDGMENTS I thank my past advisor Dr. Amanda J. Gev ens for her patience, encouragement financial support and dedication t hroughout my research program I thank my current committee chair, Dr. Jef frey 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. Mr. Jim DeValerio Mr. Mark Elliot t 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 schooli ng 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 Rahme s, 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 m ates 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 Pat hology and its

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5 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 a nd 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 v alue 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 stoppe d 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 thoug htfulness 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 fa mily: Shirley and Carl Romey, The Walters (Valerie, Chris, E than, Isaac and Caleb) and The Wises (Harriet and Buddy) 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. Witho ut Him, none of this would have been possible.

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6 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 Cucu rbitaceae ................................ ................................ ................................ .. 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 classifi cation ................................ ................................ ............ 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 Fu ngicide resistance ................................ ................................ .................. 57 3 CUCURBIT POWDERY MILDEW ISOLATE COLLECTION, MAINTENANCE AND CHARACTERIZATION ................................ ................................ ................... 64 Materials and Methods ................................ ................................ ............................ 65 Field Site Trials ................................ ................................ ................................ 65 Plant Mate rial for Field Experiments ................................ ................................ 66 Origin and Collection of Cucurbit Powdery Mildew Samples ............................ 67

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7 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 Molecu lar 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 ................................ ................................ ....................... 113 Results and Discussion ................................ ................................ ......................... 114 Powdery Mildew Identification ................................ ................................ ........ 114 Effect of Cucurbit Powdery Mildew on Detached Muskmelon Leaves ............ 114 Disease Evaluation ................................ ................................ ......................... 115 5 ................................ .................... 132 Materials and methods ................................ ................................ .......................... 133 Plant Material ................................ ................................ ................................ 134 Powdery Mild ew Disease Assessment in Field Trial ................................ ...... 135 Statistical Data Analysis ................................ ................................ ................. 137 Results and Discussion ................................ ................................ ......................... 138 Powdery Mildew Pathogen Characterization ................................ .................. 138 Disease Severity Evaluation ................................ ................................ ........... 138 6 SUMMARY AND CONCLUSIONS ................................ ................................ ........ 151 APPENDIX LIST OF POWDERY MILDEW SUSCEPTIBLE HOST ................................ ............... 157 LIST OF REFERENCES ................................ ................................ ............................. 158 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 188

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8 LIST OF TABLES Table page 2 1 Major cucurbit producing countries and production estimates for 2009 .............. 59 2 2 United States cucurbit production in 2010 ................................ .......................... 60 2 3 Comparison of production, area and value of the major U.S. states for cucurbit crops in 2010 ................................ ................................ ........................ 61 2 4 Cucurbit production in Florida for 2008 2009 seasons ................................ ....... 62 3 1 FL field sites during spring and fall of 2009. ................................ ....................... 82 3 2 Additional powdery mildew samples, from alternative locations and cucurbit hosts collected for comparison. ................................ ................................ .......... 90 3 3 Mean powdery mildew conidia and conidiophore footcell length, width and length to width ratio of isolates collected in Live Oak and Citra, FL .................... 92 3 4 Dimensions of fresh conidia of Podosphaera xanthii from leaf tissue of Cucurbita moschata .......... 99 4 1 Sub set of six Florida powdery mildew isolates, used for race typing bioassays. ................................ ................................ ................................ ......... 119 4 2 Reaction (10 days post inoculation) of some muskmelon ( Cucumis melo ) genotypes to sub set of powdery mildew isolates. ................................ ............ 119 5 1 List of 22 elite breeding lines supplied by Rupp Seeds. Cultigens were evaluated for resistance to cucurbit powdery mildew present in north Florida .. 142 5 2 Evaluation of Rupp elite breeding lines represented by percent leaf area affected and disease severity rating at Live Oak, FL during spring 2009. ........ 143 5 3 Evaluation of powdery mildew susceptible cultivar Butterbush represented by AUDPC and ave rage disease severity at Live Oak and Citra, FL ..................... 144 A 1 List of powdery mildew susceptible cucurbit hosts used throughout this research ................................ ................................ ................................ ........... 157

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9 LIST OF FIGURES Figure page 2 1 Disease cycle of cucurbit powdery mildew, illustrating the asexual stage ........ 63 3 1 Florida sites of cucurbit powdery mildew collection from spring to fall of 2009. 100 3 2 Field plot at Live Oak and Citra, FL ................................ ................................ 101 3 3 Primary leaf and cotyledon of butternut winter squash rooting in 2% water agar media ................................ ................................ .................. 101 3 4 s containing 2% water agar. ................................ ................................ ................................ 102 3 5 Cucurbit seedlings cultivated in growth room ................................ ................. 102 3 6 Leaf disks containing discrete powdery mildew colonies cut from infe cted ................................ ................................ ................ 103 3 7 Cucurbit powdery mildew isolates growing on different cucurbit hosts after periodic isolate transfer ................................ ................................ .................. 103 3 8 Characteristic powdery mildew signs and symptoms on susceptible cucurbits cultivars .. ................................ ................................ ................................ ......... 104 3 9 Characteristic Podosphaera xanthii morphological features .. .......................... 105 3 10 Electrophoretic profi le of PCR products amplified by specific primers S1/S2 and G1/G2. ................................ ................................ ................................ ....... 106 3 11 Analysis of DNA fragments amplified by a multiplex PCR with DNA extracted .. .................. 107 4 1 Set of healthy muskmelon genotypes growing in powdery mildew free greenhouse ................................ ................................ ................................ ..... 122 4 2 Demonstration of muskmelon differential inoculation process. ......................... 122 4 3 A 10 mm diameter circumference used to assess powdery mildew. ................ 123 4 4 Scale used for pathogen status classification ................................ ................. 123 4 5 Healthy leaves (mock inoculated) of 12 muskmelon genotypes, 17 days post mock inoculation with un tyledons ............................. 124 4 6 days after pathogen inoculation. ................................ ................................ ............... 124

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10 4 7 Reaction of a set of 12 muskmelon genotypes to powdery mi ldew isolate from summer squash. ................................ ................................ ....................... 125 4 8 Reactions of 12 muskmelon genotypes to P. xanthii isolate LO 6 1(A)1 from ................................ ............................. 126 4 9 Reactions of 12 muskmelon genotypes to P. xanthii isolate Ci 6 1(A)1 from ................................ ................................ .... 127 4 10 Reactions of 12 muskmelon genotypes to P. xanthii isolate 10 02 from muskmelon in greenhouse at UF, Gainesville, FL ................................ ........... 128 4 11 Reactions of 12 muskmelon genotypes to P. xanthii isolate 10 08 from squash collected in Immokalee, FL ................................ ................................ 129 4 12 Reactions of 12 muskmelon genotypes to P. xanthii isolate 10 09 from summer squash collected in Dover, FL ................................ ........................... 130 4 13 Expected reaction of 12 muskmelon genotypes to 31 known races of powdery mildew ( P. xanthii ) found i n the U.S. and around the world. ............... 131 5 1 Powdery mildew disease on 22 Rupp breeding lines ( Cucurbita spp.) and on powdery mildew susceptible cult ivars Butterbush a nd Mickey Lee. .................. 145 5 2 Detail of powdery mildew disease on the same plant of susceptible cultivar Butterbush at Live Oak, FL ................................ ................................ .............. 146 5 3 Detail of powdery mildew disease on the same plant of susceptible cultivar Butterbush at Citra, FL ................................ ................................ .................... 147 5 4 Disease severity over Live Oak and Citra, FL. ................................ ................................ ............. 148 5 5 The cumulative AUDPC of weekly powdery mildew assessment on .............................. 148 5 6 Pedigree of Rupp powdery mildew breeding lines evaluated at Live Oak, FL during field trial in spring of 2009. ................................ ................................ ..... 149 5 7 The cumulative AUDPC of powdery mi ldew on 22 elite breeding lines inoculated by natural infection at Live Oak FL. ................................ ................ 150

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11 A bstract 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 ISOLATION, I DENTIFICATION AND CHARACTERIZATION OF CUCURBIT POWDERY MILDEW IN NORTH CENTRAL FLORIDA By Gabriella Silveira Maia May 2012 Chair: Jeffrey A. Rollins Major: Plant Pathology Powdery mildew is a common and economically important foliar disease in vegetabl e production throughout the world. On cucurbitaceous crops, the disease can reduce yield by decreasing fruit size, number of fruits, and length of time fruits can be harvested. Fruit quality and marketability can also be affect ed due to premature leaf sene scence cau sing fruits to become exposed and more susceptible to sunburn Additionally, powdery mildew infection can predi spose cucurbit plants to other diseases. Cucurbit powdery mildew is most fr equently caused by two obligate fungal pathogens Podosphaera xanthii [ (Cas tagne) U. Braun & Shishkoff 2000 ] and Golovinomyces cichoracearum [ ( DC.) V.P. Heluta 1988 ] The mo st commonly identified pathogen; particularly in warmer production regions has been P. xanthii Recently, there has been an increase in occurrence and severity of the disease in Florida, resulting in heightened concern with fungicide resistance and potentially a shift or displacement of the pathogen population. In this study, we identified and characterized single colony isolates of cuc urbit powdery mildew from multiple sites, dates, and cucurbit hosts. A method for living culture maintenance of cucurbit powdery mildew isolates was determined. T wo butternut winter squash (

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12 durin g spring and fall 2009. For comparison, additional cucurbit isolates were collected from south west and north east FL. Microscopic observations of all 297 isolates sampled f rom butternut winter squash hyaline con idia, ellipsoid to ovoid in shape, with conidial dimensions of 31 44 x 15 24 67 x 10 varied from 1.4 2.6. All isolates exhibited fibrosin bodies and conidia edge lines were cr enate. Commercial winter squash resistance lines were evaluated for disease response and pathogen characterization Isolates f rom butternut winter squash ( Florida (FL) were subjected to multiplex polymerase chain reactions (PCR) with species specific primers S1/S2 (for P. xanthii ) and G1/G2 (for G. cichoracearum ). With S1/S2, a specific PCR product of 454 bp (base pairs) was amplified from genomic DNA of most isolates. In tot al based on morphological and genetic analysis, all cucurbit powdery mildew isolates were identified as Podosphaera xanthii

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13 CHAPTER 1 INTRODUCTION Historically, cucurbits (Cucurbitaceae) have been important to mankind, especially as a food source. There is great diversity among consumed cu curbitaceous crops and production regions around the world ( 276 ) Cucurbit crops continue to be developed and produced for market niches worldwide ( 290 291 ) Cucurbits include a number of species in three distinct genera in the family Cucurbitaceae. The genus Cucumis includes cucumber and many types of melon. Squash and pumpkin are found in a range of cultivated species of Cucurbita and watermelon is in the gen us Citrullus Worldwide 7 species are economically important and include: Citrullus lanatus (Thunb.) Matsum. & Nakai (watermelon), Cucumis sativus L. (cucumber), Cucumis melo L. (cantaloupe), Cucurbita pepo L. (field pumpkin), Cucurbita maxima Duchesne (w inter squash), Cucurbita moschata Duchesne. ( c rookneck squash or calabaza) and Lagenaria vulgaris (Molina) Standl. (bottle gourd) ( 263 ) In the United States the most commonly cultivated and consumed cucurbits are fresh market and processing cucumber many types of squash (acorn, butternut, kabocha, yellow squash and zucch ini), various types of melons (muskmelon cantaloupe and honeydew), pumpkin s ( Cucurbita pepo and C ucurbita maxima ) and watermelon ( 295 ) Cucurbit crops have global economic import ance. Over 1 billion tons of vegetables (including cucurbits), were produced worldwide in 2009, with a total harvested area of more than 53 million hectares and a calculated yield of over 190 thousand hectogram/hectare. Among the estimates, cucurbits inclu ding watermelon,

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14 cucumber, melons, pumpkin, squash and gourds accounted for over 206 million tons corresponding to roughly 20% of the total vegetables produced worldwide ( 81 ) In 2010, t he total U.S. average acreage of the majo r classes of cucurbits was around 185,000 hectares, yielding 4 million metric tons of produce with a value of US$ 1.5 million. The major cucurbit producing states in the U.S. are California, Florida, Georgia, Texas, Arizona, Illinois and Michigan. While California and Arizona lead production of honeydews and cantaloupe, Florida and Georgia lead in production of cucumber for fresh market and of watermelon. Michigan wa s the leader in production of processed cucumbers (f or pickles) and of squash. In most years, Illinois leads pumpkin production ( 327 ) According to 2009 data from the Florida Department of Ag riculture and Consumer Service ( FDACS ) US $290 million to the sta 14% industry ( 233 ) Cucurbit production in Florida is critical as it supplies the national demand for cucurbits during the winter months. These vine cro ps are well adapted to production in Florida during the spring, early summer, and fall seasons. During the winter production of cucurbits is limited to the warmest southern growing areas of the state ( 238 ) The causal agents of powdery mildew diseases include a diverse range of pathogenic species under the broad order Erysiphales in the phylum Ascomycota ( 27 ) Worldwide, approximately 500 powdery mildew species are able to infect over 10,000 distinct plant species ( 312 ) However, recent phylogenetic research reported the increase in the number of recognized powdery mildew species to about 820 ( 26 )

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15 Among the economically important plant species susceptible to po wdery mildew are cereals (wheat, barley), members of the families Solanaceae (to mato potato ), Cucurbitaceae ( melon, squash, cucumber, pumpkin, and watermelon ), Rosac eae (apple, strawberry, cherry), Fabaceae (pea, bean ), Asteraceae (lettuce, artichoke ), Vitaceae ( grape ) and many ornamental plants from varied plant families. Cucurbit powdery mildew is particularly prevalent in tropical and some temperate climates where it frequently causes significant reductions in product quality and yield. The disease has been reported to be the most common, widespread and easily recognized disease of both field and greenhouse grown cucurbit crops ( 214 ) This foliar fungal disease is a major cause of crop losses in cucurbit production worldwide, and is among the most intensively studied because of its economic impact on these crops ( 161 ) Powdery mildew is a common and serious disease of cucurbit crops in Florida, and the disease occurs on cucumber, melon, squash, zucchini, pumpkin, gourd and more recentl y, and increasingly on watermelon crops ( 235 ) Like other powdery mil dews, signs and symptom s are characterized by a white talcum like fungal growth on lea ves, petioles, and stems. Fruit are rarely directly infe cted by powdery mildew. Under favorable environmental conditions, fungal colonies can coalesce and the host tissu e becomes chlorotic, senescin g prematurely. This disease is frequently more severe at the end of the vegetative cycle, causi ng additional losses by shortening the length of harvest time and affecting fruit quality as a result of premature defoliation and s unscald During the growing season, fungal conidia (asexual spores) are readily disseminated and can quickly move within and among fields and inside greenhouses aerially.

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16 The two main causal agents of cucurbit powdery mildew are Podosphaera xanthii [(Cast ag.) U. Braun & N. Shish], formerly known as Sphaerotheca fuliginea (Schlecht ex Fr), and Golovinomyces cichoracearum [(DC.) V.P. Heluta] previously referred to as Erysiphe cichoracearum DC ex Merat. Studies throughout the world have shown that P. xanthii is the species most commonly identified across cucurbit hosts ( 329 ) Podosphaera xanthii is commonly detected in subtropical and tropical regions as well as in greenhouse grown crops, while G. cichoracearum occurs more frequently in temperate and cooler areas under field conditions ( 143 ) The two species can occur singly or in mixed infections on cucurbit crops ( 123 143 164 ) Potentially, all cultivated cucurbitaceous crops can be susceptible to powdery mildew ( 71 ) however tolerant and resistant cultig ens exist. Some cultivated cucurbits such as cucumber and melons, have greater resistance and are commercially available. While P. xanthii and G. cichoracearum have historically been described as pathogenic on cucurbit s recent reports indicate that G. cichoracearum has been documented to cause disease on several members of the Asteraceae family such as lettuce ( Lactuca sativa L. ) in the Czech Republic ( 160 ) gerbera daisy ( Gerbera jameso ni i Bolus ex Hook. f. ) ( 324 ) orange coneflower ( Rudb eckia fulgida Aiton. ) ( 90 ) Paris daisy ( Argyranthemum frutescens (L.) Sch. Bip. ) ( 91 ) and E nglish daisy ( Bellis perennis L. ) ( 92 ) in Italy as well as elegant zinnia ( Zin n ia elegans Cav. ) ( 243 ) and chamomile ( Matricaria chamom illa L. ) in Korea ( 242 ) and in the U .S., on crotalaria ( Crotalaria juncea L. ), a member of the Fabaceae family ( 95 ) Prior to the mid 1990 s powdery mildew was reported as an occasional problem on watermelon ( 66 120 277 ) but the incidence of powdery mildew outbreaks has

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17 increased and the disease has become an important problem in the major U.S. watermelon production areas ( 65 128 275 305 318 ) and in other parts of the world ( 46 144 239 268 ) Currently, only P. xanthi i has been reported on watermelon ( 66 ) and to date, two races of P. xanthii race 1 (1W) and race 2 (2W), have been identified on watermelon in the U.S. ( 46 65 68 201 319 ) Recent outbreaks of the disease have been confirmed in South Carolina, Georgia, Florida, Oklahoma, Texas, Maryland, New York, Arizona and California ( 65 305 ) Detection of powdery mildew on watermelon can be difficult because the presence of the pathogen is less apparent than on other cucurbit crops ( 66 ) On watermelon plants, powdery mildew is manifested as chlorotic spots with or without white mycelial and/or conidial development on leaves and stems. In addition to these symptoms, water soaked areas may appear o n petioles of highly susceptible cultigens ( 319 ) Fruit infections are rare ( 66 ) ; however they have been detected on young watermelon fruit and are characterized by small circular patches of water so aked tissue covered by white, talcum like pathogen sporulation. In late 2010, severe powdery mildew outbreak s on seedless and seeded watermelon fruit on some commercial farms in southwester n Florida were confirmed through microscopic and mole cular analysis Powdery mildew symptoms were mainly observed on immature fruit, but not on mature older fruit or leaves. Orange to dark brown chasmothecia (sexual spore bearing fruiting bodies ) containing a single ascus was detected on the surface of some fruit samples. The powdery mildew isolate from watermelon fruit was maintained on cot yledons of several cucurbit and bean cultivars and following artificia l inoculation tests, results indicated the profile of P. xanthii race 1 ( 139 )

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18 The identification of cucurbit powdery mildew fungi has not always been made accurately. The two major species defined in the literature may have been misidentified and at times, were considered as synonymous ( 195 ) The causal agents of cucurbit powdery mildew disease produce identical symptoms and can be difficult to differentiate in the absence of the perfect ( sexual or teleomorphic) stage which can be distinguished by the number of asci, ascospores and appendages and presence of the chasmothecia ( 22 ) However, morphological features of the anamorphic (asexual) stage of P. xanthii differ from those of G. cichoracearum and include: size and shape of conidia, presence of fibrosi n bodies, immature conidi a edge and germ tube morphology The two cucurbit powdery mildew fungal pathogens also differ in geographical distribution ( 10 41 71 101 144 218 221 255 ) pathogenicity on cucurbit cultivars ( 35 48 161 163 192 224 254 ) temperature requirements ( 13 144 164 294 ) and sensitivity to some fungicides ( 128 166 201 204 206 210 213 296 ) Podosphaera xanthii has been reported to be more aggressive than G. cichoracearum ( 200 ) A l ower temperature optimum has been associated with G. cichoracearum since this sp ecies is found mainly in cooler regions of the world and in the U.S. has been reported during cooler springs and early summer periods while P. xanthii appears to progress most rapidly in warmer regions during the warmest months of the year The optimum tem perature ran ges for conidial germination are reported to be between 25 30C for P. xanthii and 15 25C for G. cichoracearum ( 218 ) Podosphaera and Golovinomyces are described by broad pathogenic variability characterized as pathotypes and races ( 190 322 ) Races 1 and 2 of P. xanthii were first defined in the Imperial Valley of California in 1938 when the pathogen overcame

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19 R More than 30 years later, in 1976, race 3 of P. xanthii was detecte d in Texas. ( 193 ) To date, approximately 30 distinct physiological races of P. xanthii ( 39 190 ) and 2 races of G. cichoracearum ( 11 71 158 264 322 ) have been identified worldwide. Traditionally, c ucurbit powdery mildew races hav e bee n d efined by the disease response of the pathogen isolate on a set of muskmelon differentials ( 158 ) The most frequently used set of melon differentials includes 11 genotypes that can differentiate cucurbit powdery mildew races originating from melon and other cucurbits such as cucumber, Cucurbita spp. and watermelon ( 157 ) In spit e of the advances in genetic, chemical and biological measures of control the management of powdery mildew of cucurbit crops world wide is insufficient. Typically, the control of powdery mildew in susceptible cucurbit cultivars is achieved with use of fung icides ( 223 ) Fungicides for c ontrol of cucurbit powdery mildew include a variety of active ingredients and modes of action with pre harvest intervals ranging from 3 to 14 days ( 34 ) The repeated use of site specific fungicides over time has resulted in powdery mildew resistance to some commercial chemical compounds ( 30 112 118 166 179 203 236 326 340 341 347 ) Good fungicide coverage or fungicid es with some mobile (systemic or translaminar) activity are needed to obtain adequate protection on the underside of leaves, where conditions favor disease ( 199 ) However these fungicides generally have high risk for developing resistance becaus e of their s ingle site and s pecific modes of action ( 201 ) Natural selection within asexual populations followed by a fungicide selection process which favors resistant mutants has been widely repo rted C ucurbit powdery

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20 mildew species are known to have high evolutionary potential ( 163 196 ) and are naturally more likely to overcome plant genetic resistance and/or develop fungicide resistance ( 148 201 ) Cucurbit powdery mildew pathogens are highly variable in their pathogenicity and virulence which is evident by the existence of a large number of different pathotypes and races ( 190 ) Distinct p hysiological pathotypes and races of P. xanthii have been detected with resistance to as many as eight classes of fungicides ( 77 201 210 236 296 302 ) Presence of resistant fungal strains ha s been associated with lack of powdery mildew control and the relative ease with which fungicide resistant strains can develop in a short period of time ( 161 ) Difficulty in achieving adequate fungici de coverage public concerns for the environment, likelihood of the development of disease resistance to chemical control and shifts in pathogen virulence indicate that the best method for powdery mildew management would be provided through the use of resi stant varieties, with occasional use of chemical control when necessary ( 321 ) R esistant cucurbit varieties are being developed and are becoming an increasingly important component of powdery mildew management programs in the U.S. ( 65 123 194 319 ) and elsewhere ( 113 140 161 261 268 302 339 347 ) Additionally, integrated pest management (IPM) alternatives continue to be evaluated for efficacy and incorporation into cucurbit powdery mildew disease management programs ( 16 72 97 135 208 223 245 280 281 283 306 ) To further understand the recent increase in incidenc e, severity and host range of cucurbit powdery mildew in Florida, this thesis study undertook the following research objectives: (i) to speciate and characterize the prevailing causal agent of cucurbit

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21 powdery mildew in north central Florida through morpho logical features and DNA analysis ; (ii) to develop an efficient technique for in viv o establishment and maintenance of the fungal isolates ; (iii ) to assess the presence of physiological races within cultured isolates via bioassays using detached leaves ; an d ( i v) to evaluate the varietal reactions of Cucurbita breeding lines (genetic accessions) for susceptibility to powdery mildew under local (FL) field conditions.

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22 CHAPTER 2 LITERATURE REVIEW The Cucurbitaceae Uses and Economic Importance Cucurbitaceous crops have worldwide economic importance. Statistical data indicated that over 1 billion tons of vegetables including cucurbits were produced worldwide with a total harvested area of more than 53 million hectares and a calculated yield of over 190 thousand hectogram/hectare in 1999 Among these estimates, cucurbits including watermelon, cucumber and gherkins, melons, pumpkin, squash and gourds accounted for over 206 million tons, representing more than 20% of the tota l worldwide production of vegetables ( 81 ) (Table 2 1) Worldwide, t he most cultivated cucurbit was watermelon with a total production of about 99 million tons, followed by cucumbers and gherkins (61 million tons), melons (26 million tons) and Cucurbita spp. (22 million tons) (Table 2 2) The largest cucurbit producer, China le d the production of watermelon with 66 % of the world production. Five countries (China, Turkey, Iran, Br azil and USA) produced about 77 % (76 million tons) of the world production of watermelon. Cucumber was the second largest cucurbit produced with five c ountries (China, Turkey, I ran, Russia and USA) representing 82 % (50 million tons) of the world production. Five countries (China, Turkey, Iran, USA and Spain) produced 68 % (17 million tons) of the melons. The first five producers (China, India, R ussia, USA and Egypt) of Cucurbita spp. represented about 50% (13 million tons) of the world production ( 81 ) In 2010, t he total U.S. field production of the major classes of cucurbits was approximately 4 million metric tons on about 185,000 hectares, with a value of US $ 1.5

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23 million (Table 2 3). The leading states in cucurbit production were California, Florida, Georgia, Texas, Arizona, Illinois and Michigan ( 327 ) While California and Arizona lead production of honeydew and cantaloupe; Florida and Georgia were leaders of cucumber for fresh market and of watermelon. Furthermore, Michigan was the leader in production of processed cucumbers (for pickles) and of squash. In addition, Illinois led in U.S. pumpkin production (T able 2 4). In 2010 Florida ranked first in production of fresh market cucumber and watermelon and was the third largest producer of squash, behind Michigan and California (Table 2 4) ( 327 ) Combined, these crops contributed a value of nearly US industry (Table 2 5). The major veg etables produced in the state of Florida for the 2008 2009 season ( 79 ) are presented in Table 2 6 The total Florida production of vegetables, including the major classes of cucurbit s was approximately 2.3 million tons on 90,700 hectares (2 24,000 acres) of harvested area with a total value of US $ 1.9 million dollars (Table 2 6). Cucurbitaceous crops are cultivated around the world under different environmental conditio ns, both in fiel d and protected structures ( i.e. greenhouse and tunnels) and for several uses and purp oses. Globally, cucurbits are primarily used as a source of food, consumed fresh or cooked ( 353 ) Cucurbits are mainly cultivated as vegetables and different parts of the plants may be used, including seeds, flowers, very young shoots, tendrils and roots ( 263 ) Alternatively, cucurbits are also used for fiber, utensils, containers, floats, sponges, filters, sweeteners, musical instruments, for decoration, and also as s ucculent and ornamental plants ( 9 276 307 ) Many species

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24 are known to have medicinal value especially in Asian countries ( 9 ) Some cucurbitaceous plants, native to Asia and Australia are known to be toxic an d poisonous to humans ( 94 353 ) Additionally, several cucurbit species are considered noxious invasive weeds in the U.S. ( 133 ) and in other parts of the world ( 228 244 310 ) Cucurbits are trailing or vining, tendril bearing annuals ( 335 ) typical ly indeterminate in length and can grow up to 15 m long. Cucurbit leaves are borne singly and can be simple, th ree or five lobed and leaf sizes vary between cucurbit species. Flowers vary greatly in size, color and shape depending on the species. Cucurbits bear both perfe ct (hermaphroditic) and imperfect (pistillate or staminate) flowers and require various insects, especially honey bees ( Apis spp.), to ensure adequate pollination for both fruit and seed production ( 353 ) Cucurbitaceae are most diverse in tropical and subtro pical regions with hotsp ots in Southeast Asia, West Africa, Madagascar, and Mexico Cucurbits are of Asian origin and most likely originated in the Late Cretaceous, some 60 million years ago ( 291 ) It has been reported that long distance dispersal of seeds, between continents for at least 10,000 years, has played an important role in the biogeog raphical history of the Cucurbitaceae family ( 289 ) The f amily Cucurbitaceae forms a diverse group of species which contains several important fruits and vegetables with a variety of sizes, shapes, colors, textures and flavors ( 307 ) Cucurbit fruits, specialized berries called pep o are multi seeded. Except for a few types of winter squash and netted melons, which are rich in beta carotene and vitamin A, cucurbit fruits are gen erally low in nutritional value, though w atermelon is an excellent source of l ycopene, a red pigment known to have anticancer properties ( 335 )

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25 Cucurbitacins, compounds responsible for the bitter taste of some cucurbit species, can be highly toxic to humans. Cultivated cucurbits are low i n cucurbitacins, while some wild species have large r amounts (up to 1%) in roots and fruits. Studies have determined that these compounds are important in protecting cucurbit plants against insects and herbivores. Cucumber beetles ( Acalymma spp Barber and Diabrotica spp. Chev. Dejean ) are attracted to cucurbits plants by this class of secondary plant compounds. These beetles are induced to feeding behavior and are capable of consuming these bitter compounds ( 172 ) Insect baits and repellents have been developed from crosses between cucurbit cultivars and wild cucurbit relatives ( 353 ) Among the diversity in the Cucurbita ceae family, three genera are of the gre atest economic significance throughout the world: Cucumis, Cucurbita and Citrullus The major cultivated genera include: cucumber ( Cucumis sativus L.), several types of melons ( Cucumis L.), watermelon ( Citrullus lanatus (Thunb.) Matsum. Nakai ), and squash and pumpkin ( Cucurbita L.) ( 238 ) A few minor types of cultivated specialty cucurbits include chayote ( Sechium edule (Jacq.) Swartz), long squash or bottle gourd ( Laginaria siceraria (Mol.) Standl.), bitter melon ( Momordica charantia L.), luffa sponge gourd ( Luffa aegyptiaca Miller), Chinese o kra or luffa ridge gourd ( Luffa acutangula (L.) Roxb.), parvar or snake gourd ( Tricosanthes dioica Roxb.), wax gourd ( Benincasa hispida Thumb.), ca s sabana na ( Sicana odorifera (Vell.) and tinda ( Praecitrullus fistulosus Stocks) ( 9 238 262 353 ) Cucurbits are among the first domesticated plant species ( 263 ) and have varied centers of origin with representatives native to both the W estern and Eastern

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26 hemispheres ( 109 353 ) While Citrullus (watermelon) and Cucumis (cucumber and melon) are thought to have originated in the Eastern hemisphere Cucurbita (pumpkin and squash) originated in the Western hemisphere. Followi ng European contact, centers of diversity developed in Turkey ( Cucurbita pepo ), India and Burma ( C. maxima ), China and Japan ( C. moschata ). Cucumber originated in India; cantaloupes and melons in Africa; summer squash and butternut squash came from Mexico and Central America; winter squash from South America and watermelon from Central Africa ( 238 353 ) Cuc urbits differ in their ability to tolerate cold and heat, yet all cucurbits are sensitive to frost. The vast majority of species are vining herbaceous annuals. A few cultivars produce bush like plants ( 353 ) and a limited numb er of species are woody vines in the rainforests of Austra lia ( 291 ) A small number of species are thorny shrubs and one cucurbit specie, endemic to Yemen, grows as a tree ( Dendrosicyos socotranu s Balf. f. ) ( 289 ) Taxonomic Classification Cucurbitaceous crops belong to the order Cucurbitales and family Cucurbitaceae (Juss.). All the cultivated species are found in the subfami ly Cucurbitoide. This plant family consists of over 100 genera and more than 800 species distributed largely in tropical and subtropical regions of the world with few representatives in temperate to cooler climates ( 332 ) Recent phylogenetic research by Schaefer and Renner ( 291 ) based on molecular and morphological data, described a new classification of 95 genera and 950 to 980 species comprising t he Cucurbitaceae family.

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27 Cucurbit Breeding and Resistance to Powdery Mildew While not re cognized as modern day breeding for resistance, domestication and select ion of cucurbits by inhabitants of caves in Mexico has been date d back 7,000 to 10,000 cale ndar years before present and as suggested by arch eological records predates the domestication of corn ( Zea mays L. ) and common bean ( Phaseolus vulgaris L. ) by more than 4,000 years ( 21 308 ) Until approximately 70 years ago, Cucurbita cultivars were characterized by high genetic variab ility attributed in part by their natural tendency of outcrossing. However, demand for uniformity and selection for horticultural tr aits such as fruit size, shape and color, as well as quality and earliness resulted in high homozygosity and true breeding cultivars ( 21 ) For nearly 50 years, inbred cucurbit lines have been extensively studied and used to develop hybrids which have the advantage of being more u niform and homogeneous then previous open pollinated cultivars ( 241 ) In the U.S. early cucurbit breeding research and w ork on sources of resistance were summarized by Peterson ( 257 ) and in 2002 was updated by Jahn et al. ( 123 ) Today, many cucurbit breeding programs worldwide consider incorporating disease resistance into commercial cultivars while enhancing crop yield and quality, horticultural traits ( 123 ) as well as adaptability and market ability ( 238 ) Breeding for resistance to diseases has advanced greatly si nce the beginning of the 20 th century when Biffen (1907) discovered that a particular resistance to yellow rust in wheat plants was controlled by a single recessive gene ( 20 ) Selecting and breeding cucurbit cultivars for resistance to powdery mildew has proved to be an effective means against this disease and an alternative th at can lead to environmental and economic

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28 advantages as well as a reduction in fungicide usage, resulting in a great er benefit to cucurbits growers Worldwide, cucurbit disease resistance breeding greatly advanced with improved understanding of plant genetics. Since the 1930 s international efforts in breeding for resistance to powdery mildew in commercial varieties of melon, cucumber, pumpkin and squash has been ongoing and c onsiderable attention has been directed at breeding cucurbit cultiv ars with resistance to P. xanthii (syn. S. fuliginea ) ( 123 ) The effectiveness of cucurbit breeding programs depe nds on germplasm resources. All over the world, breeders are increasingly interested in sources of resistance to powdery mildew and have extensively investigated procedures for assessment of powdery milde w resistance ( 43 140 161 261 298 347 352 ) I nterspecific hybrids of high resistance to cucurbit powdery mildew have been obtained throug h crosses and pedigree selection between the cultivar Cucurbita moschata and wild Cucurbita species ( 37 45 87 140 141 155 168 240 ) as well as species of the genus Cucumis ( 49 149 153 156 ) and Citrullus ( 66 67 ) Particularly in the U.S., recen t outbreaks of powdery mildew on watermelon have contributed to further advances in cucurbit disease resistance breeding ( 66 68 146 319 321 ) Powdery mildew had not been considered a problem in watermelon because older cultivars were resist ant to previously described races of P. xanthii ( 64 ) In 1975, susceptibi lity to powdery mildew was demonstrated in the watermelon plant introduction ( PI 269677 ) accession ( 277 ) and was found to be controlled by a single recessiv e gene pm ( 278 ) S ince then, s creening of the USDA ARS watermelon germplasm made up of over 1500 accessions recovered 8 accessions with high levels of resistance to powdery

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29 mildew and another 86 accessions demonstrated intermediate resistan ce to P. xanthii race 1W ( 66 318 ) More recently, over 1600 cultigens (plant introductions, cul tivars and breeding lines) were screened in greenhouse tests and from those, 8 cultigens had high resistance and 21 had intermediate resistance to P. xanthii 2W ( 319 ) Extensive work in genetic linkage mapping and the understanding o f fungus host as well as fungus resistant gene interactions in cucurbit powdery mildew resistanc e mechanisms have been recognized as essential tools for genetic research and bre eding for disease resistance ( 5 89 177 178 251 302 316 346 ) Cucurbit Powdery Mildew Importance Plant diseases have had profound effects on mankind as evidenced by numerous Biblical references (about 750 B.C.) to blasts, blights and mildews of plants, fou nd in the Old Testament ( 3 220 ) Powdery mildew is one of the oldest plant diseases on record. The first historical account of the disease was recorded by the Greek writer and gardener, Theophrastus, who in his studies of bo tany and plant diseases, described powdery mildew on roses in 300 B.C. ( 3 132 ) Different powdery mildew genera infect different host plants. As an example, Erysiphe (and Golovinomyces ) spp. cause powdery mildew infections on ornamentals (begonia, chrysanthemum, and dahlia), cucurbits, legumes, crucifers, beets and tomato. Leveillula spp. G. Arnoud infects tomato and cucurbits. Podosphaera spp. infects apples, pears, stone fruits (apricot and plum), some ornamentals and cucurbits. The genus Sphaerotheca spp. Lev. is generally pathogenic on berries (strawberry, gooseberry), roses, some vegetable crops (including cucurbits), and also stone fruits. The genus Uncinula (Schwein.) Burrill causes powdery mildew of grapes. Blumeria spp.

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30 (DC.) Speer is the causal agent of powdery mildew on cereals and grasses. Microsphaera spp. affects many shade trees and woody ornamentals (azalea, lilac, and rhododendron) ( 3 ) A wide variety of vegetable crops and herbs are affected by powdery mildews, including artichoke, beans, beets, br occoli, carrot, cauliflower, collard, cucumber, eggplant, lettuce, melons, okra, parsley, parsnips, peas, peppers, potato, pumpkins radicchio, radishes, squash, tomatillo, tomatoes, and turnips ( 69 ) While pow dery mildew diseases are very common and usually distinctive these fungi have been reported to cause less significant losses than those caused by many other important g roups of plant pathogens, such as viruses, downy mildews, rusts and root rotting fungi ( 342 ) Moreover, powdery mildews are typically present every production year in varying degrees do disease pressure. s that one causal agent of powdery mildew, Uncinula necator (syn Erysiphe necator Schwein. ), identified in vineyards in France caused noteworthy economic losses and nearly destroyed grape production in that country. Young grape leaves were becoming covered with small white powdery spots and as the leaves grew and expanded, the white spots also expanded and covered most of the leaf surface. The disease spread on to the grape b erries and these developed a gra appearance, resulting in withered and cracked berries. Grape leaves would eventually turn b rown to black and die while the grape berries remained small and discolored, becoming unf it for wine production or fresh market. By 1854, French wine production was reduced by 80% due to this new disease ( 3 ) It was reported that the yield of wine decreased from about 45 million hectoliters in 1850, during the early stages of the

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31 powdery mildew epidemic, to about 10 million hectoliters in 1854 when this new disease of grapes was at its highest ( 342 ) In the U.S., powdery mildew on a cucurbitaceous crop was first noted by Jagger and Scott ( 121 ) in 1925, as a destructive disease of melons being grown in the Imperial Valley of California. While screening cantaloupe plant material from all over the world, the researchers found powdery mildew resistan ce in a seed lot (PI 78374) originated from India ( 41 186 189 ) More recently, losses to cucurbit powdery mildew have ranged from 30 50% in Chinese produced cucumbers ( 114 ) and have affected roughly 70% of the squash acreage in Florida ( 235 ) Cucurbit powdery mildew is likely the most common disease and important limiting factor in all cucurbit producing areas of the world ( 41 150 218 ) .This cucurbit disease has been reported in North America ( 82 103 189 ) Caribbean ( 101 ) South America ( 137 184 270 271 ) Africa ( 74 102 221 239 ) Asia ( 7 111 113 130 225 301 ) Middle E ast ( 4 44 117 131 286 ) Mediterranean ( 80 329 ) Europe ( 10 1 43 165 218 260 310 322 323 331 ) Australia ( 169 ) and New Zealand ( 23 107 236 ) This potentially devastating disease of cucurbits has been recognized as economically important and has been extensively studied all over the world, since the early 1800 s under field and greenhouse conditions ( 353 ) Powdery mildew has been shown to reduce yield by decreasing the size and number of fruits, and length of time these fruits can be harvested ( 214 ) Fruit qu ality and marketability are reduced due to premature leaf senescence which renders fruits exposed and susceptible to sunscald ( 353 ) Additionally, powdery mildew infection results in premature or incomplete fruit ripening causing poor flavor in melons, reduced storability in squash, and brittle and

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32 bleached stems handles in pumpkin ( 235 256 ) Other imperfections on fruit rinds such as speckling, raised indentations and edema may occur. Moreover, powdery mildew infection can predispose cucurbit plants to other important diseases such as some viruses and gummy stem bli ght caused by the fungus Didymella bryoniae ( 200 ) Causal Organisms The two powdery mildew fungi most frequently reported to cause disease on field and greenhouse cucurbit crops are Podosphaera xanthii ] and Golovinomyces cichoracearum ( 10 71 157 214 255 331 ) Both pathogens belong to the family Erysiphaceae consisting of 16 genera and approximately 650 species ( 27 ) A third endoparasitic species of cucurbit powdery mildew, Leveillula taurica has been considered of minor economic importance and occurs only in warmer areas such as countries surrounding the Mediterranean Sea ( 73 164 329 ) Powdery mildews are obligate biotrophic pathogens and cannot survive in the absence of a living host, unless in the form of chasmothecia which are the ( teleomorphic ) overwintering stage ( 200 ) Due to the obligate biotrophy of the powdery mildews, these fu ngi cannot be cultured on artificial media ( 161 ) and require living hos t tissue to grow and sporulate. These fungi are typically ectoparasites which grow on the host surface, obtaining nutrients from t he host epidermal cells through specialized structures called haustoria ( 104 ) In general, p owdery mildew fungi induce identical distinctive signs on cucurbits, however with the use of standard light microscopy; the organisms can be easily distinguished based on morphological characteristics ( 27 ) The identifica tion of cucurbit powdery mildew fungi has not always been made accurately. In early literature, the two major species may have been misidentified and

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33 were considered as synonymous ( 195 ) For example since the first report (in 1925) of cucurbit powdery mildew on melon in the Imperial Vall ey of California by Paulus and others ( 247 ) the pathogen on melon and other cucurbit species ( 248 ) in the U.S. was generally regarded as Erysiphe cichoracearum In 1968, Sphaerotheca fuliginea was named as the cause of powdery mildew without mention of E. cichoracearum in an article on control of powdery mildew on cucumber and squ ash ( 246 ) and two other reports on genetic resistance referred only to E. cichoracearum ( 77 ) In 2004, McCreight ( 189 ) published an extensive and interesting review on the change of the causal species of cucurbit powdery mildew in the U.S., demonstrating how the taxonomy and nomenclatu re of these pathogens have been unclear, and at times inaccurate. In recent literature, the nomenclature of the two main causal agents of cucurbit powdery mildew has been controversial and, not yet standardized. The organism currently designated as P. xanthii has formerly been reported as Sphaer otheca fuliginea (Schleccht. ex Fr.) Poll Other synonyms have included Sphaerotheca fusca (Fr.) Blumer emend. U. Braun ( 71 179 ) Sphaerotheca cucurbitae (Jacz.) Z.Y. Zhao ( 108 225 ) and Podosphaera fusca ( 255 325 ) The other cucurbit powdery mildew pathogen, Golovinomyces cichoracearum has previously been referred to as Erysiphe orontii Cast. Emend. U. Braun ( 71 ) Erysiphe cichoracearum (DC ex Merat) ( 142 ) and Golovinomyces orontii (Castagne) V.P. Heluta ( 235 ) Based on scanning electron microscopy of the anamorphic stages and extensive molecular and phylogenetic work, the genus Sphaerotheca has become synony mous to Podosphaera; G. cichoracearum synonymous with E. cichoracearum ( 27 41 325 )

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34 Morphology Morphological characteristics of anamorphs and teleomorphs have been widely used among researchers to distinguish between powdery mildew genera. Hammett ( 107 ) argued that conidial dimensions within the Erysiphaceae fungi overlap, and have little diagnostic value. However, conidial length/width ratios have been reported to reliably distinguish between P. xanthii and G. cichoracearum ( 269 ) Furthe rmore, since ascal structures vary with environmental conditions and do not develop on all hosts, conidial observations are more generally useful ( 322 ) Cook et al. ( 52 ) demonstrated the identification and classification of powdery mildew anamorphs using light (LM) and scanning electron microscopy (SEM) as well as host range tests The autho rs indicated that previously undescribed features on the surfaces of powdery mildew conidia, revealed by SEM, reinforced differences observed by traditional light microscopy. Description of conidia germination patterns were also proposed as reliable key to aid rapid identification of powdery mildew anamorphs ( 51 ) Both P. xanthii and G. cichoracearum produce hyaline, septate and thin walled mycelia The hyphae are relatively straight and flexuous, and the hyphal cells are uninucleate and vacuolated. Conidia are colorless (hyaline), uninucleate, one cell ed and are produced in chains ( 24 ) on the conidiophores. The conidia are readily separated from conidiophores and become airborne with moving air. Podosphaera xanthii conidiophores have foot cells of 26 86 x 10 16 m, associated with 1 3 shorter cells. Conidia are hyaline and smooth, with crenate edge lines formed by chained immature conidia ( 304 ) Conidia are primarily ellipsoid to ovoid with absolute values of length and width ranging between 20 39 x 12 22 m ( 36 304 ) Tomason and Gibson reported length/width ra tios of 1. 38 1.66 ( 322 ) while Frolov and

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35 others repor ted slightly larger ranges of 1. 32 1.76 and 1.57 1.79 for this same species ( 88 152 ) Mature conidia of P. xanthii contain well defined fibrosin bodies ( 22 ) These distinctive refractive parti cles (not present in G. cichoracearum ) appear yellow or blue when observed in polarized light ( 107 ) Fibrosin bodies are rod shaped structures measuring between 2 8 m in diameter and easily be visualized using s tandard light microscopy when fresh conidia are mounted in 3% aqueous KOH (potassium hydroxide) solution ( 24 ) .The true nature of these fibrosin bodies remains unclear, however they have been considered taxonomically relevant since first detected by Zopf in 1887 ( 27 77 ) The conidiophores of G. cichoracearum are composed of chains of conidia with sinuate edges ( 304 ) Basal foot cells are 40 140 m long and 9 15 m wide, and are usually fol lowed by 1 3 shorter cells measuring 10 30 m. Fresh conidia are ellipsoid ovoid to cylindrical doliform, with length of 25 45 m and width of 14 22 m, and do not contain fibrosin bodies ( 160 ) The germ tubes generally terminate in a club shaped aspersorium ( 51 ) Unlike, P. xan thii germ tubes are usually short and forked ( 22 23 ) The length to width ratio for G. cichoracearum has been described as being greater than 2.0 ( 127 152 ) Other authors have described ranges of 2.04 2.42 ( 322 ) and 1 .91 1.96 ( 88 ) Powdery mildew fu ngi reproduce largely asexually. Th e sexual stage is controlled by a bipolar heterothallic mechanism ( 198 ) and therefore requires two compatible hyphae of opposite mating types f or reproduction to occur and give rise to a fruiting body ( chasmothecium ) which contains one or more ascus, bearing the ascospores

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36 (sexual spores). N evertheless the role of the sexual process and epidemiology remains unclear. Still, this information could prove essential for developing more effective control strategies beca use the increased genetic diversity resulting from sexual reproduction could produce new combinations of virulence genes a nd fungicide resistance genes, making cucurbit powdery mildew management more challenging When found, the chasmothecia (formerly know n as cleistothecia) of P. xanthii and G. cichoracearum are similar. These ascomata are useful diagnostic morphological features when present. Visually, the chasmothecia are globose, dark brown to black and appear embedded superficially on the mycelium. The presence of one or several asci in side each chasmothecium as well as the morphology of its appendages are also important references and can be useful for species identification. Depending on the species of powdery mildew, a variety of appendages may occur on the surface of the chasmothecium and these appendages are thought to act like hooks or adhesive fasteners, attaching the se fruiting bodies to the host ( 333 ) In G. cichoracearum the average diameter of the chasmothecia is 85 160 m with 2 to 25 asci containing only 2 ascospores per ascus. The ascospores are one celled, primari ly straight and their size range is 18 30 x 11 20 m ( 25 ) In the case of P. xanthii chasmothecia diameter has been described as 56 80 x 56 70 m, containing a single globular ascus with dimensions of 52 70 x 44 56 m bearing eight elliptical ascospores of 12 20 x 10 16 m in size ( 36 ) Taxonomic c lassification Cucur bit powdery mildews are in the Kingdom Fungi; Phylum Ascomycota; Subdivision Pezizomycotina; Class Leotiomycetes; Order Erysiphales and F amily Erysiphaceae ( 33 ) In 1753, in his Linneaus mentioned the first

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37 binomial species referring to a powdery mildew, Mucor erysiphe (currently known as Phyllactinia guttata ) ( 342 ) Nearly 150 years later, in 1900, Salmon ( 288 ) published the pa thogens appeared more than 8 0 years later, when Braun ( 24 ) published th e si information regarding the biology, host range, distribution, phylogeny and taxonomy of the anamorphic as well as teleomorphic stages of these pathogens has increased immensely ( 26 ) especially with the advent of molecular techniques and tools. Currently, a new and updated monograph has been underway by Braun U. & Cook R. T. A., ( 26 ) In this new book, scheduled to be launched at the C BS (Fungal Biodiversity Center of the Royal Netherlands) Symposium on April 2012, the number of monograph in 1987, up to approximately 820 species including several revisions and description of new species ( 26 ) Signs and s ymptoms Signs of cucurbit powdery mildew disease are easily recognized as white to gray talcum like fungal growth primari ly composed of conidia, which may appear on leaf surfaces, petioles and stems. Fungal hyphae and conidia are produced in abundance forming a white powdery mycelium that resembles talcum powder or dust on the plant surface. These numerous conidia are easily carried to adjacent leaves and plants within a field, as well as to those at greater distance via any air movement. Symptoms usually develop first on the underside of older leaves, lower in the canopy, which is protected from direct sunlight. Initially, s mall yellow spots may form on

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38 the upper leaf surface opposite to the powdery mildew colonies. As the disease progresses, the individual fungal spots enlarge and coalesce becoming reddish brown and necrotic Heavily infected plants become chlorotic. Affecte d plants eventually senesce and drop their leaves prematurely leaving the fruits more susceptible to damages caused by sunburn Cucurbit plants in the field are often not affected until after fruit set. L eaves are reported to be most susceptible 16 23 days after unfolding ( 200 ) P owdery mildew on watermelon Althoug h all cucurbits are susceptible to powdery mildew, watermelon had been considered to be the most resistant ( 321 334 ) Except for a few isolated and minor cases of the disease on watermelon fruit (Maia, personal observation) ( 120 215 277 ) powdery mildew had been an occasional problem for watermelon production. However, since 1996 the incidence of watermelon powdery mildew outbreaks has increased and the disease has become an important problem in the majo r U.S. production areas ( 65 128 275 305 318 ) and in other parts of the world ( 46 239 268 334 ) To date, just P. xa nthii race 1 (1W) and race 2 (2W), have been identified on watermelon in the U.S. ( 46 65 66 68 319 ) Since 1996, outbreaks of watermelon powdery mildew have been confirmed in South Carolina, Georgia, Florida, Oklahoma, Texas, Maryland, New York, Arizona and California ( 63 65 128 305 319 ) Watermelon lines with resistance to powdery mildew have been demonstrated in the U.S. ( 64 66 ) and worldwide research is currently underway to find plant in troductions with new sources of resistance ( 63 66 68 140 171 268 318 319 321 )

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39 Early detection of powdery mildew on watermelon can be difficult because the presence of the pathogen is less apparent than on other cucurbit crops ( 66 ) Watermelon leaves often begin declining prior to obvious detection of fungal mycelia or conidia. On some highly susceptible cultigens, the petioles and stems can show water soaked areas ( 319 ) in addition to yellow blotching on leaves with little or no sporula tion, accompanied by a small amount of mycelia, and conidial development on either leaf surface without the characteristic chlorotic spots ( 65 ) E pidemiology and disease cycle Cucurbit powdery mild ew s have similar disease cycles. T ypically, the fungus produces hyphae and as exual spores (conidia) on lower and older leaves during the crop growing season. From infected leaves, conidia are readily disseminat ed to healthy adjacent leaves and plants by any air movement or water splashing Characteristically, in FL, in the absence of the sexual cycle (i.e. production of chasmothecia) windborne conidia land on susceptible healthy tissue, germinate, produce an abundance of mycelia and conidiophores which give rise to more conidia and the per petuation of the disease cycle (Figure 2 1) The initial source of cucurbit powdery mildew infection can be difficult to determine due to the fact that conidia are readily airborne and can travel long distances The disease spreads almost exclu sively asex ually (via conidia). P ossible sources of infection could include cucurbit crops grown earlier in the season, inoculum from greenhouse grown cucurbits, ascospores stored in chasmothecia on crop debris ( 200 ) alternate hosts ( 242 243 249 313 324 ) and Studies in the U.S. have demonstrated that p owdery mildew conidia are dispersed by wind from southern states where cucurbit are grown earlier, into northern regions ( 200 353 )

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40 U pon landing on a susceptible host, powdery mildew conidia produce a short germ tube (Figure 2 1) ending in a primary appressorium from which primary haustoria are formed inside host epidermal cell s. P rimary hypha e arise from primary appressoria or from other pole s of the conidia and form secondary appressoria from which secondary haus toria are formed. At this stage, morphologically distinct conidiophores emerge vertically (Figure 2 1) from secondary hyphae on the surface of host tissue. At the tip of each conidiophore, 5 or more conidia are produced in single chains. The abundance of h yphae and conidia forms the white mycelium on the surface of the plant tissue and this be comes the characteristic talcum like sign of powdery mildew infection ( 254 ) Chasmothecia are thought to be overwintering structures and source s of cucurbit powdery mildew inoculum ( 124 169 198 218 340 ) In a 3 year study of powdery mildew of grapes, chasmothecia were confirmed to serve as the primary source of inoculum ( 250 ) However, in the case of cucurbit powdery mildew, these structures have not been observed or are rarely found ( 353 ) in several important cucurbit producing areas in the U.S. and around the world ( 47 103 198 212 218 ) Within the family Erysipha ceae, several species are heterothallic and therefore require two compatible hyphae of opposite mating types for sexual reproduction to occur. Following the encounter of these opposite mating types, a small round and dark fruiting body termed chasmothecium (formerly kno wn as clei s tothecium), is formed which contains one or more asci bearing the ascospores (sexual spores) While chasmothecia can be obtained under laboratory conditions, their production in the field varies greatly depending on the region. For instance, in New York State (U S ), chasmothecia were observed every year from 1989 to 1994, while in other U S s tates,

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41 the occurrence was sporadic ( 198 ) The same observations were reported in Europe ( 12 181 328 ) Ecology Powdery mildew develops rapidly under favorable conditions, taking 3 7 days from infection to the appearance of the first symptoms ( 334 ) A large number of conidia can be produced in a very short time and rema in viable for 7 8 days ( 200 ) Unlike other fungal pathogens, powdery mildews are able to survive and sporulate on host tissue without presence of free water ( 344 ) In contrast to most fungal spores, powdery mildew conidia are fully hydrated and germination does not require uptake of exogenous water ( 333 ) The combination of factors such as temperature, humidity, sun light (radiation), wind, and rainfall influence dissemination and germination of conidia, mycelial growth and fungal sporulation ( 342 ) Rain and free moisture on the plant surface are unfavorable; h owever disease development occurs in presence or absence of dew ( 200 ) Powdery mildew becomes more severe during periods of low rainfall in the winter and spring months in Florida. The fungus is thought to survive between crop seasons, on wild cucurbit and other weeds year round ( 235 ) In successive cucurbit field plantings older plants planted up w ind, have been reported to serve as source of conidia ( 200 ) The role of non cucurbit hosts as source of inoculu m has been insufficiently investigated An example is a common ornamental plant in the family Verbenaceae v erbena ( Verbena spp.) which has been reported as a source of inoculum specially for cucurbits transplants grown under greenhouse conditions ( 59 200 )

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42 Greenhouses and other protective, season extension structures can also allow for the persistence of inoculum between field productions Greenhouses have been consid ered sources of powdery mildew since these structures often provide ideal conditions for powdery mildew development due to the microclimate which includes m oderate temperatures ( 20 30 C ), high relative humidity (>95%), higher plant density, shading and lower light inte nsity ( 245 ) and constant air movement which favor disease development and spread In some pa rts of the worl d such as Canada ( 76 ) Japan ( 113 224 ) Spain ( 71 ) Israel ( 272 ) and the Netherlands ( 138 293 ) where some type s of cucurbit are commercially produced in greenhouses, powdery mildew is a serious concern Temperature optimum varies for cucurbit powdery mildew pathogens. Work on conidial germination, carried out by Yarwood and Gardner ( 345 ) demonstrated that the temperature optimum for P. xanthii ranged from 9 34 C, with 22 C promoting the greatest growth. An isol ate from cantaloupe in the hot Imperial Valley of California had an optimum of 25 28 C, whereas an isolate of the same powdery mildew species found in squash in the cooler area of Colma (California) had an optimum of 15 C ( 124 ) Nagy ( 230 ) showed that the conidial germ ination of P. xanthii was observed at temperatures of 20 30 C with optimum of 22 C and the temperature range for G. cichoracearum had a larger interval of 15 30 C with optimum temperature at 25 C. Recently, laboratory experiments established that the tempe rature range of 15 25 C was optimum for conidial germination of G. cichoracearum and 25 30 C for P. xanthii ( 164 ) Daytime temperatures of 38 C or above have been reported to stop fungal development ( 200 )

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43 Dry atmospheric conditions can favor colonization, sporulation and dispersal of both pathogens, however P. xanthii tolerates higher moisture content during the infection process with the highest infection potential at 15 C and 65% relative humidity ( 14 ) High relative humidity is favorable for infection and conidial surviv al but infection can occur at levels as low as 50% ( 200 ) In the Czech Republic, G. cichoracearum i s the predominant cucurbi t powdery mildew pathogen, affecting 70% of field grown cucurbits In this same country P. xanthii has predominated on cucurbits in warmer production areas an d glasshouses ( 143 ) It has been widely shown that both cucurbit pathogens may occur either singly or together. Mixe d infections were found in 10 to 40% of the samples collected in the Czech Republic from 1995 to 2003 ( 143 ) In a subsequent study, Kristokova et al. ( 144 ) found that G. cichoracearum was found with P. xanthii as mixed infections in 28% of the locations surveyed in different parts of Europe. Infections by P. xanthii alone occurred only in up to 5% of the Czech samples during 1995 2003 periods ( 143 ) In contrast, 83 % of cucurbit powdery mildew infections detected in France were caused by P. xanthii ( 264 ) Comparatively, in the U.S., P. xanthii alone appears to be the most important cause of powdery mildew on commercial cucurbits ( 189 ) In a survey carried out from 2004 to 200 6 researchers in India reported the occurrence of cucurbit powdery mildew pathogens on 35 wild plant species ( 249 ) Host range Powdery mildew pathogens have a broad host range comprised strictly of angiosperm species. These plant pathogenic fungi have been shown to infect leaves, stems, flowers and fruits of nearly 10,000 plant species ( 98 ) Conflicting views on host

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4 4 range and on the specie s concept within the Erysiphaceae have a risen ( 27 ) with recent advances in mo lecular and phylogenetic a nalyse s ( 26 98 312 314 ) H osts of P. xanthii include economically important families such as Asteraceae (Compositeae), Cucurbitaceae, L aminaceae Fabaceae, Solanaceae and additionally Scrophulariaceae and Verbenaceae ( 78 176 255 265 ) The genus G olovinomyces has a broad and overlapping host range including the Asteraceae, Beraginaceae, Scrophulariaceae, Cucurbitaceae Solanaceae and Laminaceae F ield samples of G. cichoracearum from varied hosts belonged to six distinct RFLP haplotypes with each haplotype specific to either a single host or to a set of related host species ( 160 ) Recent studies of the phylogenetic relationship between G. cichoracearum i solates from Australia and isolat es from the northern hemisphere resulted in the distinction of 6 lineage groups based on host range, foot cell morphology and chasmothecia ( 55 ) Reports from the U.S. have also identified G. cichoracearum as the causal agent of powdery mildew on Cor e opsis spp. L. (Asteracea e ) ( 99 299 ) In 2004, researchers in California described the first occurrence of G. cichoracearum on potato (Solanaceae) plants from three fields, based on morphology and polymerase chain reaction (PCR) ( 279 ) Gevens et al. ( 95 ) described the first occurrence of G. cichoracea rum in sunn hemp ( Crotalaria juncea L. Fabaceae) in Florida based on morphology and amplification via PCR genomic sequence variation Adam et al. ( 1 ) reported a single isolate of G. cichoracearum recovered from Arabidopsis thaliana L. (Brassicaceae), to be pathogenic on five cucurbit species including cucumber, watermelon and pumpkin ( 189 ) Golovinomyces cichoracearum has also been reported on other cucurbit species such as Momordica balsamina L.

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45 (balsam apple) Trichosanthes dioicia Roxb. (pointed gourd) Lagenaria vulgaris (Molina) Standl. (bottle gourd) Coccinia cordifolia (L.) Voigt. (scarlet gourd) and Benincasa hispida (Thunb.) Cogn. ( wax gourd or winter melon) ( 322 ) Recently, reports of G. cichoracearum on important ornamentals and medicinal herbs such as zinnia ( Zinnia eleg ans Jacq.) ( 243 ) gerbera ( Gerbera jamesonnii Bolus ex Hook. f. ) ( 324 ) and chamomile ( Ma t ricaria chamomilla L. ) ( 242 ) have been confirmed. P opulation biology and genetic diversity Both G. cichoracearum and P. xanthii are characterized by great pathogenic variability represented by existence of different pathotypes and races ( 123 146 147 162 190 260 328 ) To date, the identification of races of P. xanthii and G. cichoracearum has been based upon the differin g disease responses on several muskmelon ( Cucumis melo ) cultigens ( 41 158 163 320 ) E leven muskmelon differentials have been widely used as race differentials, including : Iran H, Vedrantais, Top Mark, Ananas, PMR 45, PMR 5, WMR 29, Edisto 47, PI 414723, PI 124112 and MR 1 ( 190 ) Physiological races of P. xanthii on muskmelon were first recognized more than 70 years ago, in the Imperial Valley of California, when races 1 and 2 of the pathogen ( 121 ) I n 1976, race 3 of P. xanthii was first observed in the U.S. in Texas. ( 193 ) Since then, new races continue to be described in the U.S. ( 188 190 191 193 ) and all around the world ( 113 137 144 145 163 271 296 3 23 328 ) Currently, close to 30 distinct physiological races of P. xanthii ( 39 190 193 ) and two races of G. cichoracearum ( 11 158 162 264 322 ) have been described based on the differing responses of various muskmelon ( Cucumis melo ) cultigens to these

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46 pathogens. Worldwide, many other muskmelon cultigens have been considered as race differentials ( 41 185 188 190 298 347 ) Recently, watermelon cultigens have been investigated as potential differentials ( 319 321 ) I t will be imperative that researchers of watermelon and muskmelon powdery mildew resolve disparities in race identification. In a current review, Lebed a et al. ( 158 ) prop ose that the international cucurbit powdery mildew research, breeding, seed and production communities use a unified and uniform system of cucurbit powdery mildew determination and denomination. Additionally, they propose a uniform screening methodology ba sed on leaf disk protocol under uniform conditions ( 161 ) Two sets of differential cucurbit genotypes for the identification of cucurbit powdery mildew pathotypes and races, as well as an objective, an d comprehensive coded system for meaningful and concise designation of pathotypes (sextet code) and races (septet code) ( 158 ) The proposed set of differentials for pathotypes screening includes six cucurbit genotypes( 45, Citrullus lanatus Cucumis, Cucurbita, Citrullus ), and five species ( C. sativus, C. melo C. pepo, C. maxima, C. lanatus ), and, ultimately, six unique cucurbit genotypes. Eac h genotype is arbitrarily assigned a permanent differential order (1 6) and value (1, 2, 4, 8, 16, or 32) for a compatible (i.e. susceptible) interaction. In a given assay with a particular G. cichoracearum or P xanthii strain, the interactions are scored and then summed to yield a unique sextet code for that strain. Due to their greater number, the proposed set of race differentials is arbitrarily divided into three groups. This set comprises 21 genotypes of the single species, Cucumis melo The race diffe rentials are assigned an arbitrary permanent order with a group prefix (1 3) and

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47 value (1, 2, 4, 8, 16, 32 or 64) within each group. The binary results of any assay are then translated into a triple part, septet code and one part for each group of seven di fferentials. The three sums are then presented as a unique code in the format: sum of group 1, sum of group 2 and sum of group 3, which serves as a unique identifier for each race ( 158 ) Such detailed and standardized assessment of pathogenic variability will greatly improve our knowledge and exchange of cucurbit powdery mildew resistance. This proposed system gives basis for the application of population biology and genetic studies, which are important for both applied and theoretical research in resistance breeding. In a s urvey of the occurrence, distribution and pathogenic variability of cucurbit powdery mildew races in 84 locations in the Czech Republic during the year of 2001, Lebeda et al. ( 162 ) reported finding 22 races of G. cichoracearum and 4 races of P. xanthii Additionally, they identified 9 different pathotypes, 6 of G. cichoracearum and 3 of P. xanthii Sedlakova and Lebeda investigated the dynamics of temporal changes in cucurbit powdery mildew populations from 2 001 to 2004. Their findings indicated that among the 180 isolates examined, 16 diff erent pathotypes were found (10 of G. cichoracearum and 6 P. xanthii ); 63 races of G. cichoracearum and 26 races of P. xanthii ( 296 ) were identified. Based on their results, the r esearchers suggested that pathotypes are distinguished by host range on the most important cultivated cucurbit types a nd that races are distinguished by the level o f virulence on a set of muskmelon differentials which have different resistance factors ( 158 ) T he exact number of physiological races of P. xanthii varies greatly depending on research groups and the focus of the research progra m. Inconsistencies in the reactions

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48 of muskmelon cultigens to cucurbit powdery mildew pathogens have led to confusion and misinterpretation of resulting responses. Plant resistance responses can differ with plant age at the time of inoculation ( 49 ) purity of the pathog en isolates ( 234 ) differences in environmental conditions in the greenhouse versus the open field, cropping season ( 42 ) level or concentration of inoculum ( 234 ) and shading ( 167 ) Fur thermore, Bardin et al. ( 10 ) emphasized that race determination in powdery mildew can be confusing because most of the differential melon lines appear to possess several resistance genes, some of which h ave not yet been characterized. Additionally, it has been shown that the level of resistance varies when plants are tested under different environmental conditions and/or geographical areas. Such differences may also be attributed to variability in the vir ulence and aggressiveness of isolates present in the natural pathogen populations ( 145 ) Powdery Mildew Diagnosis and Research With the advent of molecular and DNA analysis techniques, esp ecially polymerase chain reaction (PCR) and sequencing technology, the identification and characterization of cucurbit powdery mildew, as well as other fungal plant pathogens, has greatly advanced ( 182 ) While assessment of the phenotypic (and morphological) characteristics is still necessary for identification, current technologies can be employed to investigate species identity and to discrimina te variations within species ( 54 55 ) Furthermore, molecular techniques have been particularly useful in the identi fication of obligate pathogens such as powdery mildews, as well as fungi that grow v ery slowly in the culture media ( 231 ) In contrast to conventional methods, including pathogen isolation and assessment of morphological characteristics using microscopy, molecular

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49 techniques provide accurate, consistent and reproducible results more rapidly, which facilitates early disease management ( 232 ) The development of gene specifi c primers for PCR amplification has facilitated the detection and ide nt ification of fungal plant pathogens ( 337 ) Based on sequence variations of the ITS region, species specific primers S1/S2 and G1/G2, specific for P. xanthii and G. cichoracearum respectively, were recently designed by Chen et al. ( 36 ) These primers are able to amplify a portion of rDNA of P. xanthii equivalent to 454 bp and to 391 bp for G. cichoracearum Development of these primers allowed successful identification of P. xanthii and G. cichoracearum in mixed infections by means of multiplex PCR which enable d simultaneous amplification of the different pathogens, in a single reaction, by using more than one pair of prime rs simultaneously ( 36 ) Accurate determination of the pathogen species is very important not only for dis ease management but also in plant breeding programs since different resistance genes may confer resistance to different pathogen species and pathotypes ( 77 ) With r ecent advances in biotechnology, molecular genetic markers have been used for rapid identification of some cucurbit powdery mildews ( 315 316 ) The u se of molecular characters, especially ITS (internal transcribed spacer) sequence data, has given promising results for species de termination in some powdery mildews ( 28 54 312 314 ) still to date, molecular tools have yet to differentiate species to race. Disease Management An effective method for management of powdery mildew should include IPM (Integrated Pest Management) components combining use of powdery mildew resistant or tolerant cultivars (genetic resistance), use of biorational compounds, biological control agents, use of synthetic and preventive fungicides as well as compoun ds that

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50 stimulate host defense mechanisms ( 222 ) A c curate knowle dge about the population structure of a patho gen is essential to improve the design of appropriate disease management programs as well as to reinforce resistance breeding strategies Host resistance T he use of resistant varieties is the simple st effectiv e eco compatible and economical means of controlling plant diseases ( 70 201 307 ) P owdery mildew pathogens are already present in cucur bit producing regions worldwide and the use of resistant plant cultivars would be the most suitable control strategy ( 251 ) R esistant varieties are contin uously being developed and are becoming an increasingly important component of management programs all over the world ( 161 261 302 322 323 339 347 352 ) In the U.S., cucurbit breeding programs have conducted extensive research in an eff ort to increase availability of powdery mildew resistan t lines. Most resistance squash and pumpkin varieties in the U.S. contain one or two copies of the same major resistance gene form wild cucurbit species ( 200 ) Resistance already exists in melon ( 110 190 ) gourds ( 140 ) cucumber ( 22 ) and watermelon ( 63 66 68 110 319 321 ) Temporal avoidance (avoiding high risk seasons) Adjusting planting dates to avoid con ditions favorable to the pathogen is important when managing cucurbit powdery mildew. In Florida, warmer temperatures (10 32 C) and drier conditions from fall through spring typically favor the occurrence of P. xanthii ( 330 ) Since cucurbit production occurs year round in the state of Florida, this has enabled powdery mildew to become e ndemic in the state, occurring every year to some degree ( 235 330 )

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51 Scouting and earl y prevention in high risk areas and crops P rogrammed scouting for powdery mildew symptoms is critical to determine appearance of the very first signs and symptoms of the disease. Es pecially the underside of older leaves as well as petioles and stems lower (shaded areas) in the canopy should be monitored early and frequently ( 330 ) Weekly inspections are recommended e specially af ter fruit set when plants become more susceptible to powdery mildew disease Preventive fungicide applications should start when cucurbit o produce fruit ( 200 ) E arly control of powdery mildew is the most effective strategy for effective control and maintenance of yield and quality of cucurbits ( 65 200 ) Application of fungicides Biological and o rganic fungicides : Global desire to reduce total pesticide load in the environment combined with the higher cost of fungicide applications and limitations in use of some resistant cultivars have led to extensive research into alternative methods for powdery mildew control Biorational, biological and other eco friendly strategies have been examined a n d developed all over the world ( 16 135 287 ) Greenhouse crops may offer the best opportunities for imple mentation of biological and non fungicide strategies for control of powdery mildew and other disease ( 245 ) The ability to control environmental conditions, restrictions for pesticide usage, and the higher value of greenhouse grown crops make the use of bioc ontrol and cultural methods more adequate for greenhouse and protected structures ( 217 303 ) Several biopesticides are registered for conventional and organic control powdery mildew disease in the U.S. Perhaps the most widely studied biofungicide of many powdery mildew fungi, including cucurbit powdery mildew, is a naturally occurring fungal

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52 hyperparasite Ampelomyces quisqualis ( 75 134 136 258 259 ) A commercial formulation of this hyperparasite, AQ10, must be applied preventatively along with a mineral oil or other surfactants ( 136 208 283 297 ) to be most effective. Studies of AQ10 in combination with chemical fungicides have been conducted ( 97 306 ) Another biological control agent registered for cucurbit powdery mildew control in the U.S. is the antagonistic bacteria Bacillus subtilis (strain QST 713). Commercial formulations, Serenade and Rhapsody, of this bacterial strain have demonstrated adequate contro l against P. xanthii under controlled conditions ( 97 ) Additionally, bioassay studies indicated that lipopetides extracts obtained from cultures of two antagonic strains of B. subtilis (UMAF6614 and UMAF6639) were able to arrest conidial germination of P. xanthii (syn. P. fusca ) in v itro ( 282 ) In greenhouse and field trials the m icrobial products Actinovate AG, Companion B U EXP 121 6C, and BU EXP 1216 (the first formulation containing Streptomyces lydicus and the other three, B. subtilis ) were assessed in Florida on summer squash and cantaloupe against powdery mildew Applications of these inoculants alone or alternated with a half r ate of conventional fungicide Procure 480SC (triflumizole) was evaluated In greenhouse experiments, the prod uct BU EXP 1216S significantly reduced the disease severity by nearly 70% relative to the water control but was not significantly different from t hat obtained with Procure 480SC in two of four greenhouse experiment s. T he untreated water control, BU EXP 1216C and BU EXP 1216S, when applied alternately with Procure 480SC, consistently promoted plant growth. Alternating applications of all four produc ts with Procure 480SC resulted in significantly less powdery mildew disease than in the water control alone ( 351 )

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53 Field trials conduc ted in Arizona, comparing the efficacy of the biopesticide Actinovate ( Streptomyces lydicus Waksman & Henrici ), Kaligreen (potassium bicarbonate) and Procure (triflumizole), applied alone or within a rotation program with each other, for control of powder y mildew on cantaloupe showed reduction of disease after five applications of Actinovate or Kaligreen (72 and 59%, respectively) alone at weekly intervals ( 183 ) Cucurbit powdery mildew control with oils and mineral salts has been evaluated In laboratory and g reenhouse studies, JMS Stylet Oi l significantly slowed the expansion of powdery mildew colonies from both natural infections and artificial inoculations ( 209 ) Cucumber plants treated with 0.5 and 1% olive oil and with potassium silicate respectively, demonstrated sig nificant powdery mildew control ( 252 ) Evaluation of black seed oil ( Nigela sativa L .) against powdery mild ew of cucumber reveal ed that at a concentration of 0.5%, this oil significantly reduced the severity of P. xanthii from 52% in the control plants to nearly 8% on leaves sprayed with black seed oil. Additionally, toxicity tests and microscopic examinations demonstrated that hyphal growth and conidial germination were greatly compromised ( 105 ) In greenhouse experiments, mineral oil SunSpray U ltra fine combined with 0.5% bicarbonate salts (sodium and potassium) significantly reduced powdery mildew (6.1 % and 5.9%, respectively) with inhibitory affects lasting for 10 days after treatment ( 354 ) S tudies demonstrated pre inoculation with salts (K 2 HP0 4 KH 2 P04 + KOH, KNO and NaHCO 3 ) controlled powdery mildew on leaves of greenhouse grown cucumbers as effectively as the systemic fungicide pyrifenox ( 272 ) B iocompatible products with low toxicity and adjuvants for control of powdery mildew, s pecifically on cucurbit crops, have been studied. The

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54 paraffinic oil JMS Stylet Oil suppressed powder y mildew in field grown squash Powdery mildew severity was significantly lower than non treated plants for cucurbits receiving applications of 0.75% JMS Stylet Oil initiated 5 days post inoculation in greenhouse studies and with applications initiated aft er disease detection in the field ( 208 ) The use of plant extracts and plant compounds to control cucurbit powde ry mildew has been investigated. Soil applications of bio fertilizers combined with organic mulch and foliar spray of fermented garlic were reported to reduce the incidence of powdery mildew on o rganic melon produced under greenhouse conditions ( 267 ) Tests with Milsana (leaf extracts from the giant knot weed Reynout ria sachalinensi s F. Schmidt ex Maxim. applied to powdery mildew susceptible ( Mustang ) and tolerant ( Flamingo ) cucumber cultivars, increased the accumulation of phenolic compounds in the cucumber plants and resulted in an improved antifungal activity s ignificantly reducing the incidence of P. xanthii ( 56 58 ) Similar results were obtained by Fofana et al. Plants treated with Milsana were significantly less infected compared to control plants and this protective effect against powdery mildew was maintained over time ( 86 ) Rongai et al. studied t he effects of a vegetable fungicide on cucumber powdery mildew ( G. cichoracearum ). F ormulations consisting of a dispersion of Brassicaceae meal in vegetable or mineral oils on infected muskmelon plants cultivated under plastic tunnels were tested in comparison to each oil separately. Both formulations containing B rassicaceae meals, caused 94% of powdery mildew conidia to be distorted while for the untreated controls only 2% w ere distorted ( 284 ) Numerous other studies have demonstrated the effectiveness of compounds with antifungal activities which promote

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55 host induced defenses and could potentially b e used to control cucurbit powdery mildews ( 38 106 280 283 311 ) Foliar applications of soluble silicone (Si) for the control of powdery mildew of several crops have been widely repor ted ( 227 ) An assessment of concentration ( 0, 250, 500, 750 and 1000 mg l 1 ) frequency of application (1 to 3 times per week) and runoff (covered and uncovered pots) of soluble silicon (K 2 SiO 3 ) on the severity of P. xanthii on zucchini was carried out. Results indicated that in combination with the surfactant Break Thru treatment s with Si reduced powdery mildew severity significantly. While the effect of concentration was not significant, spray frequency had a significant e ffect o n Si efficacy Using the same concentration of Si, efficacy was increased initially by 30% and almost doubled when the spray frequency was tripled An overall increase of 17% (Trial 2) and 18% (Trial 1) in disease reduction occurred on plants in unc overed pots, where Si was allowed to reach the rhizosphere, compared to the covered pots ( 317 ) The effects of s uppression of powdery mildew caused by P. xanthii (syn. S fuliginea ) on hydroponically grown cucumber by addition of silicone to nutrient solution was demonstrated to be inhibited at high temperatures (24 to 32 C) in Florida when compared to the same growing conditions in experiments conducted in Canada ( 294 ) Evaluation and whey for the control of powder y mildew in organic crop productions has been widely investigat ed M ilk based foliar sprays have been reported effective against powdery mildew ( Uncinula necator ) on grapes ( 53 ) Reports have indicated that milk based foliar sprays can effectively reduce both the signs and symptoms of natural P. xanthii in fections on pumpkin and muskmelon ( 85 ) and on tomato powdery mildew caused by Leveillula taurica in the field ( 309 ) In greenhouse

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56 experiments, milk whey sprayed twice a week at concentrations > 10% reduced severity on cucumber powdery mildew by 71 94% and zucchini powdery mildew by 81 90% in comparison to 40 and 50%, respectively, in control plants of cucumber and zucchini squash at 15 days after first application ( 19 ) Efficacy of and combinations of milk and Lactobacillus on powdery mildew of pumpkins demonstrated better suppression of P. xanthii (syn. S. fuliginea ) with raw alone ( 348 ) According to Medeiros ( 216 ) the use of milk and whey is, in general, less expensive than fungicides and has the advantage of achieving the same level of control. The viability of whey in disease control is depend ent on cost and benefits for the grower, which in turn will depend on the availability of the product and transportation costs from the dairy industries to the farm where the product will be applied ( 19 ) Conventional fungicide s : Presently, conventional fungicides are the most effective for managing cucurbit powdery mildew. There are numerous currently registered fungicides available ( 34 200 ) For conditions in Florida, fungicides should be applied immediately if powdery mildew symptoms are present, reapplying on a 7 day interval. Although f ungicides for cucurbit powdery mildew management can limit diseas e severity, th e level of protection in some crops and regions has not been sufficient Worldwide reports of reduced or lack of powdery mildew control have been indicated ( 60 83 96 108 118 166 179 201 202 296 326 340 341 ) This is likely due to the appearance of new pathogen strains insensi tive to the single site mode of action of the synthetic fungicides which consequently lose their efficacy over time ( 34 203 ) To prevent this, defense strategies that minimize the risk of de velopment of resistant

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57 strains must be adopted Therefore, the recommendation of rotation of fun gicide modes of action and limit ation on the number of treatments with the same active ingredient is highly advisable ( 34 ) Fungicide resistance It is widely known that successful management of powdery mildew in cucurbit and the appearance of new pathogenic strains. Prop er timing is critical to ensure that fungicide application s are made at appropriate times. It is important to assess efficacy of fungicides and include protectant fungicides, which are not at risk for resistance development, in a fungicide spray program ( 207 ) Fungicides with mobile (systemic, translaminar or volatile) activity are ideally recommended for protecti on and some residual activity against cucurbit powdery mildew on the underside of leaves where conditions are more favorable for early disease development ( 199 203 321 ) Tank mixing with contact fungicides has been recom mended in some regions to avoid development of fungicide resistance ( 203 ) Strains of P. xanthii have been detected with resistance to as many as eight classes of fungicides ( 77 161 201 210 236 296 302 ) I n a study on dynamics of fungicide resistance, McGrat h and Shishkoff ( 207 ) conclu ded that i t wa s not possible to predict the efficacy of fungicides based on the frequency of resistant strains the prev ious year. After one applicatio n of triadimefon and benomyl the pathogen population rap idly shifted within two weeks to predominantly r esistant strains and c onsequently, more than one application did not provide additional disease control Continued field observations are essential when implementing a fungicide program. If and when poor disease control appears following a s pray of high ri sk for

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58 resistance materials, growers should consider seeking assistance from extension crop consultants to determine if resistance has developed.

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59 Table 2 1 Major cucurbit producing countries and production estimates for 2009 Cucumber Watermelon Squash, pumpkin and gourds Melons Country Prod Country Prod. Country Prod. Country Prod. (tons) (tons) (tons) (tons) China 44,250,182 China 65,002,319 China 6,506,966 China 12,224,801 Turkey 1,735,010 Turkey 3,810,210 India 4,108,510 Turkey 1,679,190 Iran 1,603,740 Iran 3,074,580 Russia 1,123,360 Iran 1,278,540 Russia 1,132,730 Brazil 2,056,310 U.S.A. 749,879 U.S.A. 1,069,980 U.S.A. 888,180 U.S.A. 1,819,890 Egypt 700,000 Spain 1,007,000 Ukraine 883,000 Egypt 1,500,000 Iran 674,545 India 830,244 Spain 700,000 Russia 1,419,030 Mexico 577,067 Egypt 750,000 Japan 620,200 Uzbekistan 1,071,000 Ukraine 559,900 Morocco 730,000 Egypt 600,000 Algeria 1,034,720 Cuba 413,191 Mexico 552,371 Indonesia 575,995 Mexico 1,007,160 Turkey 411,942 Italy 520,800 Poland 480,553 S. Korea 900,000 Guatemala 455,556 Bangladesh 340,249 Netherlands 435,000 Spain 819,100 Brazil 402,959 Argentina 332,663 Mexico 433,644 Syria 749,695 France 301,724 S. Korea 330,000 Iraq 420,945 Greece 656,379 Pakistan 300,000 Italy 315,700 S. Korea 400,000 Morocco 650,000 Saudi Arabia 243,866 Indonesia 313,611 Total (World) 60,555,572 98,047,947 22,141,401 25,504,704 (Source: FAOSTAT)

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60 Table 2 2 United States cucurbit production in 2010 2010 Production Area harvested Value Crop (tons) (ha) ( US $1,000) Watermelons 1,866,669 53,661 492,035 Cantaloup e s 854,477 30,242 314,379 Pumpkins 481,897 19,627 203,592 Cucumbers (fresh ) 384,737 17,766 193,643 ( processed ) 542,600 39,457 180,845 Squash 296,740 17,604 116,539 Honeydews 145,331 5,949 49,608 Total (USA) 4,572,451 184,306 1,550,641 (Source: NASS/ USDA)

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61 Table 2 3 Comparison of production, area and value of the major U.S. states for cucurbit crops in 2010 2010 Prod. Area Value Crop State (tons) Rank (ha) Rank ($1000) Rank Watermelon Florida 340,330 1 9,956 2 112,545 1 Georgia 304,814 2 9,713 3 75,936 3 California 284,402 3 4,452 4 83,391 2 Texas 282,361 4 10,077 1 52,290 4 Indiana 128,820 5 2,873 6 32,376 6 S. Carolina 119,748 6 3,238 5 35,640 5 Total U.S.A. 1,866,669 53,663 492,035 Cantaloupe California 495,323 1 15,783 1 134,316 1 Arizona 221,716 2 8,418 2 97,271 2 Georgia 68,039 3 2,024 3 51,000 3 Colorado 18,960 4 890 6 7,984 5 Indiana 17,735 5 931 5 6,178 6 Texas 13,472 6 1,093 4 9,266 4 Total U.S.A. 854,477 30,243 314,379 C ucumber Florida 105,233 1 4,695 1 47,792 2 Georgia 96,388 2 3,440 2 51,000 1 Michigan 40,959 3 1,740 4 20,498 3 California 37,739 4 1,295 5 16,224 5 New Jersey 30,481 5 1,295 6 15,725 6 N. Carolina 30,436 6 2,469 3 11,743 7 Total U.S.A. 384,737 17,766 193,643 Pumpkin Illinois 193,865 1 6,111 1 15,667 5 California 84,368 2 2,509 6 18,786 2 New York 66,315 3 2,752 4 35,088 1 Ohio 50,077 4 2,792 2 16,670 3 Pennsylvania 44,089 5 2,711 5 16,524 4 Michigan 43,182 6 2,752 3 13,804 6 Total U.S.A. 481,897 19,628 116,539 Squash Michigan 59,874 1 2,671 2 12,144 6 California 54,431 2 2,428 3 34,017 3 Florida 49,532 3 3,683 1 56,784 1 New York 40,687 4 1,862 4 36,777 2 Georgia 21,772 5 1,619 5 15,360 4 New Jersey 16,874 6 1,255 7 10,304 7 Total U.S.A. 296,740 17,604 203,592 Honeydew California 107,275 1 4,452 1 31,218 1 Arizona 28,123 2 1,255 2 12,586 2 Texas 9,934 3 243 3 5,804 3 Total U.S.A. 145,331 5,949 49,608 (Source: NASS/ USDA)

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62 Table 2 4 Cucurbit production in Florida for 2008 2009 seasons 2008 2009 Production Area harvested Value Crop (tons) (ha) ( US $1,000) Watermelon 370,993 10,441 135,771 C ucumber (fresh ) 120,474 4,573 78,618 (processing ) 49,000 2,833 22,932 S quash 51,891 3,561 51,480 Total (FL) 592,358 21,408 288,801 (Source: FACS/NASS)

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63 Figure 2 1. D isease cycle of cucurbit powdery mil dew, illustrating the asexual stage.

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64 CHAPTER 3 CUCURBIT POWDERY MILDEW ISOLA TE COLLECTION, MAINT ENANCE AND CHARACTERIZATION Biotrophic fungi such as rusts, downy mildews and powdery mi ldews sporulate only on living tissue ( 285 ) As an obligate parasite the cucurbit powdery mildew fungi cannot be cultured in vitro on artificial media. These pathogens have been trad itionally cultured and maintained by periodical tra nsfers of mycelia conidia and conidiophores to fresh plant material ( 253 ) The use of leaf disk assays for obligate pathogen maintenance in plant pathology research has been a common practice for many years and in many parts of the world ( 32 115 173 175 237 266 338 ) Traditionally researchers working with cucurbit powdery mildew characterization ( 80 174 229 230 305 ) breeding programs ( 22 40 45 261 ) and fungicide resistance ( 61 108 213 219 ) have used leaf disk s as a fast and convenient bioassay However in some cases, leaf disks and whole plant inoculations have yielded inconsistent results ( 17 ) Factors such as host plant age, method of inoculation, and source of inoculum, inoculum density, relative humidity, and light intensity influence colonization and sporu lation of powdery mildew fungi H igh relative humidity (> 95%) relatively warm temperatures ( 24 30 C ) low light intensity and vigorous plant growth contribute to pathogen sporulation In general, though high relative hum idity is required, infection has been reported to occur at lower relative humidity and f ree water on plant surfaces has been considered detrimental ( 35 235 273 343 ) Through out decades of cucurbit powdery mildew research, some of the most common inoc u lum application methods h a ve been direct leaf to leaf contact ( 292 ) use of whole plants as inoculum source ( 354 ) and dusting of plants with inoculum ( 140

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65 151 226 ) Powde ry mildew inoculation originating from discrete fungal colonies maintained in vitro have been widely demonstrated and used to maintain pathogen cultures which can be considered homogeneous ( 161 ) There are contradictory reports on the harmful effects of s uspending powdery mildew conidia in water for use as inoculum ( 2 34 ) Some researchers have had successful inocu lation with spore suspensions of S. fuliginea (syn. P. xanthii ) and E. cichoracearum (syn. G. cichoracearum ) ( 116 344 ) Others have found that suspension inocu l ati on methods result in poor uniformity of spore deposition, clumping of spores, and consequently inability to accurately quantify spore deposition. Limited information is available in the literature on the effect of techniques for in vitro production and maintenance of isolates for disease establishment of P. xanthii under laboratory conditions In this research, I developed and optimized protocols for obtaining, producing and maintaining powdery mildew subculture s for assessment of pathoge n morphology, genetic variability and race identification collected from cucurbits in northern Florida Materials and Methods Field Site T rials Field experiments were conducted in n orth Florida at two University of Florida (UF) research stations. One field site was at the North Florida Research and Education Center Suwannee Valley (NFREC SV) in Live Oak in Suwanne e County FL T he second field site was at the Plant Science Research and Education Unit (PSREU) in Citra in Marion County FL ( Figure 3 1 ). At both locations, d ifferent c ucurbit types were planted in adjacent rows on raised beds covered with plastic mulch At Live Oak p lots consisted of 6 rows spaced 150 cm

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66 apart cover ed with black plastic mulch There were 16 plots per row and each plot was 275 cm long. Each plot contained five plants spaced 46 cm apart. Plot s were separate d by 92 cm between plots in the same row ( Figure 3 2 ) Two s usceptible cucurbit host s butternut winter squash ( Cucurbita moschata L. cultivar Butterbush ) and watermelon ( Citrullus lanatus (Thunb.) Matsum. cultivar Mickey Lee ) were planted at the end s of each row to serve as inoculum source s The end plots were considered buffer rows and measured 305 cm long. In addition to the susceptible hosts, 22 advanced Cucurbita breed ing lines (Rupp Seeds Inc., Wauseon, OH) were evaluated for susceptibility to powdery mildew in FL. All p lots were replicated four times in a randomized complete block design. At Citra, the field trial was comprised of 6 rows on reflective silver mulch, spaced 200 cm apart and with 3 plots per row ( Figure 3 3) Each plot was designed in the same manner as in Live Oak T he powdery mildew and were plant ed at Citra in a randomized complete block design w ith 8 replications At both locations, field soil was fumigated prior to planting and row middles were spraye d for weed control, at least one month before plant ing. During the growing season, i nsect pests and weeds were managed as needed by mechanical cultivation (no insecticides or fungicides were applied) and hand weeding of rows was done throughout the experiments The plot s were subsurface drip irrigated and fertilized as needed to maintain plant health Plant Material for Field E xperiments Two see ds of each, butternut w inter squash ( ( were direct seeded on 31 March 2009 at row ends in Live Oak Each

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67 alternating row end contained only one species of susceptible cucurbit host. On 16 April 2009, butternut win ) and watermelon ( were direct seeded into all remaining test plots. Due to lack of germination, susceptible cultivars were direct seeded again on 23 April 2009 at Live Oak At Citra were direct seeded into all plots, on 23 April 2009 in the same manner as in Live Oak Additional seeding was done on 2 June 2009 to compensate for poor germination. Origi n and C ollection o f Cucurbit Powdery Mildew S amples Cucurbit p owdery mildew isolate s used in this study originated from infected leaves of butternut collected at both research stations in north FL (Live Oak and Citra) as well as from additional cucurbit species collected from south west and north east FL (sam ples sent by Dr Pam ela Roberts and Mr. Gene McAvoy) during 2008 and 2009. Occurrence of cucurbit powdery mildew was monitored weekly at both research sites in north Florida during spring and fall of 2009. At both field sites, the pow dery mildew pathogen population was naturally occurring. Over the course of 9 weeks, during the fall of 2009, powdery mildew colonies were Florida. From each plant, 3 leaves were excised for the collection of a total of 9 powdery mildew colonies per collection day per site. At the end of a 9 week period, 126 isolates had been sampled from each field site, which represented a total of 252 cucurbit powdery mildew isolates to be maintained i n vitro at the laboratory until morphological and genetic analysis were performed

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68 Weekly s ampling was done in the spring season of 2009 during a consecutive 9 week period at Live Oak and 7 week period at Citra Leaves were remov ed only a fter assessment of powdery mildew distribution within each location and disease severity rating s had been recorded for all cucurbit plants Leaf sa mples were collected from Live Oak filed site on 19 and 26 May 2 16 and 23 June 1 10, 15 and 23 July At Citra, leaves were collected on 23 June 1 10, 15, 23 and 28 July and 3 August. A second set of samples were collected in the same manner described above, at the Citra field site during the fall of 2009 for a period of 5 consecutive weeks Forty five powdery mildew isolates were collected during this time period F all season samples were collected on 13 20 and 29 October and 3 and 13 November Each field (single colony) isolate was assigned a code detailing location and timing of collection from each field site (LO=Live Oak and Ci=Citra) during each time period (week 1 week 7 and week 8 week 12 ), according to susceptible host plant (plant 1 or 2), number of leaves sampled per plant (A, B or C) and the number of discrete colonies taken from each leaf (1, 2 or 3) (Table 3 1). Since different susceptible host plant s were grown at each field location, individual leaf samples were placed in separate labeled and sealed plastic bags and analyzed separately in the laboratory. Leaf samples were kept under refrigeration (4 C) until preparation for morphological and genetic analysis of each sample could be completed. For comparison, additional cucurbit samples infected wit h powdery mildew were collected at other locations in Florida (Table 3 2 ) and sent or br ought to our laboratory. Morphological data were collected and single colony field isolates were frozen ( 20 C) for subsequent molecular analysis

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69 Plant Material for Laboratory Experiments Since powdery mildew pathogens cannot be cultured on artificial gr owth media, it was necessary to maintain all fungal isolates on living host tissue. Traditionally, leaf disk bioassays were maintained on amended water agar to prevent early leaf senescence ( 13 40 ) In our work, rather than small leaf disks, we used detached whole leaves (cotyledons and primary leaves) as substrate for powdery mildew maintenance. Within 24 to 48h of field collection, infected onto detached leaflets and/or cotyledons of various susceptible cucurbit hosts. Preliminary tests were conducted to determine which commercially available cucurbit cultivars were most susceptib le for enhancing powdery mildew sporulation D epending on seed availability, speed of seed germination and abundance of fungal sporulation produced on host tissue different cucurbit hosts were grown for periodic isolate transfer, subculture and maintenanc e C ucurbitaceous cultivars such as musk Cucurbita evaluate d Cucurbit seedling s for all laboratory experiment s, were grown in a 24 3 C growth room in styrofoam growth trays at the University of Florida, Gainesville, FL. Trays were maintained on a bench top u nder fluorescent lighting w ith 12 hour photoperiod. S usceptible host seeds were sown every 10 to 15 days to maintain a continuous supply of fresh l eaves to be used in laboratory bioassays Styrofoam tray cells were filled with Fafard Professional 4P Mix potting media (Conrad Fafard Inc. Agawam, MA), and one

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70 seed of each cucurbit type was sown per cell. Throughout the experiments, plants were observed daily and seedlings were watered as needed Seedlings were ready for bioassays at cotyledon stage (7 to 10 days old) or at 2 or 3 true leaf stage (3 4 weeks old) depending on cultivar (Figure 3 6). To further prevent powdery mildew contamination, the air conditioner filter was cleaned regularly and structures resembling cubes made of PVC pipes and covered with organza cloth were built and place d over growing trays. Growth room doors were kept closed at all times while working inside the growth room. Optimization of Living Culture Technique for Cucurbit Powdery M ildew Detached i ntact cucurbit leaflets and cotyledon s were used rather than leaf disks to prolong the time in which cultures were viable and available for morphological and molecular characterization. During preliminary studies, we observed that detached leaf tissue could be maintained and rooted o n 2% water agar media (Figure 3 4). In initial tests, aside from maintaining isolates in Petri dishes (10cm in diameter), leaflets were also grown in 1mL test tubes containing water agar This method as successful as leaflets insert ed into test tubes rooted in to the water agar and fungal sporulation was abundant (Figure 3 5). The test tube technique required less media and was space efficient Additionally, older and larger leaves could be used for inoculations and isolate maintenan ce (Figure 3 5). Test tubes were kept inside aluminum trays (5 cm deep) lined with paper towels and had moistened cotton balls place d at each corner of the tray s to provide humidity (Figure 3 5). Each tray contained a single isolate inoculate d onto all leaves. Trays were kept at room temperature on the laboratory bench were covered with plastic bags and were sealed with twist ties. While effective and space efficient, t he use of test tubes was laborious and tubes held only a small

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71 amount of me dia which at room temperature tended to dry faster (5 7 days) than Petri Plates resulting in wilting of leaves Cu curbit Powdery Mildew Isolation, M ultiplication and M aintenance M onoconidial cultures have been used for in vitro mul tiplication of powdery m ildew isolates in past research Tradi tionally, a single powdery mildew conidium is taken from an infected sample and is transferred onto healthy sus ceptible tissue Artificial inoculation procedur es described in the literature have included methods such as dusting of con i dia ( 140 ) use of sterile brushes ( 170 223 311 ) rubbing of conidia ( 298 ) use of spore suspension ( 6 40 225 352 ) use of eyelash or hair method ( 212 ) and mass transfer of conidial chains ( 211 ) P reliminary artificial inoculation s tudies were carried out to evaluate which method would be most efficient for single spore isolation and conidia l transfer for high volume of samples Initially, an eyelash and hair affixed to a di s pos able pipette as described by McGrath et al. ( 213 ) was used without consistency Alternatively, h and made glass needles produced from a pair of Pasteur pipettes as described by Goh ( 100 ) were prepared and experimented howev er glass tips were still too thick to transfer a single conidium Additionally, a small piece of optical fiber was used as a tool to transfer a single conidium; however this material was flexuous and did not provide support for fungal transfer. The optical fiber was kindly provided by Dr. Huikai Xie (Associate Professor Department of Electrical & Computer Engineering at University of Florida) M ini ophthalmological surgical blades ( BD Micro Sharp blad e 1.5mm depth ) were investigated U nder a stereoscope t hese mini blades were used to remove a single conidium from the top of a conidiophore, and directly transfer the conidium to healthy tissue. This approach was unsuccessful because blade tips were too lar ge compared to

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72 the size of conidia. Alternativel y, t hese mini blades were used to cut out a small piece of water agar (2%) poured into small Petri plates ( 6 cm diameter) and had been coated with a suspension of powdery mildew This suspension was prepared by rinsing with sterile distilled water a small am ount of conidia from a heavily infected leaf directly onto the surface of the water agar Most likely, because water agar media and distilled water surround ed the fungal conidia, no conidia germination was observed after 7 days Powdery mildew s uspensions were prepared and applied to 3% water agar in a 10 cm Petri dish. Single conidia along with water agar media were removed with a bacterial loop and transferred to fresh tissue. This approach resulted in low conidial germination on host tissue after 7 days. After evaluating and adjusting several of the single conidial techniques described in the literature the decision was made to isolate and maintain colony level isolates rather than single conidial isolates Cucurbit p owdery mildew isolation from field sa mples Cucurbit powdery mildew isolates were obtained from a field grown susceptible cultivar (Butterbush ) and subculture d for identification of phenotypic (morphology) and genotypic (molecular) characteristics. Field i solates were subculture d on detached cucurbit leaflets in P etri dishes (10 cm in diameter) on 2% water agar as growth medium for detached host tissue Within two days after field collection was used to generate powdery mildew isolates in the laboratory. T hree discrete colonies per leaf sample were excised from each leaf with a cork borer (10 mm in diameter). To avoid isolate mixture or contamination, only one bag containing a single field leaf sample was

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73 opened at a time. The cork bore r and laminar flow ho od were disinfect ed with 70% ethanol between each isolate transfer. F reshly cut leaf disks of the same colony isolate, were placed in covered Petri plates, over moist filter paper, with fungal colony facing upward. Each leaf disk contained only one (discr ete) fungal colony (Figure 3 7) Subsequently, i nfected leaf disk s were gen tly rubbed or pressed onto detached healthy tissue (cotyledons or leaflets ) of susceptible cucurbit hosts, until mycelia could be visibly detected on the surface of healthy tissue ( Figure 3 8). When field inoculum was abundant, several subculture s were made from the same isolate which would allow multiple Petri plates of the same isolate to be available for further characterization. Fungal isolates were isolated and subcultured, in c lean bench (Labconco Corp. Kansas City, MO) under axenic conditions. M ultiplication and maintenance of cucurbit p owdery mildew isolates Powdery mildew sub cultures were incubated for 7 to 15 days in the labor atory, in enclosed containers at 22 3 C under ambient lighting, before being transferred to fresh tissue or use d in bioassays. The o ptimum period for incubation was 7days, which was when inoculated cotyledons and primary leaves were mostly covered with sporulating mycelium of the isolate to be tested. When necessary, subcultures were kept for longer than 7 days to allow enough time to prepare subculturing media or when host cotyledons were not yet ready to be used in transfers. C ultures were not kept for longer than 10 15 days before a new tran sfer was made to maintain viable plant tissue for culture maintenance When sporulation was abundant, conidia were readily transferred to multiple cotyledons and leaflets to obtain ample quantity of inoculum for bioassays and to maintain isolates.

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74 Powdery mildew living cultures were incubated at room temperature (26 2C ) under fluorescent lighting for 12 h our s photoperiod daily (Figure 3 9 ) Cucurbit P owdery Mildew Isolate C haracterization Morphological analysis Conidia and conidiophores were collected fr om individual field leaf samples for microscopic morpho logical characterization. Three randomly selected, discrete fungal colonies were taken per leaf sample. Tape strips were mounted on microscope glass slide and examined in 3% KOH (potassium hydroxide ) aqueous solution for profiling of morphological characters. C onidia l features such as shape, size, presence or absence of fibrosin bodies immature conidia edge line and conidiophores (footcell dimensions and number of conidia per chain) were recorded fo r each colony isolate Conidia size was measured for all field samples The length (L) and width (W) of 100 randomly selected conidia per fungal colony were measured by visualization with light microscope ( 40 x objectives ; Olympus BX51, Japan) with an at tached ocular reticule. Conidial length was defined as the length of the long axis of the area bounded by the outer conidia e dges and width was the length of the short axis between the outer edges For each fungal isolate, a verage dimension of conidia length (L) width (W) and length to width ratio ( L : W ) were subjected to statistical analysis. Foot cell dimensions o f length and width were also recorded and analyzed (Table 3 3) Molecular analysis Conidiophores and conidia from individual field leaf samples were also collected and frozen ( 20 C) for later molecular characterization To obtain f ungal material for DNA extraction mycelia (conidiophores and conidia) was taken from randomly selected discrete colonies, on leaves sampled from each field site A small piece of clear

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75 adhesiv e tape was gently pressed over a fungal colony and gently pulled off without removing plant tissue. The piece of adhesive tape with mycelium was put into autoclaved 1 mL snap cap micro test tube s (Eppendorf Hambur g, Germany ) and frozen at 20 C for future DNA extraction Three discrete colonies were taken from each leaf sample and e ach colony was put in a separate labeled micro tube G enomic DNA was extracted and amplified from leaf tissue using REDExtract N Amp P lant PCR Kit (Sigma Aldrich, St Louis, MO) according to the instructions supplied by the manufacturer Fungal DNA was amplified at the Department of Plant Pathology at the University of Wisconsin, Madison. Micro tubes containing previously frozen fungal samples were briefly thawed and 100 L of Extraction Solution (REDExtract N Amp Plant PCR Kit) was added to each tube and vortexed briefly making sure the piece of tape was covered by the Extraction Solution. Fungal DNA was extracted from each the piece of adhesive tape by incubation in of Extraction Solution in hot water bath at 95 C for 10 minutes. After brief cooling, 100 L of Dilution Solution (REDExtract N Amp Plant PCR Kit) was added to each tube to ne utralize inhibitory substances, and t he extracted DNA was vortexed again. The diluted DNA sample was ready for PCR (p olymerase chain reaction) or was stored at 4 C until next day For each fungal isolate, a n aliquot (4 L) of the diluted DNA extract was combined with REDExtract N Amp ReadyMi x (10 L), specific primer pairs (2 L of each) and PCR grade water (4 L) mixed gently and loaded into thin walled, 96 well PCR plates with adhesive aluminum foil cover (Bio Rad Laboratories, Hercules, CA) The provided ReadyMix (REDExtract N Amp ReadyMi x) contained the necessary buffer s olutions

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76 salts, dNTPs (deoxyribonucleotide triphosphate) Taq p olymerase enzyme JumpStart Taq antibody (used for hot start PCR to enhance specificity) and REDTaq dye to allow direct loading of PCR product onto the agaro se gel. DNA was amplified according to Chen et al. using p GGATCA TTA CTG AGC GCG AGG CCC CG CGC CGC CCT GGC GCG AGA TAC A ) and TCC GTA GGT GAA CCT GCG GAA GGA T CAA CAC CAA ACC ACA CAC ACG GCG in a multiplex reaction based on sequence variations of the ITS region and specific to P. xanthii and G. cichoracearum respectively ( 36 ) DNA a mplification was carried out in programmed thermo cycler ( Techne TC 512, Keison Products, UK ) with heated lid according to the following protocol: initial denaturing cycle at 94 C for 5 min ; followed by 30 cycles of denaturation at 94 C for 40 s. annealing at 62 C for 1 min. extension at 72 C for 90 s. and a final extension cycle at 72 C for 5 min. When samples were not amplified on the same day, PCR plates were kept at 4 C until the following day Am plified PCR products were loaded onto 2% agarose gel and were subjected to electrophoresis in TAE (Tris acetate EDTA) buffer. The amplified products were visualized with a trans illuminator (BIO RAD Universal Hood II Bio Rad Lab., Hercules, CA) after staining with SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA) A 1 kb molecular weight marker DNA ladder ( Promega, Madison, WI) was used for comparison. Sterile distilled water and DNA extracted from healthy (powdery mildew free) zucchini squash ( Cucur bita pepo cv. Dark Green Zucchini ) leaves were used as negative controls Positive controls were generated from plant tissue infected with known species of cucurbit powdery mildew In the fall season of 2008, during

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77 preliminary studies at the UF/IFAS Cowpe n Bran ch Facility in Hastings in St Jo h n s County, FL, powdery mildew pathogens were isolated from samples of summer squash ( Cucurbita pepo ) and sunn hemp ( Crotalaria juncea cv. Tropic Sun ) respectively. On the basis of morphological characteristics through microscopic examination of the asexual conidia findings were consistent with published reports of Podosphaera xanthii and Golovinomyces cichoracearum The nuclear rDNA internal transcribed spacer regions were amplified by PCR, using universal pri mers ITS1 and ITS4, and sequenced (GenBank Accession No. FJ479803) ITS sequence data indicated 100% homology with Golovinomyces cichoracearum from Helianthus ann u us L. ( Gen Bank Accession No. AB077679) and with P odosphaera xanthii Statistical Analysi s Data were summarized as means for each isolate in each study. Analysis of variance wa s performed with SAS ( SAS Institute Inc. Cary, NC). Mean separation was determined by Dunca n P =0.05 ). Results and D iscussion Cucurbit Powdery Mi ldew Isolation, Multiplication and M aintenance Detached cotyledon and leaflet inoculation s of various cucurbit hosts proved to be an efficient in vitro assay to reproduce powdery mildew symptoms observed in the field Fungal sporulation on some cucurbit occurred faster and was more abundant. These cucurbit hos ts remain ed alive f or longer periods of time after inoculation Cucumber seeds germi nated fastest, resulting in us able seedlings within appr oximat ely 7 days, yet cotyledon size was smaller compared to tissue from other hosts. Pumpkin cultivars produced larger cotyledons for fungal sporulation; however seeds took longer to germin ate. Squash

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78 cultivars ( Butterbush and Dark Green Zucchini ) were most a de quate for fungal sporulation and required 3 4 weeks for seedlings to become usable (2 3 leaf stage) for inoculation. Melon cultivars did not produce adequate fungal sporulation and were not routinely used. Watermelon cultivars were infrequently used due to leaf shape being irregular (lobed) Morphological A nalysis To investigate the powdery mildew species causing disease on cucurbit crops in north Florida, a total of 297 single colony isolates were collected from cucurbit fields in Live Oak and Citra, FL Fungal colonies on infected leaves were slightly raised and were observed on both upper and lower leaf surfaces (Figure 3 8 ). Signs and symptoms of powdery mildew pathogens on infected plants were characterized by white, powdery like m ycelium on stems, pet ioles and on leaves (Figure 3 11). Upon microscopic analyse s the following morphological features were observed: e rect conidiophores, conidia produced in chains atop 2 3 mother cell s above foot cells, immature conidia with crenate edge lines septate hyphae, ellipsoid to ovoid hyaline conidia fibrosin bodies and infrequent forked germ tubes (Figure 3 12) Conidia l length (L), width (W) and length to width ratios (L: W) were consistent with previous reports on dimensions of P. xanthii ( 36 84 144 322 ) S ome colony is olates were lost during the sub culturing and maintenance process. Of the 126 samples collected in Live Oak during spring of 2009, 94 samples were analyzed morphologically from Citra, of the total 126 samples collected during spring of 2009, 119 were analyzed For the isolates collected in Live Oak, t he mean values for conidia were 33.6 1.26 m (length), 19.1 0.79 m (width) and 1.8 0.11 for the L:W ratio. Footcell measurements were 56.1 3.61 m (length), 11.5 0.37 m (width). For

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79 the field site in Citra t he mean values for conidia were 33.5 0.81 m (length), 18.9 0.62 m (width) and 1.8 0.0 5 for the L : W ratio. As f or the 45 samples collected at Citra during fall of 2009, the mean values for conidia were 33.9 1.86 m (length), 19.0 0.41 m (width) and 1.8 0.09 for the L :W ratio. Mean dimensions of P. xanthii 4. Of the 297 colonies isolated from both field site locations during spring and fall seasons of 2009 258 col onies were morphologically characterized. C onidia had overall mean dimensions of 31 44 x 15 24 m (L x W) The overall ratio of length to width ( L:W ) varied from 1.43 2.59 and f ootcell dimensions varied from 45 67 x 10 13 m (LxW) (Table 3 4 ). The range of L:W ratios reported for P. xanthii by Frolov ( 88 ) Tomason and Gib son ( 322 ) and by Kristkova et al. ( 144 ) were of 1.0 2.6 respectively and encompassed results observed in our study Zeller ( 349 ) reported that the size of conidi a and the length to width ratio showed a high degree of phenotypic variation within population and alone sh ould not be considered significant taxonomic features S tudies by Braun ( 25 ) reported that powdery mildew conidia which develop ed on senescent leaf surfaces were smaller than those produced on healthy tissue In working with powdery mildew of eggplant, Whipps and He lyer ( 336 ) indicated that size of conidiophores and conidia were also affected by environmental conditions and by host plant Tomason and Gibson ( 322 ) reported that L:W ratio of conidia location on conidia where germination occurred (lateral or te r m inal ) and shape o f germ tube (clavate or rod ) consistently differentiated between S. fuliginea (syn. P. xanthii ) and E cichoracearum ( G. cichoracearum ) across lo cations, years and host species Nevertheless, all of these results demonstrate significant va riation in

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80 conidial morphology between different powdery mildew species Microscopic analyse s of morphological characters indicated that all isolates were Podosphaera xanthii ( 27 304 ) Molecular A nalysis Electrophoretic profiles of PCR products amplified by primer pairs S1/S2 and G1/G2 specific for P xanthii and G cichoracearum respectively were characterized Standard agarose gel electrophorese s were performed to separate and an alyze DNA fragments extracted from cucurbit field samples A 2% agarose gel showed specific PCR detection of Podosphaera xanthii from field samples of (lanes 6 and 8 ) watermelon (lanes 2, 3, 4, 5 9 and 10 ) muskmelon (lane 11) and Table acorn squash ( lane 7 ) (Figure 3 10) Fragments of expected size for P. xanthii (454 bp) we re observed in lanes 5, 6, 7, 8, 10, 11 and 12. Lane 14 sh owed specific detection of two fragment s of the expected size (double bands) corresp onding to P. xant hii and G cichoracearum as a result of multiplex PCR reaction. Lanes 1 and 17 correspond ed to 1Kb molecular marker. Lanes 2, 3, 4 and 9 d id not detect any PCR products possibly due to poor quality or low quantity of DNA extracted PCR products of DNA fra gments amplified by multiplex PCR, with DNA extracted were also separated on a 2% agarose gel using t he t wo primer pairs S1/S2 and G1/G2 (Figure 3 11) Bands corresponding to P. xanthii were observed in lanes 2 to 56. Isolates Ci 74 and Ci 75 (l anes 57 and 58 ) resulted in no bands which could possibly indicate that quality of DNA was poor or that quantity of DNA was insufficient Additionally, the powdery mildew species could have been a species other than P. xanthii or G. cichoracearum and would not have been detected using the primer pairs S1/S2 and G1/G2. Control lane 59

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81 (double bands) showed fragments of the expected size for P. xanthii and G. cichoracearum (454 bp and 391 bp respecti vely) as a result of multiplex reaction. Lanes 1, 20, 21, 40, 41 and 60 corresponded to 1 Kb molecular marker (Figure 3 11). For all samples which yielded DNA fragments as a result of molecular analysis, according to methodology described by Chen e t al. (4 0), isolates were identified as Podosphaera xanthii

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82 Table 3 1. List of powdery mildew isolates from from Live Oak and Citra FL fiel d sites during spring and fall of 2009. Field l ocation Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name Live Oak 1 5/19 1 1 A 1 1 LO 1(A)1 2 5/19 1 1 A 2 1 LO 1(A)2 3 5/19 1 1 A 3 1 LO 1(A)3 4 5/19 1 1 B 1 1 LO 1(B)1 5 5/19 1 1 B 2 1 LO 1(B)2 6 5/19 1 1 B 3 1 LO 1(B)3 7 5/19 1 1 C 1 1 LO 1(C)1 8 5/19 1 1 C 2 1 LO 1(C)2 9 5/19 1 1 C 3 1 LO 1(C)3 10 5/19 1 2 A 1 1 LO 2(A)1 11 5/19 1 2 A 2 1 LO 2(A)2 12 5/19 1 2 A 3 1 LO 2(A)3 13 5/19 1 2 B 1 1 LO 2(B)1 14 5/19 1 2 B 2 1 LO 2(B)2 15 5/19 1 2 B 3 1 LO 2(B)3 16 5/19 1 2 C 1 1 LO 2(C)1 17 5/19 1 2 C 2 1 LO 2(C)2 18 5/19 1 2 C 3 1 LO 2(C)3 19 6/02 2 1 A 1 2 LO 1(A)1 20 6/02 2 1 A 2 2 LO 1(A)2 21 6/02 2 1 A 3 2 LO 1(A)3 22 6/02 2 1 B 1 2 LO 1(B)1 23 6/02 2 1 B 2 2 LO 1(B)2 24 6/02 2 1 B 3 2 LO 1(B)3 25 6/02 2 1 C 1 2 LO 1(C)1 26 6/02 2 1 C 2 2 LO 1(C)2 27 6/02 2 1 C 3 2 LO 1(C)3 28 6/02 2 2 A 1 2 LO 2(A)1 29 6/02 2 2 A 2 2 LO 2(A)2 30 6/02 2 2 A 3 2 LO 2(A)3 31 6/02 2 2 B 1 2 LO 2(B)1 32 6/02 2 2 B 2 2 LO 2(B)2 33 6/02 2 2 B 3 2 LO 2(B)3 34 6/02 2 2 C 1 2 LO 2(C)1 35 6/02 2 2 C 2 2 LO 2(C)2 36 6/02 2 2 C 3 2 LO 2(C)3 37 6/16 3 1 A 1 3 LO 1(A)1

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83 Table 3 1. Continued Field l ocation Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name (Code) Live Oak 38 6/16 3 1 A 2 3 LO 1(A)2 39 6/16 3 1 A 3 3 LO 1(A)3 40 6/16 3 1 B 1 3 LO 1(B)1 41 6/16 3 1 B 2 3 LO 1(B)2 42 6/16 3 1 B 3 3 LO 1(B)3 43 6/16 3 1 C 1 3 LO 1(C)1 44 6/16 3 1 C 2 3 LO 1(C)2 45 6/16 3 1 C 3 3 LO 1(C)3 46 6/16 3 2 A 1 3 LO 2(A)1 47 6/16 3 2 A 2 3 LO 2(A)2 48 6/16 3 2 A 3 3 LO 2(A)3 49 6/16 3 2 B 1 3 LO 2(B)1 50 6/16 3 2 B 2 3 LO 2(B)2 51 6/16 3 2 B 3 3 LO 2(B)3 52 6/16 3 2 C 1 3 LO 2(C)1 53 6/16 3 2 C 2 3 LO 2(C)2 54 6/16 3 2 C 3 3 LO 2(C)3 55 7/01 4 1 A 1 4 LO 1(A)1 56 7/01 4 1 A 2 4 LO 1(A)2 57 7/01 4 1 A 3 4 LO 1(A)3 58 7/01 4 1 B 1 4 LO 1(B)1 59 7/01 4 1 B 2 4 LO 1(B)2 60 7/01 4 1 B 3 4 LO 1(B)3 61 7/01 4 1 C 1 4 LO 1(C)1 62 7/01 4 1 C 2 4 LO 1(C)2 63 7/01 4 1 C 3 4 LO 1(C)3 64 7/01 4 2 A 1 4 LO 2(A)1 65 7/01 4 2 A 2 4 LO 2(A)2 66 7/01 4 2 A 3 4 LO 2(A)3 67 7/01 4 2 B 1 4 LO 2(B)1 68 7/01 4 2 B 2 4 LO 2(B)2 69 7/01 4 2 B 3 4 LO 2(B)3 70 7/01 4 2 C 1 4 LO 2(C)1 71 7/01 4 2 C 2 4 LO 2(C)2 72 7/01 4 2 C 3 4 LO 2(C)3 73 7/10 5 1 A 1 5 LO 1(A)1 74 7/10 5 1 A 2 5 LO 1(A)2 75 7/10 5 1 A 3 5 LO 1(A)3 76 7/10 5 1 B 1 5 LO 1(B)1

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84 Table 3 1. Continued Field location Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name (Code) Live Oak 77 7/10 5 1 B 2 5 LO 1(B)2 78 7/10 5 1 B 3 5 LO 1(B)3 79 7/10 5 1 C 1 5 LO 1(C)1 80 7/10 5 1 C 2 5 LO 1(C)2 81 7/10 5 1 C 3 5 LO 1(C)3 82 7/10 5 2 A 1 5 LO 2(A)1 83 7/10 5 2 A 2 5 LO 2(A)2 84 7/10 5 2 A 3 5 LO 2(A)3 85 7/10 5 2 B 1 5 LO 2(B)1 86 7/10 5 2 B 2 5 LO 2(B)2 87 7/10 5 2 B 3 5 LO 2(B)3 88 7/10 5 2 C 1 5 LO 2(C)1 89 7/10 5 2 C 2 5 LO 2(C)2 90 7/10 5 2 C 3 5 LO 2(C)3 91 7/15 6 1 A 1 6 LO 1(A)1 92 7/15 6 1 A 2 6 LO 1(A)2 93 7/15 6 1 A 3 6 LO 1(A)3 94 7/15 6 1 B 1 6 LO 1(B)1 95 7/15 6 1 B 2 6 LO 1(B)2 96 7/15 6 1 B 3 6 LO 1(B)3 97 7/15 6 1 C 1 6 LO 1(C)1 98 7/15 6 1 C 2 6 LO 1(C)2 99 7/15 6 1 C 3 6 LO 1(C)3 100 7/15 6 2 A 1 6 LO 2(A)1 101 7/15 6 2 A 2 6 LO 2(A)2 102 7/15 6 2 A 3 6 LO 2(A)3 103 7/15 6 2 B 1 6 LO 2(B)1 104 7/15 6 2 B 2 6 LO 2(B)2 105 7/15 6 2 B 3 6 LO 2(B)3 106 7/15 6 2 C 1 6 LO 2(C)1 107 7/15 6 2 C 2 6 LO 2(C)2 108 7/15 6 2 C 3 6 LO 2(C)3 109 7/23 7 1 A 1 7 LO 1(A)1 110 7/23 7 1 A 2 7 LO 1(A)2 111 7/23 7 1 A 3 7 LO 1(A)3 112 7/23 7 1 B 1 7 LO 1(B)1 113 7/23 7 1 B 2 7 LO 1(B)2 114 7/23 7 1 B 3 7 LO 1(B)3

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85 Table 3 1 Continued Field location Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name (Code) Live Oak 115 7/23 7 1 C 1 7 LO 1(C)1 116 7/23 7 1 C 2 7 LO 1(C)2 117 7/23 7 1 C 3 7 LO 1(C)3 118 7/23 7 2 A 1 7 LO 2(A)1 119 7/23 7 2 A 2 7 LO 2(A)2 120 7/23 7 2 A 3 7 LO 2(A)3 121 7/23 7 2 B 1 7 LO 2(B)1 122 7/23 7 2 B 2 7 LO 2(B)2 123 7/23 7 2 B 3 7 LO 2(B)3 124 7/23 7 2 C 1 7 LO 2(C)1 125 7/23 7 2 C 2 7 LO 2(C)2 126 7/23 7 2 C 3 7 LO 2(C)3 Citra 1 6/22 1 1 A 1 1 Ci 1(A)1 2 6/22 1 1 A 2 1 Ci 1(A)2 3 6/22 1 1 A 3 1 Ci 1(A)3 4 6/22 1 1 B 1 1 Ci 1(B)1 5 6/22 1 1 B 2 1 Ci 1(B)2 6 6/22 1 1 B 3 1 Ci 1(B)3 7 6/22 1 1 C 1 1 Ci 1(C)1 8 6/22 1 1 C 2 1 Ci 1(C)2 9 6/22 1 1 C 3 1 Ci 1(C)3 10 6/22 1 2 A 1 1 Ci 2(A)1 11 6/22 1 2 A 2 1 Ci 2(A)2 12 6/22 1 2 A 3 1 Ci 2(A)3 13 6/22 1 2 B 1 1 Ci 2(B)1 14 6/22 1 2 B 2 1 Ci 2(B)2 15 6/22 1 2 B 3 1 Ci 2(B)3 16 6/22 1 2 C 1 1 Ci 2(C)1 17 6/22 1 2 C 2 1 Ci 2(C)2 18 6/22 1 2 C 3 1 Ci 2(C)3 19 7/01 2 1 A 1 2 Ci 1(A)1 20 7/01 2 1 A 2 2 Ci 1(A)2 21 7/01 2 1 A 3 2 Ci 1(A)3 22 7/01 2 1 B 1 2 Ci 1(B)1 23 7/01 2 1 B 2 2 Ci 1(B)2 24 7/01 2 1 B 3 2 Ci 1(B)3 25 7/01 2 1 C 1 2 Ci 1(C)1 26 7/01 2 1 C 2 2 Ci 1(C)2

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86 Table 3 1 Continued Field location Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name (Code) Citra 27 7/01 2 1 C 3 2 Ci 1(C)3 28 7/01 2 2 A 1 2 Ci 2(A)1 29 7/01 2 2 A 2 2 Ci 2(A)2 30 7/01 2 2 A 3 2 Ci 2(A)3 31 7/01 2 2 B 1 2 Ci 2(B)1 32 7/01 2 2 B 2 2 Ci 2(B)2 33 7/01 2 2 B 3 2 Ci 2(B)3 34 7/01 2 2 C 1 2 Ci 2(C)1 35 7/01 2 2 C 2 2 Ci 2(C)2 36 7/01 2 2 C 3 2 Ci 2(C)3 37 7/10 3 1 A 1 3 Ci 1(A)1 38 7/10 3 1 A 2 3 Ci 1(A)2 39 7/10 3 1 A 3 3 Ci 1(A)3 40 7/10 3 1 B 1 3 Ci 1(B)1 41 7/10 3 1 B 2 3 Ci 1(B)2 42 7/10 3 1 B 3 3 Ci 1(B)3 43 7/10 3 1 C 1 3 Ci 1(C)1 44 7/10 3 1 C 2 3 Ci 1(C)2 45 7/10 3 1 C 3 3 Ci 1(C)3 46 7/10 3 2 A 1 3 Ci 2(A)1 47 7/10 3 2 A 2 3 Ci 2(A)2 48 7/10 3 2 A 3 3 Ci 2(A)3 49 7/10 3 2 B 1 3 Ci 2(B)1 50 7/10 3 2 B 2 3 Ci 2(B)2 51 7/10 3 2 B 3 3 Ci 2(B)3 52 7/10 3 2 C 1 3 Ci 2(C)1 53 7/10 3 2 C 2 3 Ci 2(C)2 54 7/10 3 2 C 3 3 Ci 2(C)3 55 7/15 4 1 A 1 4 Ci 1(A)1 56 7/15 4 1 A 2 4 Ci 1(A)2 57 7/15 4 1 A 3 4 Ci 1(A)3 58 7/15 4 1 B 1 4 Ci 1(B)1 59 7/15 4 1 B 2 4 Ci 1(B)2 60 7/15 4 1 B 3 4 Ci 1(B)3 61 7/15 4 1 C 1 4 Ci 1(C)1 62 7/15 4 1 C 2 4 Ci 1(C)2 63 7/15 4 1 C 3 4 Ci 1(C)3 64 7/15 4 2 A 1 4 Ci 2(A)1 65 7/15 4 2 A 2 4 Ci 2(A)2

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87 Table 3 1 Continued Field location Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name (Code) Citra 66 7/15 4 2 A 3 4 Ci 2(A)3 67 7/15 4 2 B 1 4 Ci 2(B)1 68 7/15 4 2 B 2 4 Ci 2(B)2 69 7/15 4 2 B 3 4 Ci 2(B)3 70 7/15 4 2 C 1 4 Ci 2(C)1 71 7/15 4 2 C 2 4 Ci 2(C)2 72 7/15 4 2 C 3 4 Ci 2(C)3 73 7/23 5 1 A 1 5 Ci 1(A)1 74 7/23 5 1 A 2 5 Ci 1(A)2 75 7/23 5 1 A 3 5 Ci 1(A)3 76 7/23 5 1 B 1 5 Ci 1(B)1 77 7/23 5 1 B 2 5 Ci 1(B)2 78 7/23 5 1 B 3 5 Ci 1(B)3 79 7/23 5 1 C 1 5 Ci 1(C)1 80 7/23 5 1 C 2 5 Ci 1(C)2 81 7/23 5 1 C 3 5 Ci 1(C)3 82 7/23 5 2 A 1 5 Ci 2(A)1 83 7/23 5 2 A 2 5 Ci 2(A)2 84 7/23 5 2 A 3 5 Ci 2(A)3 85 7/23 5 2 B 1 5 Ci 2(B)1 86 7/23 5 2 B 2 5 Ci 2(B)2 87 7/23 5 2 B 3 5 Ci 2(B)3 88 7/23 5 2 C 1 5 Ci 2(C)1 89 7/23 5 2 C 2 5 Ci 2(C)2 90 7/23 5 2 C 3 5 Ci 2(C)3 91 7/28 6 1 A 1 6 Ci 1(A)1 92 7/28 6 1 A 2 6 Ci 1(A)2 93 7/28 6 1 A 3 6 Ci 1(A)3 94 7/28 6 1 B 1 6 Ci 1(B)1 95 7/28 6 1 B 2 6 Ci 1(B)2 96 7/28 6 1 B 3 6 Ci 1(B)3 97 7/28 6 1 C 1 6 Ci 1(C)1 98 7/28 6 1 C 2 6 Ci 1(C)2 99 7/28 6 1 C 3 6 Ci 1(C)3 100 7/28 6 2 A 1 6 Ci 2(A)1 101 7/28 6 2 A 2 6 Ci 2(A)2 102 7/28 6 2 A 3 6 Ci 2(A)3 103 7/28 6 2 B 1 6 Ci 2(B)1 104 7/28 6 2 B 2 6 Ci 2(B)2

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88 Table 3 1 Continued Field location Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name (Code) Citra 105 7/28 6 2 B 3 6 Ci 2(B)3 106 7/28 6 2 C 1 6 Ci 2(C)1 107 7/28 6 2 C 2 6 Ci 2(C)2 108 7/28 6 2 C 3 6 Ci 2(C)3 109 8/03 7 1 A 1 7 Ci 1(A)1 110 8/03 7 1 A 2 7 Ci 1(A)2 111 8/03 7 1 A 3 7 Ci 1(A)3 112 8/03 7 1 B 1 7 Ci 1(B)1 113 8/03 7 1 B 2 7 Ci 1(B)2 114 8/03 7 1 B 3 7 Ci 1(B)3 115 8/03 7 1 C 1 7 Ci 1(C)1 116 8/03 7 1 C 2 7 Ci 1(C)2 117 8/03 7 1 C 3 7 Ci 1(C)3 118 8/03 7 2 A 1 7 Ci 2(A)1 119 8/03 7 2 A 2 7 Ci 2(A)2 120 8/03 7 2 A 3 7 Ci 2(A)3 121 8/03 7 2 B 1 7 Ci 2(B)1 122 8/03 7 2 B 2 7 Ci 2(B)2 123 8/03 7 2 B 3 7 Ci 2(B)3 124 8/03 7 2 C 1 7 Ci 2(C)1 125 8/03 7 2 C 2 7 Ci 2(C)2 126 8/03 7 2 C 3 7 Ci 2(C)3 127 10/12 8 1 A 1 8 Ci 1(A)1 128 10/12 8 1 A 2 8 Ci 1(A)2 129 10/12 8 1 A 3 8 Ci 1(A)3 130 10/12 8 1 B 1 8 Ci 1(B)1 131 10/12 8 1 B 2 8 Ci 1(B)2 132 10/12 8 1 B 3 8 Ci 1(B)3 133 10/12 8 1 C 1 8 Ci 1(C)1 134 10/12 8 1 C 2 8 Ci 1(C)2 135 10/12 8 1 C 3 8 Ci 1(C)3 136 10/20 9 1 A 1 9 Ci 1(A)1 137 10/20 9 1 A 2 9 Ci 1(A)2 138 10/20 9 1 A 3 9 Ci 1(A)3 139 10/20 9 1 B 1 9 Ci 1(B)1 140 10/20 9 1 B 2 9 Ci 1(B)2 141 10/20 9 1 B 3 9 Ci 1(B)3 142 10/20 9 1 C 1 9 Ci 1(C)1 143 10/20 9 1 C 2 9 Ci 1(C)2

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89 Table 3 1 Continued Field l ocation Sample No. Date sampled Week sampled Plant Leaf Colony Isolate Name (Code) Citra 144 10/20 9 1 C 3 9 Ci 1(C)3 145 10/29 10 1 A 1 10 Ci 1(A)1 146 10/29 10 1 A 2 10 Ci 1(A)2 147 10/29 10 1 A 3 10 Ci 1(A)3 148 10/29 10 1 B 1 10 Ci 1(B)1 149 10/29 10 1 B 2 10 Ci 1(B)2 150 10/29 10 1 B 3 10 Ci 1(B)3 151 10/29 10 1 C 1 10 Ci 1(C)1 152 10/29 10 1 C 2 10 Ci 1(C)2 153 10/29 10 1 C 3 10 Ci 1(C)3 154 11/03 11 1 A 1 11 Ci 1(A)1 155 11/03 11 1 A 2 11 Ci 1(A)2 156 11/03 11 1 A 3 11 Ci 1(A)3 157 11/03 11 1 B 1 11 Ci 1(B)1 158 11/03 11 1 B 2 11 Ci 1(B)2 159 11/03 11 1 B 3 11 Ci 1(B)3 160 11/03 11 1 C 1 11 Ci 1(C)1 161 11/03 11 1 C 2 11 Ci 1(C)2 162 11/03 11 1 C 3 11 Ci 1(C)3 163 11/13 12 1 A 1 12 Ci 1(A)1 164 11/13 12 1 A 2 12 Ci 1(A)2 165 11/13 12 1 A 3 12 Ci 1(A)3 166 11/13 12 1 B 1 12 Ci 1(B)1 167 11/13 12 1 B 2 12 Ci 1(B)2 168 11/13 12 1 B 3 12 Ci 1(B)3 169 11/13 12 1 C 1 12 Ci 1(C)1 170 11/13 12 1 C 2 12 Ci 1(C)2 171 11/13 12 1 C 3 12 Ci 1(C)3

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90 Table 3 2 Additional powdery mildew samples, from alternative locations and cucurbit hosts collected for comparison Sample No. Location (County) Cucurbit Host Cucurbit species Cultivar Date s ampled 08 4 Putman yellow squash Cucurbita pepo unknown 10/23/08 09 1 Charlotte watermelon Citrullus lanatus Crimson 4/100 09 2 Hendry watermelon Citrullus lanatus Crimson 4/10 09 3 Collier yellow squash Cucurbita pepo unknown 4/13 09 4 Collier watermelon Citrullus lanatus Jubilee 4/20 09 5 Collier watermelon (fruit) Citrullus lanatus Jubilee 5/06 09 6 Collier watermelon Citrullus lanatus Crimson 5/12 09 7 Collier watermelon Citrullus lanatus Crimson 5/12 09 9 Marion acorn squash Cucurbita pepo Table Queen 5/20 09 20 Suwannee watermelon Citrullus lanatus Mickey Lee 6/02 09 21 Suwannee watermelon Citrullus lanatus Mickey Lee 6/10 09 22 Putman muskmelon Cucumis melo Hales Best Jumbo 6/10 09 23 Marion acorn squash Cucurbita pepo Table Queen 6/02 09 25 Suwannee watermelon Citrullus lanatus Mickey Lee 7/01 09 26 Suwannee watermelon Citrullus lanatus Mickey Lee 7/10 09 27 Marion watermelon Citrullus lanatus Mickey Lee 7/10 09 28 Marion watermelon Citrullus lanatus Mickey Lee 7/10 09 29 Suwannee watermelon Citrullus lanatus Mickey Lee 7/15 09 30 Marion watermelon Citrullus lanatus Mickey Lee 7/16 09 33 Suwannee watermelon Citrullus lanatus Mickey Lee 7/23 09 34 Marion watermelon Citrullus lanatus Mickey Lee 7/23 09 35 Marion watermelon Citrullus lanatus Mickey Lee 7/28 09 36 Marion watermelon Citrullus lanatus Mickey Lee 7/28 09 38 Marion squash (27.12.2) breeding line Not yet assigned 10/13 09 39 Marion squash (27.12.A1) breeding line Not yet assigned 10/13 09 43 Marion squash (27.12.2) breeding line Not yet assigned 10/20 09 44 Marion squash (27.12.A1) breeding line Not yet assigned 10/20 09 45 Citra squash (27.16.6 ) Rupp breeding line Not yet assigned 10/20 09 46 Citra muskm elon Cucumis melo Hales Best Jumbo 10/29

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91 Table 3 2 Continued Sample No. Location Cucurbit Host Cucurbit species Cultivar Date (sampled) 09 49 Citra squash (27.12.a1 breeding line Not yet assigned 11/03 09 50 Citra squash (27.12.2 ) breeding line Not yet assigned 11/03 09 51 Citra squash (27.16.6 ) breeding line Not yet assigned 11/03 09 52 Citra gourd C. okeechobeensis Not yet assigned 11/03 09 54 Citra gourd C. okeechobeensis Not yet assigned 11/13 09 55 Citra squash (27.12.a1 ) breeding line Not yet assigned 11/13 09 56 Citra squash (27.16.6 ) breeding line Not yet assigned 11/13 09 57 Citra squash (27.12.2 ) breeding line Not yet assigned 11/13

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92 Table 3 3 Mean powdery mildew c onidia length width length to width ratio and conidiophore foot cell length and width of isolates from collected in Live Oak and Citra, FL during spring and fall of 2009. Isolate Name (Code) Conidia l en gth (L) (n=100) Conidia width (W) (n=100) Conidia L:W (n=100) Foot cell length (n=25) Footcell width (n=25) 1 LO 1(A)1 39 15 2.59 59 11 1 LO 1(A)2 38 19 2.05 51 12 1 LO 1(B)2 33 19 1.75 53 12 1 LO 1(C)1 33 19 1.80 45 12 1 LO 1(C)2 31 19 1.64 52 11 1 LO 2(A)1 33 19 1.77 53 11 1 LO 2(B)1 34 19 1.75 60 11 1 LO 2(C)1 32 19 1.66 55 11 1 LO 2(C)2 31 19 1.64 54 12 1 LO 2(C)3 34 19 1.81 56 11 2 LO 1(A)1 34 20 1.72 55 11 2 LO 1(B)1 33 19 1.75 52 11 2 LO 1(C)2 34 20 1.71 60 11 2 LO 2(A)1 33 19 1.74 57 11 2 LO 2(A)2 38 19 1.99 57 11 2 LO 2(B)1 33 19 1.71 53 12 2 LO 2(B)3 33 19 1.73 58 11 2 LO 2(C)1 33 19 1.73 56 11 2 LO 2(C)3 34 19 1.79 54 11 3 LO 1(A)1 33 19 1.78 55 11 3 LO 1(A)2 33 19 1.73 54 12 3 LO 1(A)3 32 19 1.72 54 11 3 LO 1(B)3 36 20 1.76 58 12 3 LO 1(C)3 33 19 1.79 51 12 3 LO 2(A)2 34 19 1.75 56 11 3 LO 2(A)3 33 19 1.74 57 11 3 LO 2(B)1 33 19 1.72 57 12 3 LO 2(B)2 33 19 1.74 58 12 3 LO 2(B)3 33 18 1.91 57 11 3 LO 2(C)3 34 19 1.84 54 11 4 LO 1(A)1 34 20 1.75 58 11 4 LO 1(A)2 34 19 1.75 48 10 4 LO 1(A)3 34 19 1.76 57 11 4 LO 1(B)1 34 19 1.74 56 11 4 LO 1(B)2 34 20 1.74 62 11 4 LO 1(B)3 34 19 1.77 56 11

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93 Table 3 3 Continued Isolate Name (Code) Conidia l en gth (L) (n=100) Conidia width (W) (n=100) Conidia L:W (n=100) Foot cell length (n=25) Footcell width (n=25) 4 LO 1(C)1 34 19 1.76 61 12 4 LO 1(C)2 34 19 1.78 58 11 4 LO 1(C)3 34 20 1.72 55 11 4 LO 2(A)1 34 19 1.76 58 11 4 LO 2(A)2 35 20 1.74 57 11 4 LO 2(A)3 34 19 1.78 59 12 4 LO 2(B)1 34 20 1.74 56 12 4 LO 2(B)2 34 19 1.78 60 11 4 LO 2(B)3 34 19 1.77 58 12 4 LO 2(C)1 34 19 1.76 53 11 4 LO 2(C)2 33 19 1.76 53 11 4 LO 2(C)3 33 19 1.77 61 12 5 LO 1(A)1 34 18 1.84 55 11 5 LO 1(A)2 33 19 1.79 56 11 5 LO 1(A)3 33 19 1.78 55 12 5 LO 1(B)1 33 19 1.79 58 12 5 LO 1(B)2 34 19 1.76 55 12 5 LO 1(B)3 33 19 1.72 61 11 5 LO 1(C)1 34 18 1.86 57 12 5 LO 1(C)2 34 19 1.81 49 11 5 LO 1(C)3 34 19 1.76 58 12 5 LO 2(A)1 33 18 1.82 62 11 5 LO 2(A)2 33 19 1.74 64 12 5 LO 2(B)1 34 19 1.81 59 12 5 LO 2(B)2 33 18 1.78 55 11 5 LO 2(B)3 32 18 1.77 53 11 5 LO 2(C)1 33 19 1.75 57 11 5 LO 2(C)2 34 19 1.80 55 12 5 LO 2(C)3 33 19 1.79 59 11 6 LO 1(A)1 33 23 1.46 62 12 6 LO 1(A)2 33 19 1.72 67 12 6 LO 1(B)2 34 18 1.84 55 11 6 LO 1(B)3 33 19 1.74 60 11 6 LO 1(C)1 37 20 1.82 59 12 6 LO 2(A)1 33 19 1.79 59 12 6 LO 2(A)3 34 19 1.74 59 11 6 LO 2(B)2 36 20 1.75 55 11 6 LO 2(C)1 33 19 1.76 58 12

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94 Table 3 3 Continued Isolate Name (Code) Conidia l en gth (L) (n=100) Conidia width (W) (n=100) Conidia L:W (n=100) Foot cell length (n=25) Footcell width (n=25) 6 LO 2(C)2 36 21 1.75 56 12 6 LO 2(C)3 34 19 1.80 55 12 7 LO 1(A)1 34 19 1.78 49 11 7 LO 1(A)2 34 19 1.75 52 11 7 LO 1(A)3 33 19 1.79 52 11 7 LO 1(B)1 33 19 1.70 55 12 7 LO 1(B)2 34 19 1.80 58 12 7 LO 1(B)3 34 20 1.72 60 12 7 LO 1(C)1 33 18 1.81 58 12 7 LO 1(C)2 32 20 1.57 52 12 7 LO 1(C)3 33 20 1.68 56 12 7 LO 2(A)1 34 19 1.75 52 12 7 LO 2(A)2 34 21 1.66 55 12 7 LO 2(A)3 33 19 1.75 54 11 7 LO 2(B)1 32 18 1.73 61 12 7 LO 2(B)2 31 19 1.65 55 12 7 LO 2(B)3 33 20 1.67 54 12 7 LO 2(C)1 34 19 1.80 52 11 7 LO 2(C)2 34 19 1.79 49 11 7 LO 2(C)3 33 19 1.77 61 11 1 Ci 1(A)1 33 19 1.75 60 11 1 Ci 1(A)2 34 19 1.77 57 12 1 Ci 1(A)3 33 19 1.75 55 12 1 Ci 1(B)1 33 18 1.77 57 11 1 Ci 1(B)2 34 19 1.78 59 11 1 Ci 1(B)3 33 19 1.75 55 11 1 Ci 1(C)1 33 19 1.73 55 12 1 Ci 1(C)2 33 19 1.72 57 12 1 Ci 1(C)3 34 19 1.78 64 12 1 Ci 2(A)1 33 19 1.75 58 12 1 Ci 2(A)2 34 19 1.78 59 11 1 Ci 2(A)3 33 19 1.75 57 11 1 Ci 2(B)1 34 18 1.84 49 11 1 Ci 2(B)2 33 19 1.74 56 12 1 Ci 2(B)3 33 19 1.75 54 11 1 Ci 2(C)1 34 19 1.80 57 11 1 Ci 2(C)2 33 19 1.76 53 12 1 Ci 2(C)3 34 19 1.81 61 11

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95 Table 3 3 Continued Isolate Name (Code) Conidia l en gth (L) (n=100) Conidia width (W) (n=100) Conidia L:W (n=100) Foot cell length (n=25) Footcell width (n=25) 2 Ci 1(A)1 34 20 1.74 59 11 2 Ci 1(A)2 33 20 1.70 62 11 2 Ci 1(A)3 33 19 1.75 58 11 2 Ci 1(B)1 34 19 1.80 57 12 2 Ci 1(B)2 34 19 1.79 54 11 2 Ci 1(B)3 33 19 1.77 59 11 2 Ci 1(C)1 34 19 1.81 55 11 2 Ci 1(C)2 33 19 1.77 56 11 2 Ci 1(C)3 33 19 1.79 56 11 2 Ci 2(A)1 34 19 1.73 56 11 2 Ci 2(A)2 34 19 1.73 61 12 2 Ci 2(A)3 33 19 1.79 57 11 2 Ci 2(B)1 33 19 1.79 57 11 2 Ci 2(B)2 32 18 1.76 63 12 2 Ci 2(B)3 33 18 1.80 56 11 2 Ci 2(C)1 34 19 1.81 63 12 2 Ci 2(C)2 33 19 1.80 63 11 2 Ci 2(C)3 33 18 1.82 62 11 3 Ci 1(A)1 33 19 1.79 62 11 3 Ci 1(A)2 34 19 1.82 60 11 3 Ci 1(A)3 34 19 1.78 59 11 3 Ci 1(B)1 34 19 1.79 56 11 3 Ci 1(B)2 34 19 1.80 57 11 3 Ci 1(B)3 34 18 1.82 63 11 3 Ci 1(C)1 34 19 1.80 58 10 3 Ci 1(C)2 34 19 1.78 58 11 3 Ci 1(C)3 33 18 1.79 58 11 3 Ci 2(A)1 33 19 1.80 58 11 3 Ci 2(A)2 33 19 1.77 57 11 3 Ci 2(A)3 33 19 1.76 52 11 3 Ci 2(B)1 33 18 1.84 57 10 3 Ci 2(B)2 34 19 1.77 56 11 3 Ci 2(B)3 34 19 1.77 57 11 3 Ci 2(C)1 33 19 1.79 58 11 3 Ci 2(C)2 34 19 1.76 58 11 3 Ci 2(C)3 33 19 1.77 57 11 4 Ci 1(A)1 34 18 1.86 55 12 4 Ci 1(A)2 33 19 1.79 59 11

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96 Table 3 3 Continued Isolate Name (Code) Conidia l en gth (L) (n=100) Conidia width (W) (n=100) Conidia L:W (n=100) Foot cell length (n=25) Footcell width (n=25) 4 Ci 1(A)3 33 19 1.76 61 12 4 Ci 1(B)1 33 19 1.76 57 12 4 Ci 1(B)2 33 19 1.77 58 11 4 Ci 1(B)3 33 19 1.74 60 11 4 Ci 1(C)1 34 19 1.77 53 11 4 Ci 1(C)2 33 19 1.79 56 11 4 Ci 1(C)3 34 19 1.73 53 12 4 Ci 2(A)1 34 19 1.76 54 11 4 Ci 2(A)2 34 19 1.79 66 11 4 Ci 2(A)3 34 19 1.80 55 12 4 Ci 2(B)1 33 19 1.75 57 11 4 Ci 2(B)2 34 19 1.81 57 11 4 Ci 2(B)3 33 19 1.73 57 12 4 Ci 2(C)1 33 19 1.81 54 11 4 Ci 2(C)2 33 19 1.79 59 11 4 Ci 2(C)3 34 19 1.80 59 11 5 Ci 1(A)1 33 18 1.85 50 12 5 Ci 1(A)2 34 19 1.79 56 12 5 Ci 1(A)3 33 18 1.83 57 11 5 Ci 1(B)1 34 19 1.76 58 12 5 Ci 1(B)2 35 19 1.80 56 12 5 Ci 1(B)3 34 19 1.76 61 12 5 Ci 1(C)1 33 19 1.74 55 11 5 Ci 1(C)2 33 19 1.78 57 12 5 Ci 1(C)3 33 20 1.70 57 12 5 Ci 2(A)1 33 19 1.72 62 12 5 Ci 2(A)2 34 19 1.72 58 11 5 Ci 2(A)3 33 19 1.79 58 12 5 Ci 2(B)1 33 19 1.78 59 12 5 Ci 2(B)2 33 18 1.80 59 11 5 Ci 2(B)3 33 19 1.79 56 10 5 Ci 2(C)1 33 19 1.75 52 12 5 Ci 2(C)2 34 19 1.79 60 12 5 Ci 2(C)3 34 19 1.76 56 13 6 Ci 1(A)1 33 19 1.76 59 12 6 Ci 1(A)2 34 19 1.73 53 12 6 Ci 1(A)3 33 19 1.71 56 12 6 Ci 1(B)1 35 20 1.79 54 11

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97 Table 3 3 Continued Isolate Name (Code) Conidia l en gth (L) (n=100) Conidia width (W) (n=100) Conidia L:W (n=100) Foot cell length (n=25) Footcell width (n=25) 6 Ci 1(B)2 33 19 1.71 58 12 6 Ci 1(B)3 34 19 1.75 58 11 6 Ci 1(C)1 33 19 1.73 59 12 6 Ci 1(C)2 33 19 1.76 65 12 6 Ci 1(C)3 33 19 1.73 60 11 6 Ci 2(A)1 33 19 1.72 55 12 6 Ci 2(A)2 32 19 1.75 55 11 6 Ci 2(A)3 34 19 1.76 54 12 6 Ci 2(B)1 34 19 1.79 58 12 6 Ci 2(B)2 33 19 1.73 55 11 6 Ci 2(B)3 33 18 1.82 50 12 6 Ci 2(C)1 33 19 1.73 54 11 6 Ci 2(C)2 33 19 1.77 54 11 6 Ci 2(C)3 33 19 1.78 58 11 7 Ci 1(A)1 33 19 1.75 60 11 7 Ci 1(A)2 33 19 1.76 58 11 7 Ci 1(A)3 36 19 1.92 62 12 7 Ci 1(B)3 35 24 1.43 58 11 7 Ci 1(C)2 39 20 1.90 56 11 7 Ci 2(A)2 36 19 1.89 54 11 7 Ci 2(A)3 33 19 1.75 59 12 7 Ci 2(B)1 33 18 1.78 56 12 7 Ci 2(B)2 33 19 1.78 58 12 7 Ci 2(B)3 33 19 1.75 59 11 7 Ci 2(C)3 37 20 1.84 55 11 8 Ci 1(A)1 35 20 1.79 60 11 8 Ci 1(A)2 34 20 1.73 56 12 8 Ci 1(A)3 34 19 1.75 60 12 8 Ci 1(B)1 33 18 1.81 57 12 8 Ci 1(B)2 34 19 1.75 56 12 8 Ci 1(B)3 35 20 1.76 58 11 8 Ci 1(C)1 34 20 1.71 61 12 8 Ci 1(C)2 44 20 2.26 57 11 8 Ci 1(C)3 34 19 1.75 61 11 9 Ci 1(A)1 35 19 1.81 56 11 9 Ci 1(A)2 33 18 1.78 56 12 9 Ci 1(A)3 33 19 1.76 57 11

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98 Table 3 3 Continued Isolate Name (Code) Conidia l en gth (L) (n=100) Conidia width (W) (n=100) Conidia L:W (n=100) Foot cell length (n=25) Footcell width (n=25) 9 Ci 1(B)1 34 20 1.75 58 11 9 Ci 1(B)2 33 19 1.78 59 12 9 Ci 1(B)3 34 19 1.79 57 12 9 Ci 1(C)1 33 19 1.74 52 12 9 Ci 1(C)2 33 19 1.75 55 12 9 Ci 1(C)3 34 19 1.75 59 11 10 Ci 1(A)1 32 19 1.71 55 12 10 Ci 1(A)2 33 19 1.76 60 12 10 Ci 1(A)3 34 19 1.79 56 11 10 Ci 1(B)1 33 19 1.73 55 12 10 Ci 1(B)2 34 19 1.76 54 12 10 Ci 1(B)3 34 19 1.76 57 12 10 Ci 1(C)1 34 19 1.78 56 12 10 Ci 1(C)2 34 19 1.78 57 12 10 Ci 1(C)3 33 19 1.74 52 11 11 Ci 1(A)1 34 19 1.80 54 12 11 Ci 1(A)2 35 19 1.82 55 12 11 Ci 1(A)3 34 19 1.79 56 12 11 Ci 1(B)1 34 19 1.73 55 11 11 Ci 1(B)2 33 19 1.74 55 11 11 Ci 1(B)3 34 19 1.79 56 12 11 Ci 1(C)1 33 19 1.75 59 12 11 Ci 1(C)2 38 19 2.00 58 12 11 Ci 1(C)3 34 19 1.77 55 12 12 Ci 1(A)1 33 18 1.80 55 12 12 Ci 1(A)2 34 19 1.79 54 12 12 Ci 1(A)3 33 19 1.77 57 12 12 Ci 1(B)1 33 19 1.77 55 12 12 Ci 1(B)2 32 19 1.69 56 12 12 Ci 1(B)3 34 19 1.76 57 12 12 Ci 1(C)1 32 18 1.77 50 11 12 Ci 1(C)2 34 19 1.80 53 12 12 Ci 1(C)3 33 18 1.81 52 11

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99 Table 3 4 Dimensions of fresh conidia of Podosphaera xanthii from leaf tissue of Cucurbita moschata ( collected from Live Oak and Citra FL field sites during spring and fall of 2009 Host Species (number of samples) Size of fresh conidia L ength ( m) W idth (m) Length: width ratio (m) Live Oak, FL (spring) ( n=94 ) Mean value 33.6 19.1 1.8 Standard deviation 1.26 0.79 0.11 Minimum 39 23 2.6 Maximum 31 15 1.5 Citra, FL (spring) (n=119 ) Mean value 33.5 18.9 1.8 Standard deviation 0.81 0.62 0.05 Minimum 39 24 1.9 Maximum 32 18 1.4 Citra, FL (fall) (n=45) Mean value 33.9 19.0 1.8 Standard deviation 1.86 0.41 0.09 Minimum 44 20 2.3 Maximum 32 18 1.7 Total (both locations)(n=258) Mean value 33.6 19.0 1.8 Standard deviation 1.22 0.66 0.08 Minimum 44 24 2.6 Maximum 31 15 1.4

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100 Figure 3 1. Florida sites of cucurbit powdery mildew collection from spring to fall of 2009 Live Oak (Site 1) Citra (Site 2) A

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101 Figure 3 2 (A) Field plot at Live Oak, FL (13 May 2009) P lots consisted of 6 rows cover ed with black plastic mulch. There were 16 plots per row and each plot contained five plants/ plot. Plots were replicated 4 times in a complete randomized plot design (B) Field plot at Citra, FL (16 June 2009) Plots consisted of 6 rows covered with reflective silver plastic mulch. T here were 3 plots per row and each plot contained five plants/ plot. Plots were replicated 8 times in a complete randomized plot design Figure 3 3 Primary leaf (A) and cotyledon (B) of b utternut winter squash ( rooting in 2% water agar media. Living host tissue was maintained as substrate for powdery mildew single colony isolates. A B B A

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102 Figure 3 4 (A) Butternut winter squash ( 4 weeks old) rooting in 1mL test tub e containing 2% water agar (B) Cultivar leaflet 18 days in tube containing 2% water agar (C ) leaf maintained in 1mL test tubes, 4 days post inoculation with powdery mildew (D ) Set up of a luminum trays used to keep a si ngle powdery mi ldew isolate 7 days post inoculation Figure 3 5 (A) Cucurbit s eedlings cultivated in growth room (B ) Fresh host tissue at 1 2 true leaf stage used in periodic fungal isolate maintenance and detached leaf bioassays A B A B D C

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103 Figure 3 6 (A) Leaf disk s containing discrete powdery mildew colonies cut from infected leaf sample (B) Powdery mildew i so l a te (7 10 days old) transferred to fresh healthy tissue Signs (arrows) of white mycelia/conidia on fresh after transfer by gently touching infected leaflets (left) onto a healthy leaflet (right) Figure 3 7 Cucurbit p owdery mildew isolates growing on different cucurbit hosts a fter periodic isolate transfer (A) Overview of isolate maintenance under fluorescent lighting (B) Cotyledons inoculated with powdery mildew isolate s growing on 2% water agar media (C) Close up of leaflet and cotyledon 4 days post inoculation with isolate Ci 8 from Citra, F L B A A B C

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104 Figure 3 8 Characteristic powdery mildew signs and symptoms on susceptible cucurbits cultivars (A) Discrete fungal colonies (arrows) on watermelon fruit. (B) L infected (C ) Powdery mildew o n petioles of acorn squash (D ) Fungal colonies on ste ms of butternut winter squash (E ) Discrete fungal colonies on leaf undersurface of (F ) Heavy powdery mildew infection on l ower canopy and up per leaf surface of pumpkin At Citra field site, p remature yellowing and senescence exposing fruit to sunburn as a result of severe powdery mildew infection (G) and s evere powdery mildew infection and defoliati on (H) C E D F A B G H

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105 Figure 3 9 Characteristic Podosphaera xanthii morphological features (A) Erect conidiophores on leave surface seen under stereoscope (B) C onidia produced in chains following 2 3 mother cells above the foot cells (C) Immature conidia with crenate (arrow s ) edge s (D) Septate hyaline hyphae (arrows) and conidiophores (E) Hyaline conidia; ellipsoid to ovoid in shape and with presence of reflective fibrosin bodies (arrows) (F) Forked germ tubes (arrow) were infrequently observed. A B C E D F

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106 Figure 3 10 Electrophoretic profile of PCR products amplified by primers S1/S2 and G1/G2. S pecific PCR detection of Podosphaera xanthii (Px) from field samples of various cucurbit hosts Fragments of the expected sizes (454 bp and 391bp) are observed in lanes 12 ( P. xanthii ) and 13 ( Golovinomyces cichoracearum ) respectively. Lane 14 shows specific PCR detection of a fragment of the expected size (double bands) correspondin g to multiplex PCR product. Lanes 1 and 17 correspond to 1Kb molecular marker. Cucurbit ane 7 ). Px= P. xanthii ; Gx= G. cichoracearum and Px+Gx= multiplex reaction 2 1 7 3 1 7 6 5 4 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 500 bp 454 bp (Px) 391 bp (Gc)

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107 Figure 3 11 Analysis of DNA fragments amplifi ed by a multiplex PCR with DNA extracted from powdery mildew colonies from .. Two primer pairs (S1/S2 and G1/G2) for Podosphaera xanthii and Golovinomyces cichoracearum respectively, were used Lanes 2 56 co rrespond to P. xan thii (454 bp) Lane 59 shows two fragment s of the expected size for P. xanthii (454 bp ) and G. cichoracearum (391 bp ) respectively. Lanes 1, 20, 21, 40, 41 and 60 correspond to 1Kb molecular marker. 500 bp 500 bp 500 bp 1000 bp 1000 bp 1000 bp 2 18 3 4 8 7 6 5 9 10 11 12 13 14 15 16 17 1 19 20 Lanes 22 38 23 24 28 27 26 25 29 30 31 32 33 34 35 36 37 21 39 40 42 58 43 44 48 47 46 45 49 50 51 52 53 54 55 56 57 41 59 60 Lanes Lanes

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108 CHAPTER 4 DETERMINATION OF POWDERY MILDEW PHYSIOLOGICAL RACES Powdery mildew is an important disease of cucurbit c rops worldwide. It is primaril y caused by one of two fungi, Podosphaera xanthii or Golovinomyces cichoracearum The most commonly found pathogen, P. xanthii is known to occur in several physi ological races in the United States and world wide ( 163 190 ) These physiological races are most frequently distinguished using musk melon differential genotypes ( 188 ) And to a less er extent, other cucurbit species such as cucumber, watermelon and Cucurbita spp. have been used to differentiate cucurbit powdery mildew physiological races ( 161 180 ) W hen breeding crops for resistance to disease, it is fundamental to know the species and races of the causal agent present since resistance genes are often specific to the prevailing pathogenic race ( 158 195 ) Search for resistance genes to be used in developing resistant cucurbit breeding lines is the focus of many research programs worldwide ( 43 123 157 339 ) International effort has been dedicated to breeding for resistance to cucurbit powd ery mildew, especially P. xanthii In melon cultivar PMR 45 resistance ha s been attributed to a single dominant gene H owever, a more complex s cenario has been reported f or other cucurbit cultivars with control being governed by various combinations of several dominant, recessive and modifier gen es ( 129 ) Knowledge of the prevalence and distribution of cucurbit powdery mildew races are essential for the deployment of suitable resistant commercial lines, as well as for choice of ap propriate disease management strategies implementation of molecular techniques and tools and continued breeding resistance efforts. Screening protocols require efficient methods which can distinguish pathogenic variations among cucurbit powdery mildew pathogens at the level of pathotypes as well as races ( 10 12 ) Lebeda

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109 et al. ( 161 ) emphasized that pathotypes express variation at the host range level, while races represent the level o f virulence on a set of selected genotypes of one host species, tra ditionally, muskmelon cultigens Because pathogenic variants can impact host range and disease management, an improved understanding of powdery mildew pathogenic strains in FL was necessary For this reason I undertook the following research objectives: (1) identify and subculture the prevailing causal agent of cucurbit powdery mild ew in north central Florida; (2) characterize fungal isolates through morphologica l features and DNA analysis; (3) assess the presence of physiological races within cultured isolates via bioassa ys using detached leaves; and (4) evaluate the varietal reactions of Cucurbita breeding lines for susceptibility to powdery mildew under local (FL) field conditions. Materia ls and Methods Plant Material and Growth C onditions A differential set of t welve muskmelon ( Cucumis melo ) genotypes with varying levels of resistance to cucurbit powdery mildew were tested and used for race determination based on modifications of protocols described by McCreight J. D. ( 188 ) and Lebeda et al. ( 161 ) In our study, t he following m uskmelon lines were inoculated a sub set of powdery mildew isolate s : Edisto 47, Iran H, MR 1, PI 124111, PI 124112, PI 313 970 PI 414723, PMR 45, PMR 5, TopMark, Vedrantais, WMR 29. Seeds were kindly provided by Dr. James D. McCreight (United States Department of Agriculture) and by Dr. Eileen Kabelka ( formerly of University of Florida Department of Horticulture Science, cur rently with Harris Moran Seed Co. CA ). Muskmelon plants were grown in 3.8 liter plastic pots (Figure 4 1) containing potting medium (Fafard Professional 4P Mix), in a powdery mildew free green house.

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110 During development in the green house, plants did not receive any fungicide treatment s since this application could influence susceptibility to powdery mildew. Throughout cultivation in the greenhouse, plants were continuously monitored for unwanted powdery mildew infection Day length ranged from 10 to 12 hours and t emperature was maintained at 23 5 C. Temperature s inside the green house were recorded continuously (data not shown) over the length of the experiment using a HOBO Microstation Data Logger (Onset Computer Corp., Bourne, MA). Temperature was adjusted as needed to maintain ideal growing conditions Plants were watered and fertilized with water soluble fertilizer Peters Professional 20 10 20 Peat lite (Scotts Int ernational B.V., Netherlands) and Osmocote Plus controlled release fertilizer (S cott Sierra Horticultural Products Company, Marysville, OH) as needed. Muskmelon differentials were maintained in the green house for 6 to 8 weeks ( until 3 to 6 true leaf stage), after which older plants were discarded. Rather than using plants at the seedling stage, older cucurbit plants are recommended for resistance screening against powdery mildew because expression of resistance typically appears at more mature developmental stages ( 153 ) Seeds of e ach muskmelon type were planted every 10 to 15 days, to provide a fresh supply of host tissue for detached leaf bioassays. During laboratory experiments, h ealthy whole leaves from each muskmelon type were removed using sterile scalpel blades, and taken t o the labora tory in individual labeled and sealed plastic bags. While being harvested in the greenhouse, leaves were k ept inside a cooler to avoid premature wilting of tissue. Sample C ollection a nd P athogen I dentification To determine powdery mildew pathogen races, a sub set of powdery mildew isolates was screened using detached leaf assays. Isolates tested were selected to

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111 represent specific locations, hosts and times of year. Fully expanded muskmelon leaves (3 6 true leaf stage from 6 8 weeks old plants) were excised from the middle portion of at least two plants per muskmelon genotype. Five powdery mildew isolates (Table 4 1) used in this experiment were collected from naturally infected field or greenhouse samples as de scribed in Chapter 3. The following isolates were tested: LO isolate 10 02 from muskmelon in greenhouse at UF in Gaine sville, FL on 16 April 2010; isolate 10 08 from squash at Immokalee FL; on 10 May, 2010, and isolate10 09 from squash at Dover, FL on 6 June 2010. Prior to race typing, the identification of the powdery mildew species was confirmed by microscopic examinati on of the morphological characters of fresh conidia in 3% aqueous KOH as previously defined in Chapter 3. Cotyledons and first true leaves of susceptible cucurbit cultivars (Butterbush, Dark Green Zucchini, Mickey Lee, Straight Eight and Waltham) were use d for powdery mildew inoculum maintenance and propagation, incubated for 7 10 days as previously described in Chapter 3. P reliminary tests indicated that certain muskmelon genotypes susceptible to all powd ery mildew isolates. As such, healthy muskmelon leaves (3 8 weeks old) were also used to enhance inoculum production Host R eaction Pathogenic variation of a select sub set of fungal isolates was determined by analyzing the reactions of 12 muskmelon differential lines to each powdery mildew isolate, as described by several authors ( 18 161 190 )

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112 Detached leaf assays were based on modifications and adapt at ions of the me thod s described by McCreight et al. ( 190 ) Kristkova et al. ( 145 ) Vakalounakis and Klimonomou ( 328 ) Co hen et al. ( 40 ) and Lebeda et al ( 158 ) According to several report s the detachment of leaves rather than whole plant inoculation, is a common practice and does not seem to affect race determination In all race determination experiments each muskmelo n genotype w as represented by 2 replicates (leaf A and B). Leaves were excised from each muskmelon type and inoculated with a predetermined powdery mildew isolate. Muskmelon differential leaves were not surface sterilized prior to inoculation Each powdery mildew isolate was inoculated onto a total of 24 detached muskmelon le aves (two leaves per isolate). E ach muskmelon differential leaf was placed, adaxi al leaf surface face up, in a petri dish ( 10 or 15 c m in diameter Petri plates depending on leaf size. ) which contained a layer of 2 % water agar to provide support and moistur e (Figure 4 2 ) Each m uskmelon le af was inoculated by gently pressing infected tissue onto a healthy leaf During inoculation, musk melon genotype s were intercalated so that all genotypes of the f irs t repetition were inoculated, followed by all genotypes of the second repetition to avoid dilution of the inoculum for the subsequent different genotypes Inoculated leaves as well as controls (mock inoculat ed leaves) were incubated under fluorescent light ing ( 12 hours per day) at room temperatu re (22 to 25 C) on a laboratory bench for 10 15 days T he first powdery mildew symptoms appeared at 3 5 dp i ( days post inoculation ) Detached muskmelon leaves were observed under 60x magnification and photog raphed at 3, 7 and 10 days post i noculation QIm aging Digital camera and

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113 software Q Capture Pro (Surrey, BC, Cana da) were used for observation and analysis of infected leaf tissue Disease E valuation The development of powdery mildew hyphae and conidia on detached muskmelon leaves was evaluated at 3, 7 and 10 days post inoculation. Inoculated detached leaves were maintained in vitro for up to 15 days the time at which infected leaf tissue became too decomposed for further analysis. Disease evaluations were recorded as soon a first powdery mildew signs were observed with unaided eyes. Assessment of powdery mildew for race typing was evaluated by visually estimatin g the percent leaf area covered by the pathogen using a standard stereoscope. A pre determined circumference (10 mm diameter disk) was cut off a piece of plastic film and was placed over an infected leaf area and used as a standard template for disease sev erity and pathogen development assessment (Figure 4 3). Assessment of powdery mildew disease on detached muskmelon leaves was evaluated by the quantification of disease severity and the quality of pathogen development (pathogen status). Both measures were used to separate the response of the host (resistant or susceptible) from the reaction of the pathogen (virulent or avirulent) and eventually evaluate if both ratings correlated. Pathogen status Powdery mildew pathogen development (sporulation) on each mu skmelon detached leaf was rated using a 0 4 scale as described by McGrath et al. ( 213 ) as follows: (0) =no fungal growth (noted presence of inocul um); (1) = very little mycelial growth with no conidiophores; (2) = little mycelial growth with few conidiophores; (3) = fairly good mycelial growth with scattered conidiophores and (4) = heavy sporulation. A

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114 genotype was considered be susceptible when the isolate tested produced conidiophores (rating of 2, 3 or 4) on both inoculated leaves of the same genotype (Figure 4 4). Disease severity Disease severity (percentage of 10 mm diameter circular leaf area covered by mycelia/conidia) was classified into 5 c ategories using a modification of the scale des cribed by Kristkova et al. ( 142 ) where: 0 = no symptoms of infection on circular area 1 = less than 25% of circular area cov ered with mycelia, 2 = 2 5 50% of circular a rea covered with mycelia, 3 = 50 75% of circular area covered with mycelia and 4 = over 75% of circular area covered with mycelia. On each differential muskmelon genotype, isolates with an average severity (2 inoc ulated leaves) of mycelial growth of 0 or 1 were classified as avirulent and those with ratings of 2, 3 or 4 were considered virulent (Figure 4 4). Results and Discussion Powdery M ildew I dentification On the basis of morphological and molecular analysis, t he isolates tested in this study were determined to be Podosphaera xanthii C haracteristics f eatures such as conidia shape and dimensions (length and widt h), presence of fibrosin bodies and crenate immature conidia edge line were consistent with published reports for P. xanthii Molecular analysis through PCR confirmed presence of P. xanthii as previously described in Chapter 3 Effect of Cucurbit Powdery Mildew on D etached M uskmelon L eaves D etached leaf inoculation proved to be an efficient in vitro assay to test the pathogenic variability (race s ) of powdery mildew isolates collected from greenhouse and

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115 field samples in north Florida M ock inoculated m uskm elon leaves (negative controls) remai n ed green and healthy for u p to 20 d ays ( Figure 4 5 ) Some d etached muskmelon inoculated leaves developed comparable and reproducible disease symptoms on the upper leaf surface. When a muskmelon genotype was susceptible, p ercentages of leaf area covered by powdery mildew typically showed extensive interwoven myc elium and abundant conidiophore production (Figure 4 6 ). Previous s tudies regarding cucurbit powdery mildew race determination traditionally use leaf disks ( 161 190 192 218 ) or entire plants ( 66 319 ) L eaf disks being smaller than who le leaves allow for greater number of replications and could yield better comparison between isolates and possibly a more accurate representation of pathoge n variability. In our studies two replicates (2 leaves per muskmelon type) may have been in suffici ent for a conclusive P. xanthii race determination but were adequate for a preliminary assessment In addition, d uring this study, inoculum production decreased over time A possible explanation might be that isolates had been cultured in vitro for too long and possibly decreased virulence Furthermore toward the end of race typing studies, it had become more difficult to increase inoculum pro liferation and produce enough inoculum to infect more than 2 leaflets with each isolate Disease E valua tion A disk outline (10 mm diameter circle) was used as a standard template for disease severity and pathogen development assessment ( Figure 4 7) This method was chosen so that, for all muskmelon leaves, inoculated with each isolate, the same leaf area was assessed for comparison

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116 Reactions of 12 muskmelon genotypes to 5 powdery mildew isolate s rated 10 days post inoculation ( dpi ) indicated variability in isolate virulence ( Figures 4 8 to 4 12 ). I mages show detail of powdery mildew infection within pre determined circumference and the corresponding detached muskmelon leaf. D isease severity ratings, corresponding pathogen development (status) ratings and the type of reaction observed are summarized in Table 4 2. For each differentia l muskmelon genoty pe, isolates with severity rating of 0 or 1 were classified as avirulent and those with ratings greater than 2 were considered virulent (Figure 4 4). A genotype was considered be susceptible to the pathogen when the isolate being tested produced conidiopho res (rating 2) on both inoculated leaves (Table 4 2). The production of conidiophores and consequently fungal sporulation was an indication that infection had progressed (Figure 4 8 to Figure 4 12). There was good linear correlation (R 2 =0.80) between dis ease severity and pathogen status ratings indicating that as disease severity increased, pathogen development also increased. Isolate 10 02 presented the highest correlation coefficient (R 2 =0.93) followed by isolate LO 6 1(A ) 1 (R 2 =0.89), isolate 10 08 (R 2 =0.84) and isolate 10 09 (R 2 =0.84). However isolate Ci 6 1(A ) 1 presented the lowest correlation (R 2 =0.62) PI 124111 and PMR 5 were resistant to all 5 powdery mildew isolates tested 4 1 and PI 313970 revealed resistance to 2 out of 5 isolates; while PI 124112 and WMR 29 presented resistance to 4 out of 5 isolates and genotype PI 414723 demonstrated

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117 resistance to 3 out of 5 isolates In this evaluation, Edisto 47 PI 12411 and PMR 5 plants appeared to be among the most resi s tant of the 12 muskmelon genotypes t ested, never showing more than 25 % of leaf area covered with powdery mildew d isease high as 75 % coverage and consiste ntly had disease symptoms over 50 % of the leaf area assessed. (Table 4 2 and Figure 4 8 to Figure 4 12) A list of currently known races of P. xanthii is presented i n Figure 4 13 (p ersonal communication and graciously provided by Dr James D. McCreight of USDA ARS, Salinas, CA ). In comparison to this list, our race typin g assays indicated that we may have found race 2F as it s p rofile is consistent with the isolate from Citra (Figure 4 13 ). The isolate from Live Oak may be consistent with the profiles described for race 2Z The other 3 isolates tested (UF, IM and DO) were undefined to date and were not consistent with any of the previously described races of P. xanthii (Figure 4 13 ) In addition some of the previously defined cucurbit powdery mildew races were incompletely tested (represented by gray color) and therefore cannot be disregarded as possible matches to the results ob tained during our studies (Figure 4 13) It has been widely reported that biotic and abiotic factors may alter host response and subsequently affect race profiling. Factors such as environmental conditions (light intensity, temperature and humidity) as wel l as age of plant at time of inoculation and purity of inoculum may affect disease severity and adversely influence race identification by masking host resistance or pathogen virulence factors ( 41 190 ) In our studies, it is possible th at we had isolated mixed colonies, indicating a mixture of races. An alt ernative explanation would be that during sampling, we may

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118 have isolated a novel P. xanthii pathogenic race (or races) present in north Florida. Furthermore, differences in environmental conditions specific to Florida (field sites and greenhouse) as well a s conditions in the laboratory could have altered host response and/or pathogen virulence during our studies. Additionally, due to our low number of leaf replicates, our results may be inconclusive. Further testing, with larger number of replicates, is nec essary.

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119 Table 4 1 Sub set of six Florida p owdery mildew isolates used for race typing bio assays Isolate No. Cucurbit host Collection time Inoculation Date Origen Fungal species* Ci 6 1(A)1 Butterbush Fall 2009 5/04/10 Citra P. xanthii 10 08 Summer squash Spring 2010 5/21/10 Immokalee P. xanthii LO 6 1(A)1 Butterbush Fall 2009 5/27/10 Live Oak P. xanthii 10 02 TopMark Spring 2010 6/11/10 Gainesville P. xanthii 10 09 Summer squash Spring 2010 6/18/10 Dover P. xanthii P. xanthii isolates were confirmed by microscopic analysis of morphological features and molecular analyses Table 4 2 Reaction (10 days post inoculation) of some muskmelon ( Cucumis melo ) genotypes to sub set of powdery mildew isolates. Muskmelon genotype Powdery mildew i solate No. i noc. leaves Leaves with sympt. Inoc. presence affected leaf area (%) DS rating PS rating Type of reaction Edisto 47 LO 6 2 2 + 1 25 1 1 R Ci 6 2 2 + 1 25 1 1 R 10 02 2 2 + 1 25 1 1 R 10 08 2 2 + 1 25 1 1 R 10 09 2 2 + 1 25 1 1 R PI 124111 LO 6 2 2 + 1 25 1 1 R Ci 6 2 2 + 1 25 1 1 R 10 02 2 2 + 1 25 1 1 R 10 08 2 2 + 1 25 1 1 R 10 09 2 2 + 1 25 1 1 R PMR 5 LO 6 2 2 + 1 25 1 1 R Ci 6 2 2 + 1 25 1 1 R 10 02 2 2 + 1 25 1 1 R 10 08 2 2 + 1 25 1 1 R 10 09 2 2 + 1 25 1 1 R

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120 Table 4 2. Continued Muskmelon genotype Powdery mildew i solate No. i noc. leaves Leaves with sympt. Inoc. presence affected leaf area (%) DS rating PS rating Type of reaction Iran H LO 6 2 2 + 51 75 3 4 S Ci 6 2 2 + 51 75 3 3 S 10 02 2 2 + 51 75 3 4 S 10 08 2 2 + >75 4 4 S 10 09 2 2 + 51 75 3 3 S PMR 45 LO 6 2 2 + 1 25 1 2 S Ci 6 2 2 + 1 25 1 2 S 10 02 2 2 + 51 75 3 4 S 10 08 2 2 + 51 75 3 2 S 10 09 2 2 + 1 25 1 2 S TopMark LO 6 2 2 + >75 4 4 S Ci 6 2 2 + 26 50 2 4 S 10 02 2 2 + 51 75 3 4 S 10 08 2 2 + 51 75 3 4 S 10 09 2 2 + >75 4 4 S Vedrantais LO 6 2 2 + 51 75 4 4 S Ci 6 2 2 + 51 75 3 3 S 10 02 2 2 + 26 50 3 4 S 10 08 2 2 + >75 4 4 S 10 09 2 2 + 51 75 3 3 S MR 1 LO 6 2 2 + 1 25 1 1 R Ci 6 2 2 + 1 25 1 1 R 10 02 2 2 + 26 50 2 3 S 10 08 2 2 + 26 50 2 2 S 10 09 2 2 + 1 25 1 2 S

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121 Table 4 2. Continued Muskmelon Genotype Isolate No. Inoc. leaves Leaves with sympt. Inoc. present affected leaf area (%) DS rating PS rating Type of reaction PI 313970 LO 6 2 2 + 26 50 2 3 S Ci 6 2 2 + 1 25 1 1 R 10 02 2 2 + 1 25 1 1 R 10 08 2 2 + 1 25 1 2 S 10 09 2 2 + 26 50 2 3 S PI 124112 LO 6 2 2 + 1 25 1 1 R Ci 6 2 2 + 1 25 1 1 R 10 02 2 2 + 1 25 1 2 S 10 08 2 2 + 1 25 1 1 R 10 09 2 2 + 1 25 1 1 R WMR 29 LO 6 2 2 + 1 25 1 1 R Ci 6 2 2 + 1 25 1 2 S 10 02 2 2 + 1 25 1 1 R 10 08 2 2 + 1 25 1 1 R 10 09 2 2 + 1 25 1 1 R PI 414723 LO 6 2 2 + 1 25 1 1 R Ci 6 2 2 + 1 25 1 1 R 10 02 2 2 + 1 25 1 2 S 10 08 2 2 + 1 25 1 1 R 10 09 2 2 + 1 25 1 2 S Number of inoculated leaves= 2 += Presence of inoculum n oted on leaf surface at day of disease assessment ; DS= Disease severity rating PS=Pathogen status rating Type of reaction: R= resistant; S=susceptible

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122 Figure 4 1. Set of healthy muskmelon genotypes growing in powdery mildew free greenhouse. Figure 4 2. Demonstration of m uskmelon d ifferential inoculation process (A) H ealthy leaves from a set of twelve muskmelon genotypes awaiting inoculation in detached leaf assay ( B ) D etail of infected susceptible muskmelon leaf a powdery mildew isolate (C ) Leaf p ieces originated from whole leaf infect ed with powdery mildew inoculum used to inoculate healthy muskmelon detached leaves. A B C

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123 Figure 4 3. A 10 mm diameter circumference was visual ly assessed for powdery mildew P owdery mildew isolate [ Ci 6 1(A )1] sporulating on different muskmelon genotypes (A) TopMark (B) Iran H (C) Vedrantais (Magnification 200 x) Figure 4 4. Scale used for pathogen status classifi cation. Categories rang ed from 1 to 4 according to fungal sporulation and conidiophore production on leaf surface of different muskmelon genot ypes Isolates wer e considered virulent w ith ratings of 0 (no fungal growth) and 1and were avirulent with ratings of 2 4. A B C

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124 Figure 4 5. Healthy l eaves (mock inoculated) of 12 muskmelon genotypes 17 days post mock inoculation with un infected r cotyledons. Figure 4 6. Detached muskmelon l eaf area co vered by powdery mildew, 7 days after pathogen inoculation showing extensively interwoven mycel ium and abundant conidiophore production. ( A) Magnification of 200 x. (B) Magnification of 450 x (C) Magnification of 6 3 0 x A C B Vedrantais PI 313970 PI 12 4112 PI 414723 PMR 45 PMR 5 Edisto 47 WMR 29 TopMark Iran H MR 1 PI 124111

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125 Figure 4 7. R eaction of a set of 12 muskmelon genotypes to powdery mildew isolate from summer squa sh. Percentage ratings of d isease severity (0 to 100% ) and pathogen development status (scale 0 4) were recorded 10 days post inoculation ( Magnification 200 x )

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126 Figure 4 8 Reaction s of 12 muskmelon genotypes to P. xanthii isolate LO 6 1 (A) 1 from ( 10 d pi ) Images on the right show close up of percentage of powdery mildew infection on corresponding detached leaf ( left ) Fungal sporulation was rec orded and photographed Average d isease severity and pathogen status ratings are expressed between parenthesis (right).Disease severity was categorized by: (0) = circular leaf area without symp toms of infection; (1) = less than 25% of circular area covered with mycelia; (2) = 26 50% of circular area covered with mycelia; (3) = 51 75% of circular area covered with mycelia and (4) = over 75% of circular area covered with mycelia. Pathogen status was rated as : (0) =no fungal growth (noted presence of inoculum) ; (1) = very little mycelial growth with no conidiophores; (2) = little mycelial growth with few conidiophores; (3) = fairly good mycelial growth with scattered conidiophores and (4) = heavy sporulation. TopMark PMR 45 PMR 5 Vedrantais Iran H WMR 29 Edisto 47 PI 124111 PI 124112 PI 313970 PI 414723 MR 1 (4,4) (4,4) (1,2) (1,1) (3,4) (1,1) (1,1) (1,1) (1,1) (1,1) (1,1) (2,3)

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127 Figure 4 9 Reactions of 12 muskmelon genotypes to P. xanthii isolate Ci 6 1(A) 1 from (10 dpi) Images on the right show close up of percentage of powdery mildew infection on corresponding detached leaf (left). Fungal sporulation was recorded and photographed. Disease severity and pathogen sta tus ratings are expressed between the parenthesis (right).Disease severity was categorized by: (0) = circular leaf area without symptoms of infection; (1) = less than 25% of circular area covered with mycelia; (2) = 26 50% of circular area covered with myc elia; (3) = 51 75% of circular area covered with mycelia and (4) = over 75% of circular area covered with mycelia. Pathogen status was rated as: (0) =no fungal growth (noted presence of inoculum); (1) = very little mycelial growth with no conidiophores; (2 ) = little mycelial growth with few conidiophores; (3) = fairly good mycelial growth with scattered conidiophores and (4) = heavy sporulation. TopMark PMR 45 PMR 5 Vedrantais Iran H WMR 29 Edisto 47 PI 124111 PI 124112 PI 313970 PI 414723 MR 1 (2,4) (1,2) (3,3) (1,1) (3,3) (1,2) (1,1) (1,1) (1,1) (1,1) (1,1) (1,1)

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128 Figure 4 10 Reaction s of 12 muskmelon genotypes to P. xanthii isolate 10 02 from muskmelon with natural powdery mildew infection in greenhouse at UF, Gainesville, FL (10 dpi) Images on the right show close up of percentage of powdery mildew infection on corresponding detached leaf (left). Fungal sporulation was recorded and photographed. Disease severity and pathogen status ratings ar e expressed between the parenthesis (right).Disease severity was categorized by: (0) = circular leaf area without symptoms of infection; (1) less than 25% of circular area covered with mycelia; (2) = 26 50% of circular area covered with mycelia; (3) = 51 7 5% of circular area covered with mycelia and (4) = over 75% of circular area covered with mycelia. Pathogen status was rated as: (0) =no fungal growth (noted presence of inoculum); (1) = very little mycelial growth with no conidiophores; (2) = little mycel ial growth with few conidiophores; (3) = fairly good mycelial growth with scattered conidiophores and (4) = heavy sporulation. TopMark PMR 45 PMR 5 Vedrantais Iran H WMR 29 Edisto 47 PI 124111 PI 124112 PI 313970 PI 414723 MR 1 (1,1) (1,1) (1,2) (1,2) (2,1) (1,1) (3,4) (3,4) (3,4) (3,4) (3,4) (1,1)

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129 Figure 4 11 Reaction s of 12 muskmelon genotypes to P. xanthii isolate 10 08 from squash collected in Immokalee, FL (10 dpi) Images on the right show close up of percentage of powdery mildew infection on corresponding detached leaf (left). Fungal sporulation was recorded and photographed. Disease severity and pathogen status ratings are expressed between the parenthesis (right).Disease severity was categorized by: (0) = circular leaf area without symptoms of infection; (1) = less than 25% of circular area covered with mycelia; (2) = 26 50% of circular area covered with mycelia; (3) = 51 75% of circular area covered with mycelia and (4) = over 75% of circular area covered with mycelia. Pathogen status was rated as: (0) =no fungal growth (noted presence of inoculum); (1) = very little mycelial growth with no conidiophores; (2) = little mycelial growth with few conidiophores; (3) = fairly good mycelial growth with scattered conidiophores and (4) = heavy sporulation. TopMark PMR 45 PMR 5 Vedrantais Iran H WMR 29 Edisto 47 PI 124111 PI 124112 PI 313970 PI 414723 MR 1 (1,1) (1,1) (1,1) (1,1) (2,2) (1,2) (4,4) (3,2) (4,4) (1,2) (4,4) (1,1)

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130 Figure 4 12 Reaction s of 12 muskmelon genotypes to P. xanthii isolate 10 09 from summer squash collected in Dover, FL (10 dpi) Images on the right show close up of percentage of powdery mildew infection on corresponding detached leaf (left). Fungal sporulation was recorded and photographed. Disease severity and pathogen status ratings are expressed between the parenthesis (right).Disease severity was categorized by: (0) = circular leaf area with out symptoms of infection; (1) = less than 25% of circular area covered with mycelia; (2) = 26 50% of circular area covered with mycelia; (3) = 51 75% of circular area covered with mycelia and (4) = over 75% of circular area covered with mycelia. Pathogen status was rated as: (0) =no fungal growth (noted presence of inoculum); (1) = very little mycelial growth with no conidiophores; (2) = little mycelial growth with few conidiophores; (3) = fairly good mycelial growth with scattered conidiophores and (4) = heavy sporulation TopMark PMR 45 PMR 5 Vedrantais Iran H WMR 29 Edisto 47 PI 124111 PI 124112 PI 313970 PI 414723 MR 1 (4,4) (1,2) (3,4) (1,1) (3,3) (1,1) (1,1) (1,1) (1,2) (1,1) (1,2) (2,3)

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131 Figure 4 13. Expected reaction of 12 muskmelon genotypes to 31 known races of powdery mildew ( P. xanthii ) found in the U.S. and around the world. Different colors (red, grey, green and salmon) represent reaction of host plant to pathogen inoculation. (Table prepared by Dr. James D. McCreight USDA personal communication on 11 August, 2009). Table includes 5 powdery mildew races (far right) tested in this study. Isolates tested were: LO= LO Citra FL; isolate UF= 10 02 collected from muskmelon in greenhouse at the University of Florida in Gainesville, FL; isolate IM= 10 08 collected from squash at Immokalee FL and isolate DO= 10 09 collected from squash at Dover, FL.

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132 CHAPTER 5 Cu curbit powdery mildew pathog e n s, Podosphaera xanthii and Golovinomyces cichoracearum high risk t hese fungi hav e high evolutionary potential according to McDonald and Linde ( 196 ) and are more likely to overcome plant genetic resistance and/or develop fu ngicide resistance ( 29 197 ) Factors such as mixed reproduction cycles (asexual and sexual), asexual spores (conidi a) that are easily disseminated over long distances the possibility of gene for ge ne interactions with their host ( 30 ) and resistance to some current fungicide chemistries have enabled cucurbit powdery mildews to become hig h ly variable in the ir pathogenicity and virulence represented by the existence of several pathotypes and races ( 31 159 190 ) In cucurbits, powdery mildew disease results in moderate to severe damage to the foliage, as well as a considerable reduction in yield and fruit quality ( 214 ) The disease is adequately controlled with fungicides, nonetheless an increase in the number of reports of resistance to some recommended fungicides, and the difficulty of f ungicide ap plication on the underside of leaves where conditions are more favorable for disease development, have required more resourceful method s of disease management ( 128 159 201 ) As part of a successful integrated disease management program, b reeding cucurbit crops for resistance to powdery mildew offers a more economical and safe r method to considerably reduce disease pressure h owever, incorp orating resistance into all horticultural cucurbit types would be a n early impossible task, demanding great time and effort. Thus the use of plant disease resistance is not a viable option for all cucurbit

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133 growers. In addition, resistant cultivars ten provide complete disease control when used as the sole management practice. Breeding of cucurbit crops for powdery mildew resistance has been effective especially in resources of race specific resistance in muskmelon ( 8 77 110 137 149 190 305 347 ) S ources of resistance have also been described in pumpkin, gourds and squash ( 15 119 123 140 154 155 352 ) and in cucumber ( 5 22 153 156 177 178 ) Additionall y sources of resistance to powdery mildew in watermelon have been identifi ed ( 63 66 68 171 318 319 321 350 ) Genes for resistance to powdery mildew in melon have been widely studi ed. PMR 45 has a single dominant gene for powdery mildew resistance ( 77 122 ) M oreover, m ost reported genotypes of melon resistant to powdery mildews include several genes ( 187 264 ) and the exact number of genes involved differs according to the study ( 77 129 ) and to the strain (race) of cucurbit powdery mildew tested ( 195 ) In most cases, monogenic or digenic dominant control has been reported ( 251 ) In watermelon, findings suggest that resistance is controlled by multiple genes which are expressed as degrees of tolerance ( 62 64 ) The development of cucurbit varieties displaying resistance to powdery mildew (mainly P. xanthii ) has been one of the major aims of cucurbit breeding programs worldwide. In this study we evaluated the powdery mildew resistance response of 22 elite breeding lines developed by Rupp Seeds which contained two sources ( Cucurbita lundelliana Bailey and C. okeechobeensis (Small) Bailey ) of resistance to powdery mildew. Resistance to powd ery mildew in the wild species, C. lundelliana is conferred by a single dominant gene ( 274 ) and can be transferred to C. moschata Resistance in

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134 the other wild species C. okeechobeensis was reported to be conferred by a single dominant or incomplete dominant gene which is su bject to the influence of modifier genes affecting the level of resistance ( 50 ) Resistance in the cultivated species C. moschata is conferred by either of two genes, designated pm 1 L and pm 2 S ( 2 ) The pedigree of the Rupp breeding lines presented in Figure 5 6 provides additional information about the parentage of each line. Cucurbita lundelliana and C. okeechobeensis are wild Cucurbita species, known to have natural resistance to powd ery mildew disease. Each Rupp breeding line with both sources of resistance has 12.5% of C. lundelliana genome and 12.5% of C. okeechobeensis genome. The rest of the genome of each of the breeding lines is 75% Cucurbita moschata (winter butternut squash ty pe). Each line contained different proportions of each of the two wild species. The purpose of this study was to assess the resistance of Rupp cucurbit breeding material to powdery mildew in comparison to two cucurbit cultivars (Butterbush and Mickey Lee) known to be highly susceptible to powdery mildew in Florida. Weekly evaluations, on leaf and whole plants, were recorded based on disease severity (percentage of infection) after the initial disease find in field plots. Materials and m ethods Plant M aterial Field experiment s were conducted at the North Florida Research and Education Center Suwannee Valley (NFREC SV) in Live Oak, FL. D uring spring of 2009, s eedlings of 22 Cucurbita Rupp elite breeding lines were planted in adjacent rows on raised bed s covered with black plastic mulch, as descried in C hapter 3 Additionally, two susceptible cultivars (Butterbush and Mickey Lee), were planted at Live Oak to promote

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135 disease and serve as inoculum sources. The same two susceptible cultivars were also plant ed at Citra for disease assessment and comparison. Seeds of each breeding line were kindly supplied by Mr Duane Bell cucurbit breeder with Rupp Seeds Inc A list of the breeding lines is presented in Table 5. Seeds were direct seeded into styrofoam growing trays on 25 March 2009. One seed of each breeding line was planted into each tray cell using potting media Fafard Professional 4P Mix (Fafard Company, Agawam, MA). A total of 25 seeds of each breeding line were sown. Seedlings were g rown in a greenh ouse at UF, for 30 45 days (1 to 2 true leaf stages ). Subsequently, seedlings were acclimatized for one week in a shaded and natural ly ventilated greenhouse at Live Oak field station before transplant into field plots During acclimatizati on, seedlings were watered as needed. Five seedling s of each breeding line were transplanted into field plots on 23 April 2009 S ome seedlings were re planted on 29 April 2009 due to lack of plant g rowth All plots were replicated four times in a r andomized complete block design as previously described in Chapter 3. Powdery M ildew D isease A ssessment in Field Trial were not assessed at Citra due to lack of seed germination when this cultivar was direct seeded in the field. Consequently, disease occurrence was monitored only on Butterbush plants (at each location) based on weekly evaluations of leaves, until disease o n set. Powdery mildew disease severity of the 22 elite breeding lines was assessed for a period of 9 consecutive weeks at Live Oak. Disease evaluations were based on average percentage of leaf area infected on both adaxial (upper) and abaxial (lower) leaf surfaces. Three leaves per plant per plot were assessed each week The average

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136 disease severity for whole plants, for each breeding line, was record ed based on the individual leaf assessments (3 leaves per plant) and on total appearance of plant health (gr een, senescing or necrotic). Disease severity ratings of all b reeding lines were compared to both susceptible culti vars Butterbush and Mickey Lee at Live Oak. Disease severity e valuations for both susceptible cultivars were recorded from two plants per plo t at each location on two randomly selected lower leaves per plant Disease severity ratings at Live Oak, were recorded on 13 19 and 26 May, 2, 10, 16 and 23 June 1 10, 15 and 23 July 2009. Ratings at Citra were not ed on 26 May, 2 10, 16 and 22 June 1 10, 15, 23 and 28 July and 3 August 2009. The time from trans planting in the field to first disease s ymptoms was approximately 3 0 days. P owdery mildew disease symptoms appeared first at the Live Oak site on 13 May 200 9 and at Citra symptoms were first noted on 16 June 2009. P owdery mildew severity evaluations were conducted starting from the time of appearance of the first signs of disease and upon confirmation of powdery mildew presence via microscopic examination of leaf samples as describe d in C hapter 3 First disease symptoms were defined as having at least one visible (unaided eye) fungal colony on any leaf of any susceptible plant, indicating naturally occurring presence of the pathogen i n each field site. Each week, i nfected Butterbush and Mickey Lee leaf samples were randomly collected from each location, on the same day as disease severity ratings were recorded. Individual leaves were placed in separate labeled and sealed plastic bags, kept in coolers and taken t o the laboratory for morphological analysis and powdery mildew species identification within 24 to 48 hours as described in Chapter 3.

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137 W eekly assessment of powdery mildew disease severity was evaluated by visual observations based on disease grad ing scale with 11 categories ranging from 0 100% total leaf area infected (covered by mycelia) as follows: (0) = 0% leaf area infected; (1) =1 to 9%; (2) =10 to 19%; (3) =20 to 29%; (4) =30 to 39%; (5) =40 to 49%; (6) =50 to 59%; (7) =60 to 69%; (8) =70 t o 79%; (9) =80 to 89%; (10) =90 to 99% and (11) =100% of leaf area infected. Breeding lines with severity ratings 4 were considered susceptible to powdery mildew. Lines with ratings of 3 were considered resistant to powdery mildew Statistical D ata A naly sis Data were summarized as means (of 5 plants per plot) for each Rupp breeding line and for two susceptible standards (Table 5 2 ). Analysis of variance (ANOVA) was performed with SAS (SAS Institute, Cary, NC). Numerical data were compared using the Dunc test ( =0.05). In the breeding line screening experiment, disease severity was scored each week up to 9 consecutive weeks starting from first appearance of disease symptoms. The AUDPC (area under the disease progress curve) is an integrated measure which can be calculated directly from observed data or from the logistic function of estimated parameters It is an approach for assessing plant disease epidemic as well as a useful tool for evaluating treatment effectiveness and for the development of di sease control strategies ( 126 ) Additionally, AUDPC has served for the assessment of quantitative resistance in breeding programs ( 125 ) and has been reported as a reliable parameter to estimate and rank the performance of host genotypes according to their ability to retard the rate of disease development ( 93 ) In this study, AUDPC was calculated directly from observed field data (disease severity) and was chosen as a way

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138 to summarize disease progress over time The total plant disease severity (% leaf area affected assessed on 2 leaves per plant and 5 plants per plot) was calculated as the average of weekly ratings starting from time of appearance of first di sease symptoms at each location AUDPC value s were calculated according to Equation 5 1 (5 1 ) Where t is time ( in days ) at the i th observation (rating) y is the percentage of affected foliage (powdery mildew severity) at the i th observation and n is the total number of observations ( 300 ) Results and D iscussion Powdery Mildew Pathogen C haracterization Upon morphological and molecular analysis of powdery mildew samples collected randomly, each week, from suscepti ble types ( Butterbush and Mickey Lee ) and from some of the Rupp breeding lines isolates tested in this study were determined to be Podosphaera xanthii Characteristic morphological features such as erect conidiophores, immature conidia with crenate edge lines, ellipsoid to ovoid hyaline conidia presence of fibrosin bodies and conidia dimensions (length and width ) were consistent with published reports for P. xanthii and comparable to observations made previously in Chapter 3. Disease Severity E valuation Powdery mildew infection at both field sites in north Florida (Live Oak and Citra) was characterized by a wide range of reactions. Symptoms ranged from little fungal sporulation to 100% coverage of mycelia on susceptible plant s ; moderate yellowing to senescing leaves, and premature plant senescence and defoliation which caused fruit to

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139 be exposed and suffer sun damage Powdery mildew disease symptoms progr essed over a period of 9 we ek s at Live Oak and of 6 weeks at Citra Highly suscept ible cultivars (Butterbush and Mickey Lee ) became infected first at Live Oak on 13 May 2009 (Figure 5 2) and four weeks later (16 June 2009) disease symptoms were observed at Citra (Figure 5 3) Initial d is ease severity ratings, for bo th field locations, were visually assesse d and recorded at the appearance of first disease symptoms observed There were no significant differences ( P > 0.05) in disease severity (% leaf area in Live Oak and Citra (Figure 4 5). Overall, ratings taken on a weekly basis were continually increasing to harvest. At both locations, disease increased from 0 to 76% leaf area infected, with variability in rate of disease progress. In contrast, cumulati ve AUDPC values were significantly different ( P <0.05) between locations (Table 5 3 and Figure 5 5 and ) This may have been explained by the fact that AUDPC values represent a buildup of disease at each location over time (9 or 6 weeks for Live Oak and Citra respectively) and is not based on single ratings Dissimilarities in plant maturity when powdery mildew infection initially occurred and the presence of downy mildew ( Pseudoperonospor a cubensis (Berk. & M.A. Curtis) Rostovzev ) and aphids ( Myzus spp Sulzer and Aphis spp. ) at Citra may have led to premature plant decline and affected disease severity assessment B efor e powdery mildew disease peaked at Live Oak, B utterbush p lants thrived and were not severely impacted by downy mildew and aphid s in the same way as the plants at the other location younger when first disease symptoms were observed at Citra S usceptible plants were

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140 healthier and survived longer at Live Oak which could explain why there was gradual disease pressure build up at Live Oak compared to Citra (Figure 5 5) Of the 22 Rupp breeding lines evaluated, a total of 19 lines were monitored. Some Rupp plots could not be assessed during the 9 we ek period due to severe downy mildew infection, confirmed via microscopic examination of leaf samples which killed plants prematurely No data were collected from three Rupp lines ( lines 13, 14 and 15) which were omitted due to poor seed germination and/o r viability. Upon a nalysis of disease severity for each Rupp line, 10 breeding line s had disease severity 6 represented by ratings of at least 5 0 % of mycelia coverage on plants and indicating susceptibility to powdery mildew Nine breeding lines had disease severity ratings of 4 or 5 indicated by 30 to 49% leaf area covered by mycelia Two breeding lines were statistically different from the rest ( P<0.05 ) and had disease severity rating which represented some resistance to the pathogen ( Table 5 2 ). According to disease severity ratings, susceptible cultivars (Butterbush and Mickey Lee) were not statistically different ( P>0.05 ) f rom some Rupp breeding lines ( Table 5 2). According to our calculated AUDPC values, most breeding line s were not significantly different ( P>0.05 ) in disease from susceptible control Butterbush and Mickey Lee (Figure 5 5) Breeding line s 12 ( 08 CvU3124 ) and 5(08 CqU2659 6) were statistically different ( P<0.05 ) from the rest and displayed the highest level of res istance to powdery mildew The identification of powdery mildew resistance in Rupp lines 5 and 12 indicates that deployment of these lines under loca l (north Florida) conditions could result in a healthier crop with less reliance on fungicide applications. A few cultigens (lines 3, 7, 1 and 9 ) demonstrated some level of resistance that was better than the

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141 susceptible controls but not statistically significan t ( P >0.05) No signifi cant difference ( P>0.05 ) was detected in disease severity between Butterbush and Mickey Lee suggesting that these plants were good control checks in this study. However, line s 21(SB368 3) and 2 (06 CvU2029 29 7) showed statistically higher ( P<0.05 ) susceptibility to powdery mildew compared to all other culti gen s tested (Figure 5 5) If a particular line was considered susceptible to powdery mildew ( in Live Oak, FL ) it may have b e e n that it did not carry the correct wild genome segment ( the 12.5% from either wild parent ). Additionally the susceptibility of a particular line may also have be en explained by the fact that the not to a specific powdery mildew race present in Florida at the time of disease assessments The powdery mildew pathogen characterized in this study may have differed in pathogenicity and virulence to the breeding lines evaluated at Live Oak. W e expected to find one or more of the breeding lines to be resistant or tolerant to the pathogen populat ion present in the field during the s pring of 200 9 however more studies are needed to definitely confirm which race or races of cucurbit powdery mildew are present in Florida and to establish if and which specific plant resistance genes were effective agai nst single or select genotypes of powdery mildew pathogens

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142 Table 5 1. List of 22 elite breeding lines supplied by Rupp Seeds. Cultigens were evaluated for resistance to cucurbit powdery mildew present in north Florida No. Breeding Line s Cucurbit Specie 1 01 CsC460 1 5 4 4 3 9 Cucurbita sp. 2 06 CvU2029 29 7 Cucurbita sp. 3 08 CqU2659 3 Cucurbita sp. 4 08 CqU2659 5 Cucurbita sp. 5 08 CqU2659 6 Cucurbita sp. 6 08 CqU2659 7 Cucurbita sp. 7 08 CqU2659 8 Cucurbita sp. 8 08 CqU2659 9 Cucurbita sp. 9 08 CvU3090 Cucurbita sp. 10 08 CvU3091 Cucurbita sp. 11 08 CvU3123 Cucurbita sp. 12 08 CvU3124 Cucurbita sp. 13 08 CvU3125 Cucurbita sp. 14 08 CvU3139 Cucurbita sp. 15 08 CvU3163 Cucurbita sp. 16 08 CvU3164 Cucurbita sp. 17 08 CvU3253 Cucurbita sp. 18 08 CzU3089 Cucurbita sp. 19 08 CzU3130 Cucurbita sp. 20 SB359 7 Cucurbita sp. 21 SB368 3 Cucurbita sp. 22 SD358 Cucurbita sp. 23 SQ4 10 ( Butterbush ) Cucurbita moschata 24 WM23 20 ( Mickey Lee ) Citrullus lanatus C ommercially available c ultivars Butterbush (23) and Mickey Lee (24) are known to be highly susceptible to powdery mildew in Florida.

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143 Table 5 2. E valuation of Rupp elite breeding lines represented by percent leaf area affected and disease severity rating caus ed by po wdery mildew at Live Oak, FL during spring 2009. Breeding Line Cultigen s % leaf area affected Average d isease severity r ating 21 72 24.82 A 8 2 69 23.73 AB 7 20 58 22.92 ABC 6 8 56 14.08 BCD 6 22 54 20.28 BCDE 6 18 54 20.61 BCDE 6 16 54 19.96 BCDE 6 23 ) 54 17.7 BCDE 6 11 52 23.06 CDEF 6 17 52 22.6 CDEF 6 19 49 19.24 CDEF 5 6 48 17.68 CDEF 5 10 48 14.36 CDEFG 5 24 ) 43 17.96 CDEFG 5 4 43 14.59 CDEFG 5 3 41 18.45 CDEFG 5 7 40 15.86 DEFG 5 1 37 14.42 EFG 4 9 36 12.53 FG 4 5 30 12.82 GH 3 12 18 10.85 H 2 Data represents the mean of n=32 Values followed by the same letters are not statistically significant (Duncan, = 0.05). Disease severity ratings are v isual estimates taken based on grading scale with 11 categories where : (0)= 0% leaf area infected; (1)=1 to 9%; (2)=10 to 19%; (3)=20 to 29%; (4)=30 to 39%; (5)=40 to 49%; (6)=50 to 59%; (7)=60 to 69%; (8)=70 to 79%; (9)=80 to 89%; (10)=90 to 99%; (11)=100% of l eaf area infected Breeding lines with severity ratings 6 were considered susceptible to powdery mildew. Lines with ratings of 4 or 5 were considered moderately resistant and lines with ratings of 3 were resistant to powdery mildew. Description of each c ultigen is presented in Table 5 1. Values represent % leaf area affected followed by the standard deviation.

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144 Table 5 3. Evaluation of powdery mildew susceptible cultivar Butterbush represented by AUDPC (area under disease progress curve) and average d isease severity (% leaf area affected) at Live Oak and Citra, FL during spring 2009. Cultivar Butterbush (location) AUDPC Average disease severity (% leaf area affected) 23 ( Live Oak ) 2735 478.7 A 5 3.6 17.7 A 2 3 ( Citra ) 1951 206.4 AB 4 7 .0 32.6 A Data represents the mean of n=32 for Live Oak and n=56 for Citra. Values followed by the same letters are not statistically significant (Duncan, =0.05). Average disease severity based grading scale with 11 categories ranging from 0 to 100% AUD PC was calculated directly from observed field data (disease severity) and was chosen as a way to summarize disease progress over time The total plant disease severity (% leaf area affected assessed on 2 leaves per plant and 5 plants per plot) was calcula ted as the average of weekly ratings starting from time of appearance of first di sease symptoms at each location. Values represent AUDPC and average disease severity followed by the standard deviation.

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145 Figure 5 1 Powdery mildew d isease on 22 Rupp breeding lines ( Cucu r bita spp.) and on powder y mildew susceptible cultivars Butterbush and Mickey Lee at Live Oak FL field site during spring 2009 Increase p owdery mildew disease resulted in premature plant senescence, defoliation and exposure of fruit to sunscald. Disease severity assessed on (A) 26 May (B) 10 June (C) 16 June (D) 23 June (E) 01 July (F) 23 July. E F D C A B

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146 Figure 5 2 Detail of p owdery mildew disease on the same plant of susceptible cultivar Butterbush at Live Oak FL field site during spring 2009. Increase powdery mildew disease resulted in premature plant senescence, defoliation and exposure of fruit to sunscald Dis ease severit y assessed on (A) 13 May (B) 26 May (C) 10 June (D) 23 June (E) 10 July (F) 23 July 2009 A B C D E F

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147 Figure 5 3 Detail of p owdery mildew disease on the same plant of susceptible cultivar Butterbush at Citra FL, field site during spring 2009. Increase powdery mildew disease resulted in premature plant senescence, defoliation and death. In addition to powdery mildew, plants at Citra were heavily affected by downy mildew ( Pseudoperonospor a cubensis ) and by aphid pests ( Myzus spp and Aphis spp. ) before fruit set. Di sease severity assessed on (A) 16 June (B) 23 June (C) 01 July (D) 10 July (E) 23 July (F) 2 8 July 2009 A B C D E F

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1 48 Figure 5 4. Disease severity (percent leaf area infected) of powdery mildew on (Citra). At Live Oak, day 0 corresponded to 13 May 2009 and at Citra day 0 was 16 June 2009. Live Oak values are represented by dotted line and Citra by solid line. Figure 5 5. The cumulative AUDP C (area under disease progress curve) of weekly powdery mildew assessment on susceptible Butterbush infected at Live Oak (dotted li ne) and at Citra (solid line). AUDPC v alues were calculated from disease severity data presented in Figure 5 4. Time 0 ( z ero ) represents the week prior (no symptoms) to powdery mildew appearance at each field site. 0 20 40 60 80 100 0 10 20 30 40 50 60 70 % leaf area infected time (days) 0 500 1000 1500 2000 2500 3000 0 10 20 30 40 50 60 70 Cumulative AUDPC time (days)

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149 Figure 5 6. Pedigree of Rupp powdery mildew breeding lines evaluated at Live Oak, FL during field trial in spring of 2009 (Pedigree prepared by Dr. Eileen Kabelka, formerly of UF Department of Horticulture Science, cur rently with Harris Moran Seed Co., CA) C. lundelliana PI 438542 C. moschata 01 CsC460 C. okeechobeensis ssp. martinezzi PI 532363 C. moschata 01 CsC460 X X 06 CvU2029 50% C. lundelliana 50% C. moschata 05 CzU1358 50% C. okeechobeensis 50% C. moschata X 07 CqU2434 25% C. lundelliana 25% C. okeechobeensis 50% C. moschata X C. moschata SB 359 7 08 CqU2659 5 12.5% C. lundelliana 12.5% C. okeechobeensis 75% C. moschata

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150 Figure 5 7 The cumulative area under th e disease progress curve of powdery mildew on 22 elite breeding lines (Rupp Seeds Inc.) inoculated by natural infection at Live Oak, FL during spring of 2009. Data represen ts the mean of n=4 Values with similar letters are not sign ificantly different (Duncan, = 0.05). The b lack bar (cultigen 23) represents susceptible winter butte and white Area under th e disease progress curve was calculated using the formula where t is time in days between each evaluation, y is the percentage of af fected foliage at each rating time and n is the number of ratings. Description of each cultigen is presented in Table 5 1. a ab bc cd cde cde cde cde cde cdef cdefg cdefg cdefg defgh defgh efgh efgh fgh gh hi i 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 21 2 20 8 16 18 22 23 11 17 19 6 10 4 24 3 7 1 9 5 12 AUDPC Cultigens

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151 CHAPTER 6 SUMMARY AND CONCLUSIONS Cucurbit powdery mildew caused by two obligate fungal pathogens, Podosphaera xanthii and Golovinomyces cichoracearum has been reported to cause crop losses in cucurbit production worldwide ( 218 ) The disease can significantly reduce yield by decreasing fruit size, number of fruits, and length of time fruits can be harvested ( 353 ) Fruit quality and marketability can also be affected due to premature leaf senescence causing fruits to become exposed to sunscald ( 214 ) Recently, there has been an increase in occurrence and severity of powdery mildew in Florida, resulting in heightened concern with fungicide resistance and potentially a change in the race composition. Cucurbit powdery mildew occurs on cucumber, melon, squash, zucchini, pumpkin, gourd and more recently, on watermelon where the incidence of outbreaks has increased and the disease has become an important problem in the major U.S. production areas. This study focuse d on investigating the recent increase in incidence, severity and host range of cucurbit powdery mildew in Florida. An efficient technique for in vivo establishment and maintenance of cucurbit powdery mildew isolates collected throughout Florida was develo ped. The first objective was to identify and characterize the prevailing causal agent of cucurbit powdery mildew in north central Florida through morphological features and DNA analysis. The second objective was to assess the presence of physiological race s within cultured isolates using detached muskmelon leaf bioassays. The third objective was to evaluate the varietal reactions of 22 Cucurbita elite breeding lines (Rupp Seed Inc.) for susceptibility to powdery mildew under local (Florida) field conditions

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152 Cucurbit Powdery Mildew Isolate Collection, Maintenance and Characterization The causal agents of cucurbit powdery mildew disease produce identical symptoms and can be difficult to differentiate in the absence of the teleomorphic stage. However morpholog ical features of P xanthii differ from those of G. cichoracearum at the anamorphic (asexual) stage and include: size and shape of conidia, presence of fibrosin bodies, immature conidia edge morphology and germ tube morphology ( 22 24 36 304 ) In this study, we identified and characterized single colony isolates of cucurbit powdery mildew from multiple field sites, dates, and cucurbit host s. Two butternut winter squash cultivar Butterbush fields in north central Florida (Live Oak and Citra) were sampled and additional cucu rbit iso lates were collected from southwest and north east conidia, ellipsoid to ovoid in shape, with conidial dimensions of 31 44 x 15 100) and footcells of 45 6 7 x 10 and conidia edge lines were crenate. Isolates were subjected to multiplex polymerase chain reactions (PCR) with species specific primers S1/S2 (for P. xanthii ) and G1/G2 (for G. cichoracearum ) a nd a specific PCR product of 454 bp was amplified from genomic DNA of most isolates. Based on morphological and molecular analyses, all cucurbit powdery mildew isolates were identified as P. xanthii Additionally, in this study, a method for culture mainte nance of cucurbit powdery mildew isolates on cotyledons and first leaflets of some cucurbit types was developed. Previous studies had demonstrated that isolates can be maintained using leaf disks ( 161 192 218 ) or whole plants ( 66 319 ) and w e showed that detac hed leaflets can be an efficient substrate for powdery mildew isolate maintenance and for in vivo bioassays.

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153 Determination of Powdery Mildew Physiological Races To date, approximately 30 distinct physiological races of P. xanthii ( 39 190 ) and 2 races of G. cichoracearum ( 71 158 322 ) have been identified worldwide. Traditionally, cucurbit powdery mildew races have been defined by the disease response of the pathogen isolate on a set of muskmelon differentials which can differentiate cucurbit powdery mildew races originating from melon, cucumber, Cucurbita spp. and watermelon ( 157 ) In this study we evaluated the reactions of 12 muskmelon genotypes to 5 powdery mildew isolates (10 d ays post inoculation). Our observations indicated variability in isolate virulence. When a muskmelon genotype was susceptible to a certain isolate, percentages of leaf area covered by powdery mildew typically showed extensive interwoven mycelium and abunda nt conidiophore production. Three muskmelon genotypes were resistant to all 5 powdery mildew isolates tested, exhibiting less than 25% of leaf area infected. In contrast, three other genotypes were susceptible to all fungal isolates and had ratings as high as 75% leaf area covered with mycelia. Based on currently recognized races of P. xanthii using a differential profile on muskmelon (Dr. James D. McCreight, personal communication) our isolates did not belong to any of the currently described races of P. x anthii Our results indicated that we may have found race 2F or race 2Z or race 3. Three isolates tested (UF, IM and DO) were undefined to date and not consistent with any o f the previously described race profiles of P. xanthii A different P. xanthii path ogenic race may be present in the field sites sampled throughout Florida or a mixed race colonies were isolated during our sampling process Further replication and careful monitoring or environmental conditions during inoculation experiments are suggested approaches for future work. Further characterization studies

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154 of the powdery mildew pathogen population in Florida should be performed to d etermine if other pathogenic strains could be present at different times of the year and in different regions. By identifying these pathogen populations researchers will be able to distinguish and recommend appropriate control strategies for powdery mildew disease in Florida and better understand and match host resistance to local pathogen populati o ns This information is important for cucurbit breeders, plant pathologists and extensionists, as well as cucurbit growers. T ypically, the control of powdery mildew in susceptible cucurbit cultivars is achieved with the use of fungicides. However, the repeated use of site specific products over time has resulted in powdery mildew resistance to some commercial chemical compounds. Distinct physiological pathotypes and races of P. xanthii have been detected with resistance to as many as eight classes of fungicides ( 302 ) Presence of resistant fungal strains has been associated with lack of powdery mildew control ( 161 ) Resistant cucurbit varieties are being developed and are becoming an increasingly important component of powdery mildew management programs in the U.S. ( 123 ) and elsewhere ( 347 ) In this study, we evaluated the powdery mildew resistance response of 22 elite breeding lines developed by Rupp Seed Inc. (Wauseon, OH) which contained parentage from two wild cucurbit species ( Cucurbita lundelliana and C. okeechobeensis ) with known resistance to powdery mildew. Disease response was measured by pathogen development, disease severity and AUDPC ( area under the disease progress curve). Powdery mildew infection at two field sites in north central Florida (Live Oak and Citra) was characterized by little fungal sporulation to 100%

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155 coverage of mycelia on susceptible plants; moderate yellowing to senesc ing leaves, and premature plant senescence and defoliation which caused fruit to be exposed to sunscald. Analysis of disease severity indicated that 10 breeding lines had disease severity greater than 50% indicating susceptibility to powdery mildew and two breeding lines were statistically different from the rest and demonstrated some level of resistance to powdery mildew. Susceptible cultivars (Butterbush and Mickey Lee) known to be highly susceptible to powdery mildew in Florida, were not statistically different from some of the susceptible breeding lines. If a particular breeding line was considered susceptible to powdery mildew it may have been that it did not carry the correct wild genome segment (from either wild parent) or it may have been because the source of resistance was not to a specific powdery mildew race present in Florida at the time of disease assessments. AUDPC values for most breeding lines were not significantly different from the susceptible cultivars. Two breed ing lines presented lower calculated AUDPC and were statistically different from the rest. Two other lines showed statistically higher AUDPC compared to all other cultigens tested. A possible explanation would be that AUDPC values represent a buildup of di sease over time (9 or 6 weeks for Live Oak and Citra respectively) and is not based on a single rating. Dissimilarities in plant maturity and the presence of downy mildew and aphids at Citra may have led to premature plant decline and adversely affected po wdery mildew disease assessments. were not severely impacted by other disease or pests in the same way as plants at the other location. Butterbush plots were planted later an d plants were younger when first disease symptoms were observed at Citra. Susceptible plants were healthier and

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156 survived longer at Live Oak which could explain why there was gradual disease pressure build up compared to Citra. Additional studies with less variation which caused plants to senesce prematurely, before complete powdery mildew assessment would be recommended for a more comparable evaluation between locations. Ideally plants evaluated at each location should have been planted at the same time so that possible effects such as plant maturity and interference of other diseases would be reduced. Further studies are needed to confirm which race or races of cucurbit powdery mildew are present in Florida and to establish which specific plant resistance g enes would be effective against single or select genotypes of powdery mildew pathogens. Discovery, deployment, and adoption of host resistance to powdery mildew will potentially advance the profitability and reduce reliance upon fungicides in Florida cucur bit crops.

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157 APPENDIX LIST OF POWDERY MILD EW SUSCEPTIBLE HOST Table A 1. List of powdery mildew susceptible cucurbit hosts used throughout this research PI (Plant Introduction) Cultivar Cucurbit specie Cucurbit type SQ4 10 Butterbush Cucurbita moschata butternut squash WM23 20 Mickey Lee Citrullus lanatus watermelon All Sweet Citrullus lanatus watermelon PMR 45 Ames 26811 Cucumis melo muskmelon PMR 5 Ames 26809 Cucumis melo muskmelon MR 1 Ames 8578 Cucumis melo muskmelon PI 414723 Cucumis melo muskmelon Edisto 47 NSL 3 4600 Cucumis melo muskmelon Topmark NSL 30032 Cucumis melo muskmelon Athena Hybrid Cucumis melo cantaloupe Best Jumbo Cucumis melo cantaloupe SQ72 20 Table Ace Cucurbita pepo acorn squash PM2 10 Big max Cucurbita pepo pumpkin Long Island Cheese Cucurbita moschata pumpkin CU57 20 Poinsett 76 Cucumis sativus cucumber CU34 20 Straight 8 Cucumis sativus cucumber SMR 58 Wisconsin Cucumis sativus cucumber Waltham Cucurbita moschata butternut squash

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158 LIST OF REFERENCES 1. Adam, L., Ellwood, S., Wilson, I., Saenz, G., Xiao, S., Oliver, R. P., Turner, J. G., and Somerville, S. 1999. Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens. Mol. Plant Microbe Interact. 12:1031 1043. 2. Adeniji, A. A., and Coyne, D. P. 1983. Genetics and mature resistance to powdery mildew in crosses of butternut with calabaza squash and 'Seminole pumpkin' Journal of the American Society for Horticultural Science 108:360 368. 3. Agrios, G. N. 1997. Plant Pathology. 4th ed. Academic Press, San Diego, CA. 4. Al Kherb, S. M. 1 994. Prevalent races of cucurbit powdery mildews in Riyadh region of Saudi Arabia. Assiut Journal of Agricultural Sciences 25:249 252. 5. Alfandi, M., Ji, Y., Shen, L., Qi, X., Xu, Q., and Chen, X. 2010. Construction of genetic linkage map and localization of QTLs for powdery mildew resistance in cucumber ( Cucumis sativus L.). Acta Horticulturae 871:33 42. 6. Alvarez, J. M., Gomez Guillamon, M. L., Tores, N. A., Canovas, I., and Floris, E. 2000. Virulence differences between two Spanish isolates of Sphaerot heca fuliginea race 2 on melon. 67 69 In: Proceedings of the 7th EUCARPIA Meeting on Cucurbit Breeding and Genetics, Cucurbitaceae 2000 Acta Horticulturae, Ma'ale Ha Hamisha, Israel. 7. Amano, K. 1986. Host Range and Geographical Distribution of the Powd ery Mildew Fungi. Japan Scientific Societies Press, Tokyo, Japan. 8. Anagnostou, K., Jahn, M., and Perl Treves, R. 2000. Inheritance and linkage analysis of resistance to zucchini yellow mosaic virus watermelon mosaic virus papaya ringspot virus and powd ery mildew in melon. Euphytica 116:265 270. 9. Andres, T. C., and Decker Walters, D., Cucurbitaceae. Cucurbit Network. Retrieved 6 July 2011, from http://www.cucurbit.org/family.html 10. Bardin, M., Carli er, J., and Nicot, P. C. 1999. Genetic differentiation in the French population of Erysiphe cichoracearum a causal agent of powdery mildew of cucurbits. Plant Pathology 48:531 540. 11. Bardin, M., Dogimont, C., Nicot, P., and Pitrat, M. 1997. Genetic analysis of resistance of melon line PI 124112 to Sphaerotheca fuliginea and Erysiphe cichoracearum studied in recombinant inbred lines. 163 168 In: Proceedings of the First International S ymposium on Cucurbits International Society of Horticulture Science, Adana, Turkey.

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188 BIOGRAPHICAL SKETCH Gabriella Silveira Maia, was born in Vicosa, MG Brazil, in 1977 to Jaime Maia dos Santos and Arlete Silveira. Gabriella has a younger sister, Fabiola Silveira Maia. Gabriella graduated from UNESP (Universidade Estadual Paulista in Jaboticabal, Sao Paulo, B razil) in 2003, with a bachelor degree in Agriculture Engineering. On August 1999, she was awarded a Fulbright Scholarship and spent one year at Texas A&M University as an undergraduate student in Agronomy. On August 2002, Gabriella came to the University of Florida (UF) in Gainesville for a 3 month internship in the Plant Pathology Department. She worked in the laboratory of Dr. Raghavan Charudattan and assisted in research trials concerning biological control of weeds. On July 2003, Gabriella went to Del emont, Switzerland, where she worked with biological management of invasive weeds and insect pests, in the laboratory of Dr. Andre Gassman, at CABI Europe, until November 2003. On June 2004 in Vicosa ,Minas Gerais, Brazil, Gabriella married Alberto Azered o, a Food Engineer. They moved to Brasilandia de Minas and worked at Fuchs Gewrze until February 2005. During this time, Gabriella managed an insect breeding laboratory which focused on biological control of insect pests. Gabriella returned to Gainesvill e, in 2005 to work as a research assistant at the Plant Pathology Department at UF. She again, worked in the laboratory of Dr. Charudattan until she became a graduate student in 2008. In August 2010, Gabriella and Alberto had their first daughter, Anna Aze redo. Gabriella finished her MS degree in Plant Pathology in May 2012. She has a strong interest in plant pathology extension and in research, and intends to continue her studies and pursue a Ph.D. degree.